SEED UPGRADE AND GERMINATION STRATEGIES FOR ALNUS TENUIFOLIA AND BETULA OCCIDENTALIS BY CINDY LEE JONES, B.S. A Thesis submitted to the Graduate School in partial fulfillment of the requirements for the degree Master of Science Major Subject: Horticulture Minor Subject: Experimental Statistics New Mexico State University May 2000
120
Embed
SEED UPGRADE AND GERMINATION STRATEGIES BETULA ...
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
A Thesis submitted to the Graduate School
in partial fulfillment of the requirements
for the degree
Master of Science
Major Subject Horticulture
~ Minor Subject Experimental Statistics
New Mexico State University
May 2000
Seed Upgrade and Germination Strategies for Alnus tenuifoha and
Betula occidentahs a thesis prepared by Cindy Lee Jones in partial
fulfillment of the requirements for the degree Master of Science has
been approved and accepted by the following
Chair of the Examining Committee
Committee in charge
Dr John T Harrington Chair
Dr David R Dreesen
Dr Leigh Murray
Dr Geno A Picchioni
11
DEDICATION
This work is dedicated to my mother Eula1a Jones who
supported me unwaveringly in this eifort and who sacrificed and
worked as much as I to accomplish the end result and to my late
father Earl Jones who never doubted me even when I doubted
myself
ill
ACKNOWLEDGMENTS
I wish to thank my advisor Dr John T Harrington for his
assistance and support in every phase of this study and for allowing
me the use of the facilities at the Mora Research Center in Mora
New Mexico
I wish to thank Dr Leigh Murray for her extensive assistance in
the data analysis of this project and for her professional informative
and helpful manner
Thanks also to Dr David Dreesen for his guidance in
evaluating ideas for the study and to Dr Geno Picchioni for his
support guidance and excellent instruction over the years
My special thanks to Molycorp for the funding which made this
study possible
lowe a greatdebt ofgratitude to my cousin Eugenia Shepan
and her husband Don who opened their home to me and gave me
their love and support and to my supervisor at University Hospital
Virginia Nymeyer for her faithful friendship
IV
VITA
October 8 1957 Born at Clayton New Mexico
1975 Graduated from Belen High School Belen New Mexico
1984-1987 Medical Technologist Santa Fe Medical Labs Santa Fe New Mexico
1987-present Medical Technologist Tricore Reference Laboratories at University Hospital Albuquerque New Mexico
1997--present Research Assistant Department ofAgronomy and Horticulture New Mexico State University
PROFESSIONAL AND HONORARY SOCIETIES
American Society for Horticultural Science
American Society of Clinical Pathologists
American Society for Clinical Laboratory Science
Phi Kappa Phi
FIELD OF STUDY
Major Field Horticulture
Minor Field Experimental Statistics
v
ABSTRACT
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
Master of Science in Horticulture
New Mexico State University
Las Cruces New Mexico 2000
Dr John T Harrington Chair
Little is known about the propagation of thinleaf alder (Alnus
tenuifolia) and water birch (Betula occidentalis) These species
native to New Mexico have the potential to be useful trees for
rehabilitation of disturbed lands and possibly landscaping An
efficient and economical method for propagation is needed Birch
and alder share many common seed characteristics including small
V1
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
Seed Upgrade and Germination Strategies for Alnus tenuifoha and
Betula occidentahs a thesis prepared by Cindy Lee Jones in partial
fulfillment of the requirements for the degree Master of Science has
been approved and accepted by the following
Chair of the Examining Committee
Committee in charge
Dr John T Harrington Chair
Dr David R Dreesen
Dr Leigh Murray
Dr Geno A Picchioni
11
DEDICATION
This work is dedicated to my mother Eula1a Jones who
supported me unwaveringly in this eifort and who sacrificed and
worked as much as I to accomplish the end result and to my late
father Earl Jones who never doubted me even when I doubted
myself
ill
ACKNOWLEDGMENTS
I wish to thank my advisor Dr John T Harrington for his
assistance and support in every phase of this study and for allowing
me the use of the facilities at the Mora Research Center in Mora
New Mexico
I wish to thank Dr Leigh Murray for her extensive assistance in
the data analysis of this project and for her professional informative
and helpful manner
Thanks also to Dr David Dreesen for his guidance in
evaluating ideas for the study and to Dr Geno Picchioni for his
support guidance and excellent instruction over the years
My special thanks to Molycorp for the funding which made this
study possible
lowe a greatdebt ofgratitude to my cousin Eugenia Shepan
and her husband Don who opened their home to me and gave me
their love and support and to my supervisor at University Hospital
Virginia Nymeyer for her faithful friendship
IV
VITA
October 8 1957 Born at Clayton New Mexico
1975 Graduated from Belen High School Belen New Mexico
1984-1987 Medical Technologist Santa Fe Medical Labs Santa Fe New Mexico
1987-present Medical Technologist Tricore Reference Laboratories at University Hospital Albuquerque New Mexico
1997--present Research Assistant Department ofAgronomy and Horticulture New Mexico State University
PROFESSIONAL AND HONORARY SOCIETIES
American Society for Horticultural Science
American Society of Clinical Pathologists
American Society for Clinical Laboratory Science
Phi Kappa Phi
FIELD OF STUDY
Major Field Horticulture
Minor Field Experimental Statistics
v
ABSTRACT
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
Master of Science in Horticulture
New Mexico State University
Las Cruces New Mexico 2000
Dr John T Harrington Chair
Little is known about the propagation of thinleaf alder (Alnus
tenuifolia) and water birch (Betula occidentalis) These species
native to New Mexico have the potential to be useful trees for
rehabilitation of disturbed lands and possibly landscaping An
efficient and economical method for propagation is needed Birch
and alder share many common seed characteristics including small
V1
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
DEDICATION
This work is dedicated to my mother Eula1a Jones who
supported me unwaveringly in this eifort and who sacrificed and
worked as much as I to accomplish the end result and to my late
father Earl Jones who never doubted me even when I doubted
myself
ill
ACKNOWLEDGMENTS
I wish to thank my advisor Dr John T Harrington for his
assistance and support in every phase of this study and for allowing
me the use of the facilities at the Mora Research Center in Mora
New Mexico
I wish to thank Dr Leigh Murray for her extensive assistance in
the data analysis of this project and for her professional informative
and helpful manner
Thanks also to Dr David Dreesen for his guidance in
evaluating ideas for the study and to Dr Geno Picchioni for his
support guidance and excellent instruction over the years
My special thanks to Molycorp for the funding which made this
study possible
lowe a greatdebt ofgratitude to my cousin Eugenia Shepan
and her husband Don who opened their home to me and gave me
their love and support and to my supervisor at University Hospital
Virginia Nymeyer for her faithful friendship
IV
VITA
October 8 1957 Born at Clayton New Mexico
1975 Graduated from Belen High School Belen New Mexico
1984-1987 Medical Technologist Santa Fe Medical Labs Santa Fe New Mexico
1987-present Medical Technologist Tricore Reference Laboratories at University Hospital Albuquerque New Mexico
1997--present Research Assistant Department ofAgronomy and Horticulture New Mexico State University
PROFESSIONAL AND HONORARY SOCIETIES
American Society for Horticultural Science
American Society of Clinical Pathologists
American Society for Clinical Laboratory Science
Phi Kappa Phi
FIELD OF STUDY
Major Field Horticulture
Minor Field Experimental Statistics
v
ABSTRACT
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
Master of Science in Horticulture
New Mexico State University
Las Cruces New Mexico 2000
Dr John T Harrington Chair
Little is known about the propagation of thinleaf alder (Alnus
tenuifolia) and water birch (Betula occidentalis) These species
native to New Mexico have the potential to be useful trees for
rehabilitation of disturbed lands and possibly landscaping An
efficient and economical method for propagation is needed Birch
and alder share many common seed characteristics including small
V1
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
ACKNOWLEDGMENTS
I wish to thank my advisor Dr John T Harrington for his
assistance and support in every phase of this study and for allowing
me the use of the facilities at the Mora Research Center in Mora
New Mexico
I wish to thank Dr Leigh Murray for her extensive assistance in
the data analysis of this project and for her professional informative
and helpful manner
Thanks also to Dr David Dreesen for his guidance in
evaluating ideas for the study and to Dr Geno Picchioni for his
support guidance and excellent instruction over the years
My special thanks to Molycorp for the funding which made this
study possible
lowe a greatdebt ofgratitude to my cousin Eugenia Shepan
and her husband Don who opened their home to me and gave me
their love and support and to my supervisor at University Hospital
Virginia Nymeyer for her faithful friendship
IV
VITA
October 8 1957 Born at Clayton New Mexico
1975 Graduated from Belen High School Belen New Mexico
1984-1987 Medical Technologist Santa Fe Medical Labs Santa Fe New Mexico
1987-present Medical Technologist Tricore Reference Laboratories at University Hospital Albuquerque New Mexico
1997--present Research Assistant Department ofAgronomy and Horticulture New Mexico State University
PROFESSIONAL AND HONORARY SOCIETIES
American Society for Horticultural Science
American Society of Clinical Pathologists
American Society for Clinical Laboratory Science
Phi Kappa Phi
FIELD OF STUDY
Major Field Horticulture
Minor Field Experimental Statistics
v
ABSTRACT
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
Master of Science in Horticulture
New Mexico State University
Las Cruces New Mexico 2000
Dr John T Harrington Chair
Little is known about the propagation of thinleaf alder (Alnus
tenuifolia) and water birch (Betula occidentalis) These species
native to New Mexico have the potential to be useful trees for
rehabilitation of disturbed lands and possibly landscaping An
efficient and economical method for propagation is needed Birch
and alder share many common seed characteristics including small
V1
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
VITA
October 8 1957 Born at Clayton New Mexico
1975 Graduated from Belen High School Belen New Mexico
1984-1987 Medical Technologist Santa Fe Medical Labs Santa Fe New Mexico
1987-present Medical Technologist Tricore Reference Laboratories at University Hospital Albuquerque New Mexico
1997--present Research Assistant Department ofAgronomy and Horticulture New Mexico State University
PROFESSIONAL AND HONORARY SOCIETIES
American Society for Horticultural Science
American Society of Clinical Pathologists
American Society for Clinical Laboratory Science
Phi Kappa Phi
FIELD OF STUDY
Major Field Horticulture
Minor Field Experimental Statistics
v
ABSTRACT
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
Master of Science in Horticulture
New Mexico State University
Las Cruces New Mexico 2000
Dr John T Harrington Chair
Little is known about the propagation of thinleaf alder (Alnus
tenuifolia) and water birch (Betula occidentalis) These species
native to New Mexico have the potential to be useful trees for
rehabilitation of disturbed lands and possibly landscaping An
efficient and economical method for propagation is needed Birch
and alder share many common seed characteristics including small
V1
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
ABSTRACT
SEED UPGRADE AND GERMINATION STRATEGIES
FOR ALNUS TENUIFOLIA AND
BETULA OCCIDENTALIS
BY
CINDY LEE JONES BS
Master of Science in Horticulture
New Mexico State University
Las Cruces New Mexico 2000
Dr John T Harrington Chair
Little is known about the propagation of thinleaf alder (Alnus
tenuifolia) and water birch (Betula occidentalis) These species
native to New Mexico have the potential to be useful trees for
rehabilitation of disturbed lands and possibly landscaping An
efficient and economical method for propagation is needed Birch
and alder share many common seed characteristics including small
V1
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
size prolific seed production with low viability and seed dormancy
These characteristics make propagation by seed problematic
Stratification is used to break dormancy in many species including
alder Vegetative propagation is often difficult for alder and birch
The most likely method for propagation is seed in view of the need
for genetic diversity in plants used in restoration Problems with seed
propagation might be solved by refining or upgrading the seed and
the use of stratification to break seed dormancy
The LDS method developed by Milan Simak (1983) for
conifer seeds was evaluated for its effectiveness in refining thinleaf
alder and water birch seeds LDS involves imbibing the seeds
partially re-drying to leave a residue of moisture and separating by a
density method The viable seeds should retain moisture while the
non-viable should not thus creating a density differential between
viable and non-viable seeds
Thinleaf alder and water birch seeds were subjected to simple
density separation by the specific gravity method with and without
IDS treatment Untreated dry seeds untreated imbibed seeds and
the floating and sinking IDS treated seed fractions were subjected to
VII
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
germination tests Three levels of stratification were used in
combination with the LDS study to evaluate the presence of
physiological dormancy in thinleaf alder and water birch
Seed refinement was determined to be useful in improving
germination of thin leaf alder and water birch LDS methods were
found to be useful in the case of thinleaf alder while water birch
germination benefitted most from a simple density separation in
ethanol Twenty~eight days of stratification improved water birch
germination but the actual gain in percentage was small
Stratification was not shown conclusively to be useful in improving
thinleaf alder germination
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
TABLE OF CONTENTS
Page
LIST OF TABLES XlI
LIST OF FIGURES XVI
INTRODUCTION 1
LITERATURE REVIEW 3
Revegetation and Reconstruction 3
Species Selection 4
Planting Methods 5
Birch and Alder Suitability in Reconstruction 7
Production of Stock Plants 9
Seed Dormancy and Methods to Overcome It 10
Germination Requirements 14
Thinleaf Alder 15
Water Birch 17
Seed Quality Improvements 18
OBJECTIVES OF THIS STUDY 20
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
METHODS AND MATERIALS
Page
21
Sources 21
Separation Media 23
Seed Refinement 25
Thinleaf Alder 25
Water Birch 28
Germination Requirements 30
Thinleaf Alder 30
Water Birch 33
DATA ANALYSIS 36
RESULTS 42
Seed Refinement 42
Thinleaf Alder Fill Enhancement 42
Thinleaf Alder Recovery 47
Water Birch Fill Enhancement 49
Water Birch Recovery 54
Germination Requirements 55
Thinleaf Alder 55
x
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
Page
Water Birch 65
DISCUSSION 76
Seed Refinement 76
Germination Requirements 85
Thinleaf Alder 85
Water Birch 90
General Observations 92
LITERATURE CITED 95
Xl
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
LIST OF TABLES
Table Page
221 Seed Source Locations and Elevations
2 Alder Preparation Protocols for Seed Refinement 26
3 Birch Preparation Protocols for Seed Refinement 29
4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder 32
5 Treatment Combinations for Experimental Layout of Randomized Complete Block--Water Birch 35
6 Analysis of Variance Table for Thinleaf Alder Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 42
7 Thinleaf Alder Percentage ofFilled Seeds in Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced by Preparation Protocol 45
8 Thinleaf Alder Percentage of Filled Seeds as Influenced by Separation Fraction 46
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
Table Page
9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 47
10 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Preparation Protocol 48
11 Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source 48
12 Analysis ofV ariance Table for Water Birch Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source--Factorial Analysis 49
13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction 51
14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol and Seed Source--Factorial Analysis 54
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
Table Page
17 Thinleaf Alder Percentage Germination as Influenced by Separation--Factorial Analysis 57
18 Analysis of Variance Table for Thinleaf Alder Percentage Germination as Influenced By Treatment Combination and Seed Source--Augmented Factorial 61
19 Thinleaf Alder Analysis of Contrasts--Augmented Factorial 61
20 Thinleaf Alder Analysis ofV ariance Table--Factorial Analysis without Red River Canyon Seed Source 64
21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source--Factorial Analysis 64
22 Water Birch Percentage Germination Analysis of Variance Table--Factorial Analysis 66
23 Water Birch Percentage Germination as Influenced by Stratification--Factorial Analysis 66
24 Water Birch Percentage Germination as Influenced by Separation--Factorial Analysis 67
XIV
Table Page
25 Water Birch Percentage Gennination as Influenced by Seed Source--Factorial Analysis 67
26 Analysis ofVariance Table for Water Birch Percentage Gennination as Influenced By Treatment COInbination and Seed Source--Augmented Factorial 73
27 Water Birch Analysis of Contrasts--Augmented Factorial 73
LIST OF FIGURES
PageFigure
1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction 44
2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction 52
3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction 53
4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source 58
5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification 59
6 Alder Percentage Germination as Influenced by Imbibition and Seed Source 62
7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source 68
8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction 69
9 Birch Percentage Germination as Influenced by Stratification and Seed Source 70
10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification 71
XVI
Figure Page
11 Birch Percentage Germination as Influenced by Imbibition and Seed Source 75
12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 81
13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 83
INTRODUCTION
Birch (Betula) and alder (Alnus) are two genera of Betulaceae
trees found in riparian areas throughout New Mexico The presence
ofbirch and alder in riparian zones of New Mexico has been noted in
many early surveys of the region (Britton 1908 Sargent 1901 1905
Wooton and Standley 1915) Water birch (Betula ocddentalisHook
formerly B fontinalis Sarg) is found in the northern mountains of the
state (Martin and Hutchins 1980) New Mexico has two species of
alder Arizona alder (Alnus oblongifolia Torr) which is found in the
mountains of southwest New Mexico (Martin and Hutchins 1980
Vines 1960) and thinleaf alder (Alnus tenuifolia Nutt) designated by
Carter (1997) as Alnus incana ssp tenuifolia Nutt found in the
northern and western mountains (Martin and Hutchins 1980 Vines
1960) Until recently existence of these species has been of interest
mainly from a botanical standpoint However with increasing landshy
use in the western United States these trees may have a further
purpose in the revegetation of degraded riparian areas and as oasis
plants for those interested in native landscapes (Phillips 1995)
Successful revegetation of degraded areas is influenced by many
1
factors including the site conditions commonly encountered and the
chosen plant material Desirable plant material should be wellshy
adapted to the site have high survival and be economical to obtain or
produce
LITERATURE REVIEW
Revegetation and Reconstruction
Strategies for revegetation of disturbed lands are generally
divided into three categories restoration reclamation and
rehabilitation Restoration is the complete replication of the original
conditions species habitat and function of the area Reclamation
involves returning the area to a condition that is habitable by the
organisms that were originally present or organisms that approximate
the original inhabitants Rehabilitation involves returning the land to
a form and function which conforms to a prior land-use plan
including a stable ecological state that does not contribute
substantially to environmental deterioration and is consistent with
surrounding aesthetic values (Allen 1988 National Academy of
Sciences 1974) These three categories have been collectively termed
reconstruction by Allen (1988) Complete restoration is often not
practical as certain requisite intermediate conditions of varying
durations maybe necessary In the arid western United States
natural succession is slow and dependence on natural process risks
further site degradation (National Academy of Sciences 1974)
3
Reclamation and rehabilitation may be more workable concepts A
practical guiding philosophy would be the objective to create a stable
ecosystem that is compositionally and functionally similar to that
which existed prior to human disturbance with the realization that
such a goal is not completely attainable (Burton et al 1988)
Species Selection
It has long been the philosophy of those involved in
reconstruction efforts that the use ofnative and diverse species is
desirable rather than dependence on a few proven species (Daniel
et al 1979 Harker et al 1993 Nielson and Peterson 1973) The
rationale is that native species are better adapted to adverse site
conditions such as low moisture and high surface temperatures and
exposure (Nielson and Peterson 1973) Only native species survived
in a European study even though exotic species examined also
possessed characteristics which were well-adapted to the site (Herrera
et al 1993) Use of diverse native plant species can enhance
reconstruction efforts and sustain more diverse wildlife populations
(Harker et al 1993) Using plant material of local provenance (origin
of seed) to maximize survival is also important (Albers and Carpenter
4
1979 Burton et al 1988 Daniel et al 1979 Hobbs 1984) Species of
plants evolve within their habitat to site conditions including edaphic
topographic and climatic conditions such as temperature (Bewley and
Black 1994) photoperiod (Currie 1990) and growing season A plant
with origins in southern latitudes may not properly harden off for
winter in time to avoid early frost when grown in northern latitudes
with longer day1engths while a plant from northern latitudes may not
have optimal shoot growth in the shorter day1ength of southern areas
(Fowells 1965 Lane 1993)
PlantingMethods
Natural colonization processes can take anywhere from ten to
hundreds ofyears depending on site conditions (National Academy
of Sciences 1974) Planting methods used in reconstruction include
direct seeding wildling transplants and use ofbare-root or
containerized transplant material (Schubert et al 1970) Direct
seeding is often the least expensive planting method but success with
woody species is frequently limited Predation of seed germination
failure and adverse conditions for germinants can result in planting
failure (Fowells 1965 Haeussler et al 1995 Hibbs et al 1994
5
Monsen 1984 Pratt 1986) Wildling transplants may have poor
survival ifplanting is not timed properly and done carefully (Schubert
et al 1970) Use ofnursery grown seedlings bare-root or
containerized can improve survival rates relative to other
reconstruction efforts (Hobbs 1984) The ability to match stock type
(source physiological and morphological condition) to the site
known as the target seedling concept (Rose et al 1990) and greater
latitude in planting conditions (timing) can contribute to improved
transplant success of nursery stock relative to wildlings Combining
direct seeding ofnon-woody plants and nursery-grown seedlings can
be the most efficient and economical method of reconstruction when
costs ofproducing container stock can be kept low (Belcher 1982
Dunlap and Barnett 1984 Rose et al 1990) The success of
reconstruction efforts is heavily dependent on site conditions and the
quality of the plant material used (Monsen 1984) In tum quality of
plant material is dependent on well-developed germination and
culture protocols The economic feasibility of stock propagation for
reconstruction work is dependent on finding methods to efficiently
upgrade seed quality (proportion ofgerminable seeds) and optimize
6
germination capacity and seedling survival (Belcher 1982 Bonner
1984)
Birch and Alder Suitability in Reconstruction
Montane riparian vegetation zones are contained in areas where
the supply ofwater is constant (perennial) as well as areas with an
ephemeral (intermittent) water supply Riparian zones contain both
obligate and facultative riparian species Facultative riparian species
are also found in surrounding open spaces and in high cool nonshy
riparian locations (Dick-Peddie 1993) Riparian vegetation follows an
elevational gradient from the source to the mouth of the drainage
perpendicular to the zone of upland vegetation (Dick-Peddie 1993)
Other habitats where water may be caught but are not part of a true
drainage are termed pseudoriparian Pseudoriparian habitats include
gullies roadside ditches and the bottoms of talus slopes (Dick-Peddie
1993) Most of the obligate riparian species found in riparian and
pseudoriparian areas are adapted to flood conditions with the ability
to rapidly reproduce and colonize a devastated area Characteristics
ofobligate riparian species include prolific seed production efficient
7
seed dispersal fast growth short life-cycles and rapid attainment of
reproductive stage (Dick-Peddie 1993)
Birch and alder species are generally confined to montane
riparian zones (Elias 1980) Members ofboth genera have properties
indicative of obligate riparian species including fast growth prolific
seed production and short life-cycle these properties also make
members of these genera suitable candidates for use in reconstruction
efforts (Elias 1980) Birch and alder are known as pioneer species
which can successfully establish on denuded areas (Young and Young
1992) and which prefer mineral soil for germination and early growth
(Haeussler et al 1995 Schalin 1968) In addition most alder species
including thirlleaf alder and Arizona alder have the ability to fix
atmospheric nitrogen via a symbiotic relationship with root-nodule
forming species of Frankia actinomycetes (Bond 195519711976
Virtanen 1957) Many researchers believe the formation of a dynamic
rhizosphere of this type is critical to the rehabilitation of degraded
lands (Herrera et al 1993 Whitford 1988) Biological nitrogen
fixation in conjunction with the production of large amounts of litter
has been shown to help build up organic matter nitrogen and
8
improve soil structure in deficient soils such as glacial till (Bollen and
Lu 1968 Crocker and Major 1955 Tarrant and Trappe 1971)
Biological nitrogen fixation can also improve conditions for other
non-nitrogen fixing species (Tarrant 1961) and enhance species
diversity (Franklin and Pechanec 1968)
The use ofthese deciduous trees with the objective of improving
the site conditions (ie shade nutrients and organic matter) for other
species (Albers and Carpenter 1979) is a valuable strategy in the
reconstruction of disturbed areas such as mine spoils
Production ofStockP1ants
Efficient propagation ofnursery stock from seed requires
extensive knowledge of the germination requirements and cultural
methods needed for the particular species Little is known about the
propagation requirements for the two species used in this study
thinleaf alder and water birch This deficit is due in part to a lack of
demand for these species in the past Extensive work has been done
on the propagation of other species within the Alnus and Betula
genera specifically those species of commercial value to the timber
industry such as red alder (A rubra Bong) and paper birch (B
9
papyrifera Marsh) Information generated from propagation studies
on these species has elucidated some universal seed characteristics
and germination requirements for members ofBetulaceae Seeds aremiddot
characteristically very small and light and may have a winged
integument to aid in wind dispersal Average seed density for B
ocddentalis is about 2500 seeds per gram while A tenuifolia
averages about 1488 seeds per gram (Vines 1960) Seed quality and
germination capacity are often very low as it is difficult to separate
sound from empty seeds when size and weight are so low (Brinkman
1974 Schopmeyer 1974) Seed quality may vary considerably from
harvest to harvest (Bjorkbom et al 1965) Within species
germination requirements may differ with provenance (Fowler and
Dwight 1964 Wilcox 1968) or even within a provenance (Bjorkbom
et al 1965 Schopmeyer 1974) In some instances the requirements
for germination may be met but germination does not occur a
condition referred to as dormancy
Seed Dormanqr and Methods to Overcome It
Dormancy in seeds is defined as the condition where seeds will
not germinate even when environmental conditions (water
10
temperature and aeration) are permissive for germination (Bewley
and Black 1994 Hartmann et al 1997) This mechanism ensures that
germination does not take place in less than optimum conditions or at
the wrong time (Bewley and Black 1994 Thompson 1971) For
example in some species seeds of southern provenance require
longer stratifications (Fowler and Dwight 1964) probably to prevent
germination in areas where there are intermittent periods ofwarm
weather followed by frost Seed dormancy results from a
combination ofgenetic and environmental conditions and it is not
always possible to predict the dormancy of a particular species from
characteristics of other species within the genus (Schopmeyer 1974)
There are different systems for classifying dormancy but the
condition may be divided into four basic types exogenous
endogenous double or combinational and secondary (Hartmann et
al 1997) The seed dormancy exhibited by birch and alder falls under
the category of endogenous dormancy a dormancy imposed by
embryonic factors This includes morphological dormancy (an
underdeveloped embryo) and physiological dormancy ofvarying
degrees (non-deep intermediate and deep) Non-deep physiological
11
dormancy is characterized by the need for after-ripening or exposure
to red light (photodormancy) Intermediate physiological dormancy
is characterized by the need for moderate periods of cold stratification
(up to 56 days) Deep physiological dormancy requires long periods
of cold stratification more than 56 days (Hartmann et al 1997)
Seeds ofboth Alnus and Betula exhibit varying degrees of
dormancy in most cases broken by coolmoist stratification andor
germination under red light (Brinkman 1974 Dirr and Heuser 1987
Schopmeyer 1974 Young and Young 1992) In some species of these
genera chemical treatments such as potassium nitrate have been
effective to overcome dormancy (Bradbeer 1988 Hartmann et al
1997 Young et al 1984) Many birch species are known to possess a
phytochrome light detection system which prevents germination
when seeds are buried too deep to allow seedling survival after
germination (Bewley and Black 1994 Black and Wareing 1955
Bradbeer 1988) Where the phytochrome detection mechanism is
present exposure to red light during germination is required for
breaking dormancy Most species of birch and alder have seeds that
ripen in late summer or early fall fall germination would result in
12
seedling loss over the winter so an after-ripening or stratification
requirement decreases the possibility of fall germination Joseph
(1929) found non-stratified birch seeds had a higher temperature
requirement for germination The current theory is that stratification
causes phase changes in membrane fluidity and triggers membraneshy
related signal transduction pathways activating enzymes and
hormones thus allowing dormancy release (Bewley and Black 1994
Ross and Bradbeer 1971)
Leaching of certain chemical inhibitors from seeds can also
break dormancy it maybe that this is part of the mechanism by
which photo dormancy is broken by moist stratification as only small
amounts of moisture are needed (Brad beer 1988) Research indicates
that the testa and pericarp of the seeds are involved in dormancy not
because they contain the inhibitor but because they prevent leaching
of the inhibitor (Villiers and Wareing 1964 Webb and Wareing
1972) Ru40lf (1950) found that cold-soaking might in some cases be
an acceptable substitute for stratification in some conifer species this
might be due to the leaching mechanism
13
The role ofpotassium nitrate in breaking dormancy has not
been clarified but there is speculation that the nitrogen supplied or
the oxygenating properties of the nitrate are involved (Brad beer
1988) Biswas et al (1972) found that the chemical treatment
enhanced the effect of stratification but did not necessarily replace it
Hilton (1985) found the germination-stimulating properties ofnitrate
depend on the presence of light nitrate in the presence of red light is
believed to be a cofactor to the phytochrome system which is involved
in the synthesis ofgibberellins that promote germination (Hilhorst et
al 1986)
Germination Requirements
General requirements for germination include moisture
favorable temperatures adeq-qate gas exchange and for some species I)
light In the presence of these conditions the quiescent seed can
imbibe water causing the seed to swell and the seed coat to split or
break Enzymatic activity within the seed accelerates increasing
respiration and use of stored energy resulting in the commencement
of growth processes within the seed (Bewley and Black 1994
14
Pretreatment requirements for germination of alder seed are
quite variable both between and within species For many species of
alder cold stratification periods of60-180 days are recommended
(Dirr and Heuser 1987) In one study ofthinleaf alder prechilling
(stratification) did not improve germination percentage while in
European speckled alder 180 days of stratification did improve
percentage germination (Young and Young 1992) Several other
treatments including light freezing and potassium nitrate
independently and with stratification have been shown to enhance
germination ofalders In red alder stratification was not necessary
when seed was germinated in light (Kenady 1978 Radwan and
DeBell 1981) Evidence of a phytochrome-regulated dormancy was shy
subsequently found in this species (Bormann 1983) Several general
horticultural texts recommend a pretreatment with 0200 potassium
nitrate (wv) to enhance stratification effects (Hartmann et al 1997
Young and Young 1992) In one study stratification followed by
freezing of seed for 3 days at -20degC enhanced germination (Schalin
1968)
16
Water Birch
Birch species are widely distributed in the northern hemisphere
found further north than alders can grow in various habitats and are
tolerant of a wide range of soils and moisture levels but are sensitive
to drought (Ashburner 1993 deJong 1993) Birch species are thought
to be more resistant to drought than alder species (McVean 1956) B
ocddentaJis Hook occurs as a shrub or small tree along streams or in
moist canyons and occasionally in dryer sites of the mountain West
( at elevations of 1500-2700 meters (Foxx and Hoard 1995 Vines
1960) It is known in the vernacular as water birch red birch and
black birch A small tree it is not used for lumber but can be used as
firewood posts browse by livestock or wildlife and sometimes as a
landscape tree (BrenzeI1995 Elias 1980 Preston 1968 Vines 1960)
Germination requirements for species of Betula generally
include stratification or red light treatment (Brinkman 1974)
indicating the presence ofphytochrome far-red inhibition (Bevington
1986 Bevington and Hoyle 1981 Schopmeyer 1974) Occasionally
both red light and stratification are recommended to improve
germination rate (Dirr and Heuser 1987) Potassium nitrate 02
17
pretreatment is recommended for birch species by Hartmann et al
(1997) Seeds of this species are considered to have a fairly shallow
dormancy (Lane 1993)
Seed Quality Improvements
Methods to upgrade seed quality (separate viable from nonshy
viable seeds) have been developed for different species Conventional
seed separation techniques are based on density such as air column or
liquidseparation or by size and shape such as with screens
Separation ofviable and non-viable seeds is extremely problematic
with very light winged seeds like those of alder and birch Air
separation techniques may not be practical for winged light-weight
seed Flotation techniques often employ lighter-than-water solvents
but some of these substances may have adverse effects on seed
viability (Barnett 1971 McLemore 1965) Widescale use of some
solvents is not considered desirable because of health and safety
concerns
A method of seed refinementupgrade originally developed in
Sweden by Milan Simak called the LDS method (Incubation
Drying Separation) shows promise for separating live and dead seeds
18
(cited in Bonner 1984 Downie and Wang 1992 Simak 1983
Sweeney et al 1991) Seeds are imbibed for several hours then
incubated at cool temperatures (15~or several hours in 100
relative humidity Seeds are then dried for several hours at 35
relative humidity at cool temperatures (timing and relative humidity
must be adjusted for the particular species) During the drying
dead seeds will lose most of the water previously imbibed while live
seeds should retain most of their imbibed water This differential
moisture content would make separation by flotation and other
density separation methods potentially feasible Similar methods of
conditioning have been shown to improve seed quality in lettuce
tomato and onion (Hill et al 1989) It has also been shown that
drying of stratified seeds for storage or for separation from
stratification medium need not result in loss of viability (Danielson
and Tanaka 1978 Schopmeyer 1974)
19
OBJECTIVES OF THIS STUDY
The purpose of this study is to determine the effectiveness of the
LDS seed refinement technique and othi separation procedures in
increasing the percentage of live seeds in a seed lot and to develop
germination strategies for water birch and thinleaf alder investigating
the use of stratification Secondly this study will examine the within-
species variability of different seed lots in their response to LDS and
stratification treatments
METHODS AND MATERIALS
Sources
Alder strobiles were collected in October and November of
1998 in Catron County New Mexico near the towns of Luna and
Reserve in the Cottonwood Canyon Campground and in the Head of
the Ditch Campground and in Taos County New Mexico in the
Red River Canyon near the Molycorp molybdenum mine Table 1
shows the seed source elevations and locations Strobiles were kept
cool and allowed to dry for several weeks Seeds were separated from
the opening strobiles by rubbing on a coarse screen
Birch strobiles were collected in October and N overrtber of 1998
in Taos County in the Red River Canyon near the Mo1ycorp
molybdenum mine (Table 1) Strobiles were kept cool and allowed to
dry for several weeks allowing the release of seeds from the bracts
In addition commercial seed sources ofbirch and alder were
purchased in the summer of 1999 (collected in the fall of 1998) The
seed lots collected in 1998 (Table 1) were used in the seed refinement
study providing four seed lots for that study For the final seed
refinement-germination study the two Red River Canyon seed lots of
21
Table 1 Seed Source Locations and Elevations
Species Source Lot Baseline Description Elevation Latitude Notes No Fill (meters) Longitude
Thinleaf Alder Luna NA 234 Head ofthe Ditch CG 2134 N 33deg49 W 108deg59
t+
Reserve NA 268 Cottonwood Canyon 1829 N 33deg37 W 108deg55
t+
RRC-l 98108 08 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
RRC-2 98109 09 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 537 W of Poncha Springs CO 2438 N 38deg31 W 106deg05
I
Water Birch RRC-3 98104 69 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Moly-1 98106 39 Molycorp Tailings Rd 2469 N 36deg41 W 105deg29
t+
Moly-2 98107 52 Molycorp Low Dump 2469 N 36deg41 W 105deg29
t+
Mo1y-3 98105 56 Molycorp Front Dump 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 297 W ofPoncha Springs CO
t used in Experiment 1--Seed Refinement I used in Experiment 2--Germination Requirements sectpooled for use in Experiment 2-Germination Requirements
2377 N 38deg31 W 106deg05
I
alder and the Red River Canyon and Moly 3 seed lots ofbirch were )
pooled due to limited amounts of seed The seed lots collected in
1998 and the 1998 purchased seed lots from Chaffee County
Colorado provided four seed lots of each species for that study
All seed sources were evaluated for percentage of filled seeds by
means ofdissection performed under a dissecting microscope at 30X
magnification (Berry and Torrey 1985) Alder species baseline
percentage of filled seeds was estimated using 25 samples of 100 seeds
pooled into one percentage response for each seed source Birch
species baseline percentage of filled seeds was determined using 15
samples of 50 seeds pooled into one percentage response for each seed
source Baseline percentage fill (Table 1) is the estimate of the
percentage of filled seed in the entire seed collection for each source
Separation Media
Ethanol and water were not particularly effective in separation
ofthinleaf alder seeds either using IDS methods or when separating
dry seed It was necessary to choose a fluid with a lower specific
gravity than ethanol (SG=O 79) in order to separate filled and empty
seeds with very low densities Falleri and Pacella (1997) found that
23
low-density London plane tree (Platanus x acerifolia [Aid Willd)
seeds could not be separated using water as the separation medium
due to the very small density differences between sound and empty
seeds and chose petroleum ether as a separation medium Petroleum
ether was chosen for the separation of thinleaf alder seeds because of
its low specific gravity (SG middot060) its relative stability low
reactivity and rating as a slight health risk Contact with skin may
cause dryness and irritation but no chronic systematic effects have
been reported with industrial use (Mallinckrodt Baker Inc 1997a)
As observed previously for thinleaf alder seeds the simple
specific gravity method using water was not effective for separating
water birch seeds In preliminary studies ethanol and petroleum
ether were found to be effective in separation of dry water birch seeds
and petroleum ether ethanol and water were somewhat effective in
separation of water birch seeds treated by the LDS method but
ethanol was chosen as the separation medium because of its lower
cost greater effectiveness and availability
Denatured ethanol is actually rated a greater health risk than
petroleum ether because ingestion is more likely to result in death or
24
permanent damage and prolonged skin contact may affect the
nervous system and other organ systems of the body Ethanol also
has a higher reactivity rating Gloves goggles and lab coat (personal
protective equipment) proper ventilation avoidance of ingestion and
proper fire safety measures should prevent problems with use of either
solvent (Mallinckrodt Baker Inc 1997a 1997b)
Seed Refinement
Thinleaf Alder
Separation treatments examined includeddensity separation of
dry seed samples in petroleum ettter (the control) and imbibed seed ~
samples treated with the IDS method at 0 1 18 and 24 hour drying
times followed by density separation in petroleum ether (Table 2)
Seeds were imbibed for 24 hours by submersion in a 10-gallon glass
aquarium filled with distilled water and equipped with an aeration
pump and filter Seeds were packaged in filter paper then the
packages were enclosed in wire cages (purchased tea balls were used
for this purpose) weighted with marbles to keep them submerged At
the end of the imbibition period seeds were removed from the cages
thoroughly blotted and placed on clean filter paper The drying
25
incubation was performed in a closed chamber with a constant
humidity obtained by the use ofCaC12middot6H20 salt in a saturated
solution prepared by adding SOOOg CaClzmiddot6HzO to 30 liters of
distilled water (Slavik 1974 Young 1967) Imbibed seeds were placed
on filter paper and suspended on a screen above the calcium chloride
solution Humidity was monitored using an hygrometer and held
steady at 50 in the presence of the wet seeds and filter paper
Table 2 Alder Preparation Protocols for S~d Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) 0 0
2
3
24
24
0
1 )
4 24 18
5 24 24
At the end of the appropriate drying incubation the seeds were
placed in petroleum ether and briefly and vigorously stirred to
separate seeds adhering to one another Floating seeds were removed
from the surface of the petroleum ether by means of a small net
andor a spatula placed on clean moistened filter paper and placed in
26
a labeled plastic bag to await counting The sinking seeds were
strained through the net and packaged in a similar manner Five
repetitions were performed for each of the five treatments using 100
seeds per repetition Percentage of filled seeds contained in each
fraction was determined by means of dissection tests performed on the
floating and sinking fractions using a scalpel and a dissecting
microscope with 30X magnification
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that particular repetition
of filled seeds in the sinking fraction X 100=percentage recovery
of filled seeds in the sinking fraction + of filled seeds in the floating fraction
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product of percentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
27
Water Birch
Separation treatments included density separation of dry seed in
95 ethanol (the control) and seed samples treated with the IDS
method at 005 1 and 2 hour drying times followed by density
separation in 95 ethanol (Table 3) Seeds were imbibed for 12 hours
by submersion in a 10-gallon glass aquarium filled with distilled water
and equipped with an aeration pump and filter Seeds were packaged
in filter paper then the packages were enclosed in wire cages
(purchased tea balls were used for this purpose) weighted with
marbles to keep them submerged At the end of the imbibition
period seeds were removed from the cages thoroughly blotted and
placed on clean filter paper The drying incubation was performed in
a closed chamber with a constant humidity obtained by the use of
CaCI2middot6H20 salt in a saturated solution prepared as described in the
previous section (Slavik 1974 Young 1967) Imbibed seeds were
placed on filter paper and suspended on a screen above the calcium
chloride solution Humidity was monitored using an hygrometer and
held steady at 50 in the presence of the wet seeds and filter paper
28
Table 3 Birch Preparation Protocols for Seed Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) o o
2 12 o
3 12 05
4 12 1
5 12 2
At the end of the appropriate drying incubation the seeds were
placed in 9500 ethanol and briefly and vigorously stirred to separate
seeds adhering to one another Floating seeds were removed from the
surface of the ethanol by means of a small net andor a spatula
placed on clean moistened filter paper and placed in a labeled plastic
bag to await counting The sinking seeds were strained through the
net and packaged in a similar manner Three repetitions were
performed for each of the five treatments using 50 seeds per
repetition Percentage of filled seeds contained in each fraction was
determined by means of dissection tests performed on the floating and
sinking fractions using a scalpel and a dissecting microscope with
30X magnification
29
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that repetition (as given in the previous equation)
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product ofpercentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
Germination Requirements
Thinleaf Alder
Separations were performed using the separation method
chosen from the seed refinement study alder preparation protocol 4
24-hour imbibition followed by I8-hour drying time and separation in
petroleum ether as described in the seed refinement experiment
(Table 2) Unseparated imbibed seeds and seeds from both the
floating and sinking fractions were subsequently treated with 028
and 56 days of stratification In addition an unseparated nonshy
stratified control of dry seeds was tested for germination Seeds for
stratification treatments were placed in layers ofpaper towel
30
moistened with 25 m1 of distilled water and placed in sealed zip-lock
plastic bags The bags were placed in a cooler at I-5degC (average
temperature 50degC) for periods of 28 or 56 days Initiation of
stratification treatments was staggered so that all treatments came out
ofstratification at the same time
Following stratification the seeds were sown in Ray-Leach
Super Cells (Steuwe amp Sons Inc Corvalis OR) containing a 2 1 1
ratio ofpeatmossperlitevermiculite (vvv) with OsmocoteR 14-14-10
slow release fertilizer at a rate of 4007 gm3bull Five seeds were sown
per tube Treatments were distributed in a randomized complete
block design consisting of4 blocks (locations on the greenhouse
bench) with each block containing the 10 treatment combinations for
each of four seed lots (Table 4) Each repetition contained 20 tubes
repetitions were placed in random order four repetitions to a rack ten
racks to each block Each repetition for each treatment contained 100
seeds therefore 100 seeds were used for each seed source by
treatment by block combination pooled to one measurement for the
response variable germination percentage Racks were placed in a
greenhouse for germination Germination conditions included
31
ambient light and 70 relative humidity with average daily
temperature 243degC (daytime temperature range 200-272degC) and
average night temperature 216degC (nighttime temperature range 206shy
239degC) Tubes were watered at 2 hour intervals six times a day
Germination was recorded at weekly intervals 7 1421 and 28 days
after planting
Table 4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder
Treatment Number Stratification (Days) Separation Fraction
1 56 None
2 56 Floating
3 56 Sinking
4 28 None
5 28 Floating
6 28 Sinking
7 0 None
8 0 Floating
9 0 Sinking
blocks Qocations on the greenhouse bench) with each block
containing the 10 treatment combinations for each of four seed lots
(Table 5) Each repetition contained 20 tubes repetitions were placed
in random order four repetitions to a rack ten racks to each block
Each repetition for each treatment contained 100 seeds therefore 100
seeds were used for each seed source by treatment by block
combination pooled to one measurement for the response variable
germination percentage Racks were placed in a greenhouse for
germination Germination conditions included ambient light and
7000 relative humidity with average daily temperature 243 degC
(daytime temperature range 200-272degC) and average night
temperature 216degC (nighttime temperature range 206-239degC)
Tubes were watered at 2 hour intervals six times a day Germination
was recorded at weekly intervals 7 1421 and 28 days after planting
34
Table 5 Treatment Combinations for Experimental Layout of Randomized Complete Block-Water Birch
Treatment Number Stratification (Days) Separation Fraction
56 None
2 56 Floating
3 56 Sinking
4 21 None
5 21 Floating
6 21 Sinking
7 0 None
8 0 Floating
9 0 Sinking
10 0 None
DATA ANALYSIS
The seed refinement experiment was performed to determine
the mostadvantageous separation technique for use in the
germination studies with the percentage of filled seeds present in the
sinking fractions (percentage fill) and proportion of filled seeds
recovered from the total filled seeds available in the sample
(percentage recovery) as response variables and the preparation
protocols and seed sources as independent variables
The second experiment utilized the chosen seed refinement
method with levels of stratification seed separation fraction and seed
source as independent variables (or in the augmented factorial
treatment combination as the independent variable) with germination
percentage measured as the response variable Germination rate was
also recorded however the rapid germination between the time of
sowing and the first sampling (at 7 days) prevented meaningful
analysis of this da~
Data was analyzed by using categorical data modeling analysis
as found in the SAScopy statistical program The PROC CATMOD
procedure can perform analysis and giveanalysis of variance in the
36
general sense that it analyzes the response functions fits linear models
to functions of response frequencies and partitions the variation
among those functions into various sources (SAS Institute 1989)
CATMOD analyzes data that can be represented in a two-
dimensional contingency table with the rows corresponding to
populations or samples defined by one or more independent variables
and the columns corresponding to one or more dependent (response)
variables The frequencies in the table are assumed to follow a
product multinomial distribution with a simple random sample taken
for each population The probability for the response ofeach cell is
estimated and the vector (P) of these proportions is transformed into a
vector of functions F =F(P) If It denotes the vector of true
probabilities for the table then the functions of the true probabilities
F(It) are assumed to follow a linear model
I
where EA denotes asymptotic expectation X is the design matrix
containing fixed constants and Pis a vector ofparameters to be
37
estimated CA TMOD provides two estimation methods the
maximum-likelihood method and the weighted-least-squares method
which was used in this analysis (SAS Institute 1989)
Hypotheses about linear combinations of the parameters can be
tested these statistics are approximately distributed as chi-square for
sufficiently large sample sizes (SAS Institute 1989)
All of the response variables considered had a binomial type of
probability distribution (seed filled or not filled seed germinated or
not germinated) All treatments ofboth experiments were analyzed
using the PROC CATMOD procedure to examine the general model
as well as planned comparisons using contrast statements where ~
appropriate The PROC MEANS procedure was used to calculate
marginal percentages (main effect and interaction combinations)
along with standard errors Pairwise Z-tests were used to separate
percentages in those effects which were determined to be significant
by categorical modeling at the observed significance level laquo=005 this
method of percentage separation is analogous to Fishers LSD for
separating means
38
Categorical modeling of the seed refinement experiments used
two models one for the percentage of filled seeds attained in the
sinking and floating fractions and one for the percentage of filled
seeds recovered from those available in the baseline sample The
treatment structure for both of these seed refinement studies was a 5
X 2 X 4 factorial (preparation protocol by separation fraction by seed
source)
The model in CATMOD for the percentage of filled seeds
attained in the fractions is as follows
fill = source prep fraction sourceprep sourcefraction prepfraction sourceprepfraction
where fill is the response variable the number of filled seeds
source is the seed source prep is the LDS treatment protocol
(imbibition plus one of four drying times or no treatment) and
fraction is the separation fraction (floating or sinking)
The CATMOD model for the percentage of filled seed
recovered in the floating or sinking fractions is as follows
39
rec = source prep sourceprep
where rec is the response variable (number of filled seeds floating or
sinking) source is the seed source and prep is the IDS
treatment protocol
The treatment structure for the germination studies was a 3 X 3
factorial (stratification by separation) with an additional control (no
treatment) for 4 seed sources termed an augmented factorial design
by Lentner and Bishop (1986) The PROC CATMOD procedure
was used to analyze the data without the no treatment control as a
simple 3 X 3 X 4 factorial (stratification by separation by seed source)
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
Table Page
25 Water Birch Percentage Gennination as Influenced by Seed Source--Factorial Analysis 67
26 Analysis ofVariance Table for Water Birch Percentage Gennination as Influenced By Treatment COInbination and Seed Source--Augmented Factorial 73
27 Water Birch Analysis of Contrasts--Augmented Factorial 73
LIST OF FIGURES
PageFigure
1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction 44
2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction 52
3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction 53
4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source 58
5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification 59
6 Alder Percentage Germination as Influenced by Imbibition and Seed Source 62
7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source 68
8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction 69
9 Birch Percentage Germination as Influenced by Stratification and Seed Source 70
10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification 71
XVI
Figure Page
11 Birch Percentage Germination as Influenced by Imbibition and Seed Source 75
12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 81
13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 83
INTRODUCTION
Birch (Betula) and alder (Alnus) are two genera of Betulaceae
trees found in riparian areas throughout New Mexico The presence
ofbirch and alder in riparian zones of New Mexico has been noted in
many early surveys of the region (Britton 1908 Sargent 1901 1905
Wooton and Standley 1915) Water birch (Betula ocddentalisHook
formerly B fontinalis Sarg) is found in the northern mountains of the
state (Martin and Hutchins 1980) New Mexico has two species of
alder Arizona alder (Alnus oblongifolia Torr) which is found in the
mountains of southwest New Mexico (Martin and Hutchins 1980
Vines 1960) and thinleaf alder (Alnus tenuifolia Nutt) designated by
Carter (1997) as Alnus incana ssp tenuifolia Nutt found in the
northern and western mountains (Martin and Hutchins 1980 Vines
1960) Until recently existence of these species has been of interest
mainly from a botanical standpoint However with increasing landshy
use in the western United States these trees may have a further
purpose in the revegetation of degraded riparian areas and as oasis
plants for those interested in native landscapes (Phillips 1995)
Successful revegetation of degraded areas is influenced by many
1
factors including the site conditions commonly encountered and the
chosen plant material Desirable plant material should be wellshy
adapted to the site have high survival and be economical to obtain or
produce
LITERATURE REVIEW
Revegetation and Reconstruction
Strategies for revegetation of disturbed lands are generally
divided into three categories restoration reclamation and
rehabilitation Restoration is the complete replication of the original
conditions species habitat and function of the area Reclamation
involves returning the area to a condition that is habitable by the
organisms that were originally present or organisms that approximate
the original inhabitants Rehabilitation involves returning the land to
a form and function which conforms to a prior land-use plan
including a stable ecological state that does not contribute
substantially to environmental deterioration and is consistent with
surrounding aesthetic values (Allen 1988 National Academy of
Sciences 1974) These three categories have been collectively termed
reconstruction by Allen (1988) Complete restoration is often not
practical as certain requisite intermediate conditions of varying
durations maybe necessary In the arid western United States
natural succession is slow and dependence on natural process risks
further site degradation (National Academy of Sciences 1974)
3
Reclamation and rehabilitation may be more workable concepts A
practical guiding philosophy would be the objective to create a stable
ecosystem that is compositionally and functionally similar to that
which existed prior to human disturbance with the realization that
such a goal is not completely attainable (Burton et al 1988)
Species Selection
It has long been the philosophy of those involved in
reconstruction efforts that the use ofnative and diverse species is
desirable rather than dependence on a few proven species (Daniel
et al 1979 Harker et al 1993 Nielson and Peterson 1973) The
rationale is that native species are better adapted to adverse site
conditions such as low moisture and high surface temperatures and
exposure (Nielson and Peterson 1973) Only native species survived
in a European study even though exotic species examined also
possessed characteristics which were well-adapted to the site (Herrera
et al 1993) Use of diverse native plant species can enhance
reconstruction efforts and sustain more diverse wildlife populations
(Harker et al 1993) Using plant material of local provenance (origin
of seed) to maximize survival is also important (Albers and Carpenter
4
1979 Burton et al 1988 Daniel et al 1979 Hobbs 1984) Species of
plants evolve within their habitat to site conditions including edaphic
topographic and climatic conditions such as temperature (Bewley and
Black 1994) photoperiod (Currie 1990) and growing season A plant
with origins in southern latitudes may not properly harden off for
winter in time to avoid early frost when grown in northern latitudes
with longer day1engths while a plant from northern latitudes may not
have optimal shoot growth in the shorter day1ength of southern areas
(Fowells 1965 Lane 1993)
PlantingMethods
Natural colonization processes can take anywhere from ten to
hundreds ofyears depending on site conditions (National Academy
of Sciences 1974) Planting methods used in reconstruction include
direct seeding wildling transplants and use ofbare-root or
containerized transplant material (Schubert et al 1970) Direct
seeding is often the least expensive planting method but success with
woody species is frequently limited Predation of seed germination
failure and adverse conditions for germinants can result in planting
failure (Fowells 1965 Haeussler et al 1995 Hibbs et al 1994
5
Monsen 1984 Pratt 1986) Wildling transplants may have poor
survival ifplanting is not timed properly and done carefully (Schubert
et al 1970) Use ofnursery grown seedlings bare-root or
containerized can improve survival rates relative to other
reconstruction efforts (Hobbs 1984) The ability to match stock type
(source physiological and morphological condition) to the site
known as the target seedling concept (Rose et al 1990) and greater
latitude in planting conditions (timing) can contribute to improved
transplant success of nursery stock relative to wildlings Combining
direct seeding ofnon-woody plants and nursery-grown seedlings can
be the most efficient and economical method of reconstruction when
costs ofproducing container stock can be kept low (Belcher 1982
Dunlap and Barnett 1984 Rose et al 1990) The success of
reconstruction efforts is heavily dependent on site conditions and the
quality of the plant material used (Monsen 1984) In tum quality of
plant material is dependent on well-developed germination and
culture protocols The economic feasibility of stock propagation for
reconstruction work is dependent on finding methods to efficiently
upgrade seed quality (proportion ofgerminable seeds) and optimize
6
germination capacity and seedling survival (Belcher 1982 Bonner
1984)
Birch and Alder Suitability in Reconstruction
Montane riparian vegetation zones are contained in areas where
the supply ofwater is constant (perennial) as well as areas with an
ephemeral (intermittent) water supply Riparian zones contain both
obligate and facultative riparian species Facultative riparian species
are also found in surrounding open spaces and in high cool nonshy
riparian locations (Dick-Peddie 1993) Riparian vegetation follows an
elevational gradient from the source to the mouth of the drainage
perpendicular to the zone of upland vegetation (Dick-Peddie 1993)
Other habitats where water may be caught but are not part of a true
drainage are termed pseudoriparian Pseudoriparian habitats include
gullies roadside ditches and the bottoms of talus slopes (Dick-Peddie
1993) Most of the obligate riparian species found in riparian and
pseudoriparian areas are adapted to flood conditions with the ability
to rapidly reproduce and colonize a devastated area Characteristics
ofobligate riparian species include prolific seed production efficient
7
seed dispersal fast growth short life-cycles and rapid attainment of
reproductive stage (Dick-Peddie 1993)
Birch and alder species are generally confined to montane
riparian zones (Elias 1980) Members ofboth genera have properties
indicative of obligate riparian species including fast growth prolific
seed production and short life-cycle these properties also make
members of these genera suitable candidates for use in reconstruction
efforts (Elias 1980) Birch and alder are known as pioneer species
which can successfully establish on denuded areas (Young and Young
1992) and which prefer mineral soil for germination and early growth
(Haeussler et al 1995 Schalin 1968) In addition most alder species
including thirlleaf alder and Arizona alder have the ability to fix
atmospheric nitrogen via a symbiotic relationship with root-nodule
forming species of Frankia actinomycetes (Bond 195519711976
Virtanen 1957) Many researchers believe the formation of a dynamic
rhizosphere of this type is critical to the rehabilitation of degraded
lands (Herrera et al 1993 Whitford 1988) Biological nitrogen
fixation in conjunction with the production of large amounts of litter
has been shown to help build up organic matter nitrogen and
8
improve soil structure in deficient soils such as glacial till (Bollen and
Lu 1968 Crocker and Major 1955 Tarrant and Trappe 1971)
Biological nitrogen fixation can also improve conditions for other
non-nitrogen fixing species (Tarrant 1961) and enhance species
diversity (Franklin and Pechanec 1968)
The use ofthese deciduous trees with the objective of improving
the site conditions (ie shade nutrients and organic matter) for other
species (Albers and Carpenter 1979) is a valuable strategy in the
reconstruction of disturbed areas such as mine spoils
Production ofStockP1ants
Efficient propagation ofnursery stock from seed requires
extensive knowledge of the germination requirements and cultural
methods needed for the particular species Little is known about the
propagation requirements for the two species used in this study
thinleaf alder and water birch This deficit is due in part to a lack of
demand for these species in the past Extensive work has been done
on the propagation of other species within the Alnus and Betula
genera specifically those species of commercial value to the timber
industry such as red alder (A rubra Bong) and paper birch (B
9
papyrifera Marsh) Information generated from propagation studies
on these species has elucidated some universal seed characteristics
and germination requirements for members ofBetulaceae Seeds aremiddot
characteristically very small and light and may have a winged
integument to aid in wind dispersal Average seed density for B
ocddentalis is about 2500 seeds per gram while A tenuifolia
averages about 1488 seeds per gram (Vines 1960) Seed quality and
germination capacity are often very low as it is difficult to separate
sound from empty seeds when size and weight are so low (Brinkman
1974 Schopmeyer 1974) Seed quality may vary considerably from
harvest to harvest (Bjorkbom et al 1965) Within species
germination requirements may differ with provenance (Fowler and
Dwight 1964 Wilcox 1968) or even within a provenance (Bjorkbom
et al 1965 Schopmeyer 1974) In some instances the requirements
for germination may be met but germination does not occur a
condition referred to as dormancy
Seed Dormanqr and Methods to Overcome It
Dormancy in seeds is defined as the condition where seeds will
not germinate even when environmental conditions (water
10
temperature and aeration) are permissive for germination (Bewley
and Black 1994 Hartmann et al 1997) This mechanism ensures that
germination does not take place in less than optimum conditions or at
the wrong time (Bewley and Black 1994 Thompson 1971) For
example in some species seeds of southern provenance require
longer stratifications (Fowler and Dwight 1964) probably to prevent
germination in areas where there are intermittent periods ofwarm
weather followed by frost Seed dormancy results from a
combination ofgenetic and environmental conditions and it is not
always possible to predict the dormancy of a particular species from
characteristics of other species within the genus (Schopmeyer 1974)
There are different systems for classifying dormancy but the
condition may be divided into four basic types exogenous
endogenous double or combinational and secondary (Hartmann et
al 1997) The seed dormancy exhibited by birch and alder falls under
the category of endogenous dormancy a dormancy imposed by
embryonic factors This includes morphological dormancy (an
underdeveloped embryo) and physiological dormancy ofvarying
degrees (non-deep intermediate and deep) Non-deep physiological
11
dormancy is characterized by the need for after-ripening or exposure
to red light (photodormancy) Intermediate physiological dormancy
is characterized by the need for moderate periods of cold stratification
(up to 56 days) Deep physiological dormancy requires long periods
of cold stratification more than 56 days (Hartmann et al 1997)
Seeds ofboth Alnus and Betula exhibit varying degrees of
dormancy in most cases broken by coolmoist stratification andor
germination under red light (Brinkman 1974 Dirr and Heuser 1987
Schopmeyer 1974 Young and Young 1992) In some species of these
genera chemical treatments such as potassium nitrate have been
effective to overcome dormancy (Bradbeer 1988 Hartmann et al
1997 Young et al 1984) Many birch species are known to possess a
phytochrome light detection system which prevents germination
when seeds are buried too deep to allow seedling survival after
germination (Bewley and Black 1994 Black and Wareing 1955
Bradbeer 1988) Where the phytochrome detection mechanism is
present exposure to red light during germination is required for
breaking dormancy Most species of birch and alder have seeds that
ripen in late summer or early fall fall germination would result in
12
seedling loss over the winter so an after-ripening or stratification
requirement decreases the possibility of fall germination Joseph
(1929) found non-stratified birch seeds had a higher temperature
requirement for germination The current theory is that stratification
causes phase changes in membrane fluidity and triggers membraneshy
related signal transduction pathways activating enzymes and
hormones thus allowing dormancy release (Bewley and Black 1994
Ross and Bradbeer 1971)
Leaching of certain chemical inhibitors from seeds can also
break dormancy it maybe that this is part of the mechanism by
which photo dormancy is broken by moist stratification as only small
amounts of moisture are needed (Brad beer 1988) Research indicates
that the testa and pericarp of the seeds are involved in dormancy not
because they contain the inhibitor but because they prevent leaching
of the inhibitor (Villiers and Wareing 1964 Webb and Wareing
1972) Ru40lf (1950) found that cold-soaking might in some cases be
an acceptable substitute for stratification in some conifer species this
might be due to the leaching mechanism
13
The role ofpotassium nitrate in breaking dormancy has not
been clarified but there is speculation that the nitrogen supplied or
the oxygenating properties of the nitrate are involved (Brad beer
1988) Biswas et al (1972) found that the chemical treatment
enhanced the effect of stratification but did not necessarily replace it
Hilton (1985) found the germination-stimulating properties ofnitrate
depend on the presence of light nitrate in the presence of red light is
believed to be a cofactor to the phytochrome system which is involved
in the synthesis ofgibberellins that promote germination (Hilhorst et
al 1986)
Germination Requirements
General requirements for germination include moisture
favorable temperatures adeq-qate gas exchange and for some species I)
light In the presence of these conditions the quiescent seed can
imbibe water causing the seed to swell and the seed coat to split or
break Enzymatic activity within the seed accelerates increasing
respiration and use of stored energy resulting in the commencement
of growth processes within the seed (Bewley and Black 1994
14
Pretreatment requirements for germination of alder seed are
quite variable both between and within species For many species of
alder cold stratification periods of60-180 days are recommended
(Dirr and Heuser 1987) In one study ofthinleaf alder prechilling
(stratification) did not improve germination percentage while in
European speckled alder 180 days of stratification did improve
percentage germination (Young and Young 1992) Several other
treatments including light freezing and potassium nitrate
independently and with stratification have been shown to enhance
germination ofalders In red alder stratification was not necessary
when seed was germinated in light (Kenady 1978 Radwan and
DeBell 1981) Evidence of a phytochrome-regulated dormancy was shy
subsequently found in this species (Bormann 1983) Several general
horticultural texts recommend a pretreatment with 0200 potassium
nitrate (wv) to enhance stratification effects (Hartmann et al 1997
Young and Young 1992) In one study stratification followed by
freezing of seed for 3 days at -20degC enhanced germination (Schalin
1968)
16
Water Birch
Birch species are widely distributed in the northern hemisphere
found further north than alders can grow in various habitats and are
tolerant of a wide range of soils and moisture levels but are sensitive
to drought (Ashburner 1993 deJong 1993) Birch species are thought
to be more resistant to drought than alder species (McVean 1956) B
ocddentaJis Hook occurs as a shrub or small tree along streams or in
moist canyons and occasionally in dryer sites of the mountain West
( at elevations of 1500-2700 meters (Foxx and Hoard 1995 Vines
1960) It is known in the vernacular as water birch red birch and
black birch A small tree it is not used for lumber but can be used as
firewood posts browse by livestock or wildlife and sometimes as a
landscape tree (BrenzeI1995 Elias 1980 Preston 1968 Vines 1960)
Germination requirements for species of Betula generally
include stratification or red light treatment (Brinkman 1974)
indicating the presence ofphytochrome far-red inhibition (Bevington
1986 Bevington and Hoyle 1981 Schopmeyer 1974) Occasionally
both red light and stratification are recommended to improve
germination rate (Dirr and Heuser 1987) Potassium nitrate 02
17
pretreatment is recommended for birch species by Hartmann et al
(1997) Seeds of this species are considered to have a fairly shallow
dormancy (Lane 1993)
Seed Quality Improvements
Methods to upgrade seed quality (separate viable from nonshy
viable seeds) have been developed for different species Conventional
seed separation techniques are based on density such as air column or
liquidseparation or by size and shape such as with screens
Separation ofviable and non-viable seeds is extremely problematic
with very light winged seeds like those of alder and birch Air
separation techniques may not be practical for winged light-weight
seed Flotation techniques often employ lighter-than-water solvents
but some of these substances may have adverse effects on seed
viability (Barnett 1971 McLemore 1965) Widescale use of some
solvents is not considered desirable because of health and safety
concerns
A method of seed refinementupgrade originally developed in
Sweden by Milan Simak called the LDS method (Incubation
Drying Separation) shows promise for separating live and dead seeds
18
(cited in Bonner 1984 Downie and Wang 1992 Simak 1983
Sweeney et al 1991) Seeds are imbibed for several hours then
incubated at cool temperatures (15~or several hours in 100
relative humidity Seeds are then dried for several hours at 35
relative humidity at cool temperatures (timing and relative humidity
must be adjusted for the particular species) During the drying
dead seeds will lose most of the water previously imbibed while live
seeds should retain most of their imbibed water This differential
moisture content would make separation by flotation and other
density separation methods potentially feasible Similar methods of
conditioning have been shown to improve seed quality in lettuce
tomato and onion (Hill et al 1989) It has also been shown that
drying of stratified seeds for storage or for separation from
stratification medium need not result in loss of viability (Danielson
and Tanaka 1978 Schopmeyer 1974)
19
OBJECTIVES OF THIS STUDY
The purpose of this study is to determine the effectiveness of the
LDS seed refinement technique and othi separation procedures in
increasing the percentage of live seeds in a seed lot and to develop
germination strategies for water birch and thinleaf alder investigating
the use of stratification Secondly this study will examine the within-
species variability of different seed lots in their response to LDS and
stratification treatments
METHODS AND MATERIALS
Sources
Alder strobiles were collected in October and November of
1998 in Catron County New Mexico near the towns of Luna and
Reserve in the Cottonwood Canyon Campground and in the Head of
the Ditch Campground and in Taos County New Mexico in the
Red River Canyon near the Molycorp molybdenum mine Table 1
shows the seed source elevations and locations Strobiles were kept
cool and allowed to dry for several weeks Seeds were separated from
the opening strobiles by rubbing on a coarse screen
Birch strobiles were collected in October and N overrtber of 1998
in Taos County in the Red River Canyon near the Mo1ycorp
molybdenum mine (Table 1) Strobiles were kept cool and allowed to
dry for several weeks allowing the release of seeds from the bracts
In addition commercial seed sources ofbirch and alder were
purchased in the summer of 1999 (collected in the fall of 1998) The
seed lots collected in 1998 (Table 1) were used in the seed refinement
study providing four seed lots for that study For the final seed
refinement-germination study the two Red River Canyon seed lots of
21
Table 1 Seed Source Locations and Elevations
Species Source Lot Baseline Description Elevation Latitude Notes No Fill (meters) Longitude
Thinleaf Alder Luna NA 234 Head ofthe Ditch CG 2134 N 33deg49 W 108deg59
t+
Reserve NA 268 Cottonwood Canyon 1829 N 33deg37 W 108deg55
t+
RRC-l 98108 08 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
RRC-2 98109 09 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 537 W of Poncha Springs CO 2438 N 38deg31 W 106deg05
I
Water Birch RRC-3 98104 69 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Moly-1 98106 39 Molycorp Tailings Rd 2469 N 36deg41 W 105deg29
t+
Moly-2 98107 52 Molycorp Low Dump 2469 N 36deg41 W 105deg29
t+
Mo1y-3 98105 56 Molycorp Front Dump 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 297 W ofPoncha Springs CO
t used in Experiment 1--Seed Refinement I used in Experiment 2--Germination Requirements sectpooled for use in Experiment 2-Germination Requirements
2377 N 38deg31 W 106deg05
I
alder and the Red River Canyon and Moly 3 seed lots ofbirch were )
pooled due to limited amounts of seed The seed lots collected in
1998 and the 1998 purchased seed lots from Chaffee County
Colorado provided four seed lots of each species for that study
All seed sources were evaluated for percentage of filled seeds by
means ofdissection performed under a dissecting microscope at 30X
magnification (Berry and Torrey 1985) Alder species baseline
percentage of filled seeds was estimated using 25 samples of 100 seeds
pooled into one percentage response for each seed source Birch
species baseline percentage of filled seeds was determined using 15
samples of 50 seeds pooled into one percentage response for each seed
source Baseline percentage fill (Table 1) is the estimate of the
percentage of filled seed in the entire seed collection for each source
Separation Media
Ethanol and water were not particularly effective in separation
ofthinleaf alder seeds either using IDS methods or when separating
dry seed It was necessary to choose a fluid with a lower specific
gravity than ethanol (SG=O 79) in order to separate filled and empty
seeds with very low densities Falleri and Pacella (1997) found that
23
low-density London plane tree (Platanus x acerifolia [Aid Willd)
seeds could not be separated using water as the separation medium
due to the very small density differences between sound and empty
seeds and chose petroleum ether as a separation medium Petroleum
ether was chosen for the separation of thinleaf alder seeds because of
its low specific gravity (SG middot060) its relative stability low
reactivity and rating as a slight health risk Contact with skin may
cause dryness and irritation but no chronic systematic effects have
been reported with industrial use (Mallinckrodt Baker Inc 1997a)
As observed previously for thinleaf alder seeds the simple
specific gravity method using water was not effective for separating
water birch seeds In preliminary studies ethanol and petroleum
ether were found to be effective in separation of dry water birch seeds
and petroleum ether ethanol and water were somewhat effective in
separation of water birch seeds treated by the LDS method but
ethanol was chosen as the separation medium because of its lower
cost greater effectiveness and availability
Denatured ethanol is actually rated a greater health risk than
petroleum ether because ingestion is more likely to result in death or
24
permanent damage and prolonged skin contact may affect the
nervous system and other organ systems of the body Ethanol also
has a higher reactivity rating Gloves goggles and lab coat (personal
protective equipment) proper ventilation avoidance of ingestion and
proper fire safety measures should prevent problems with use of either
solvent (Mallinckrodt Baker Inc 1997a 1997b)
Seed Refinement
Thinleaf Alder
Separation treatments examined includeddensity separation of
dry seed samples in petroleum ettter (the control) and imbibed seed ~
samples treated with the IDS method at 0 1 18 and 24 hour drying
times followed by density separation in petroleum ether (Table 2)
Seeds were imbibed for 24 hours by submersion in a 10-gallon glass
aquarium filled with distilled water and equipped with an aeration
pump and filter Seeds were packaged in filter paper then the
packages were enclosed in wire cages (purchased tea balls were used
for this purpose) weighted with marbles to keep them submerged At
the end of the imbibition period seeds were removed from the cages
thoroughly blotted and placed on clean filter paper The drying
25
incubation was performed in a closed chamber with a constant
humidity obtained by the use ofCaC12middot6H20 salt in a saturated
solution prepared by adding SOOOg CaClzmiddot6HzO to 30 liters of
distilled water (Slavik 1974 Young 1967) Imbibed seeds were placed
on filter paper and suspended on a screen above the calcium chloride
solution Humidity was monitored using an hygrometer and held
steady at 50 in the presence of the wet seeds and filter paper
Table 2 Alder Preparation Protocols for S~d Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) 0 0
2
3
24
24
0
1 )
4 24 18
5 24 24
At the end of the appropriate drying incubation the seeds were
placed in petroleum ether and briefly and vigorously stirred to
separate seeds adhering to one another Floating seeds were removed
from the surface of the petroleum ether by means of a small net
andor a spatula placed on clean moistened filter paper and placed in
26
a labeled plastic bag to await counting The sinking seeds were
strained through the net and packaged in a similar manner Five
repetitions were performed for each of the five treatments using 100
seeds per repetition Percentage of filled seeds contained in each
fraction was determined by means of dissection tests performed on the
floating and sinking fractions using a scalpel and a dissecting
microscope with 30X magnification
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that particular repetition
of filled seeds in the sinking fraction X 100=percentage recovery
of filled seeds in the sinking fraction + of filled seeds in the floating fraction
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product of percentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
27
Water Birch
Separation treatments included density separation of dry seed in
95 ethanol (the control) and seed samples treated with the IDS
method at 005 1 and 2 hour drying times followed by density
separation in 95 ethanol (Table 3) Seeds were imbibed for 12 hours
by submersion in a 10-gallon glass aquarium filled with distilled water
and equipped with an aeration pump and filter Seeds were packaged
in filter paper then the packages were enclosed in wire cages
(purchased tea balls were used for this purpose) weighted with
marbles to keep them submerged At the end of the imbibition
period seeds were removed from the cages thoroughly blotted and
placed on clean filter paper The drying incubation was performed in
a closed chamber with a constant humidity obtained by the use of
CaCI2middot6H20 salt in a saturated solution prepared as described in the
previous section (Slavik 1974 Young 1967) Imbibed seeds were
placed on filter paper and suspended on a screen above the calcium
chloride solution Humidity was monitored using an hygrometer and
held steady at 50 in the presence of the wet seeds and filter paper
28
Table 3 Birch Preparation Protocols for Seed Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) o o
2 12 o
3 12 05
4 12 1
5 12 2
At the end of the appropriate drying incubation the seeds were
placed in 9500 ethanol and briefly and vigorously stirred to separate
seeds adhering to one another Floating seeds were removed from the
surface of the ethanol by means of a small net andor a spatula
placed on clean moistened filter paper and placed in a labeled plastic
bag to await counting The sinking seeds were strained through the
net and packaged in a similar manner Three repetitions were
performed for each of the five treatments using 50 seeds per
repetition Percentage of filled seeds contained in each fraction was
determined by means of dissection tests performed on the floating and
sinking fractions using a scalpel and a dissecting microscope with
30X magnification
29
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that repetition (as given in the previous equation)
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product ofpercentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
Germination Requirements
Thinleaf Alder
Separations were performed using the separation method
chosen from the seed refinement study alder preparation protocol 4
24-hour imbibition followed by I8-hour drying time and separation in
petroleum ether as described in the seed refinement experiment
(Table 2) Unseparated imbibed seeds and seeds from both the
floating and sinking fractions were subsequently treated with 028
and 56 days of stratification In addition an unseparated nonshy
stratified control of dry seeds was tested for germination Seeds for
stratification treatments were placed in layers ofpaper towel
30
moistened with 25 m1 of distilled water and placed in sealed zip-lock
plastic bags The bags were placed in a cooler at I-5degC (average
temperature 50degC) for periods of 28 or 56 days Initiation of
stratification treatments was staggered so that all treatments came out
ofstratification at the same time
Following stratification the seeds were sown in Ray-Leach
Super Cells (Steuwe amp Sons Inc Corvalis OR) containing a 2 1 1
ratio ofpeatmossperlitevermiculite (vvv) with OsmocoteR 14-14-10
slow release fertilizer at a rate of 4007 gm3bull Five seeds were sown
per tube Treatments were distributed in a randomized complete
block design consisting of4 blocks (locations on the greenhouse
bench) with each block containing the 10 treatment combinations for
each of four seed lots (Table 4) Each repetition contained 20 tubes
repetitions were placed in random order four repetitions to a rack ten
racks to each block Each repetition for each treatment contained 100
seeds therefore 100 seeds were used for each seed source by
treatment by block combination pooled to one measurement for the
response variable germination percentage Racks were placed in a
greenhouse for germination Germination conditions included
31
ambient light and 70 relative humidity with average daily
temperature 243degC (daytime temperature range 200-272degC) and
average night temperature 216degC (nighttime temperature range 206shy
239degC) Tubes were watered at 2 hour intervals six times a day
Germination was recorded at weekly intervals 7 1421 and 28 days
after planting
Table 4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder
Treatment Number Stratification (Days) Separation Fraction
1 56 None
2 56 Floating
3 56 Sinking
4 28 None
5 28 Floating
6 28 Sinking
7 0 None
8 0 Floating
9 0 Sinking
blocks Qocations on the greenhouse bench) with each block
containing the 10 treatment combinations for each of four seed lots
(Table 5) Each repetition contained 20 tubes repetitions were placed
in random order four repetitions to a rack ten racks to each block
Each repetition for each treatment contained 100 seeds therefore 100
seeds were used for each seed source by treatment by block
combination pooled to one measurement for the response variable
germination percentage Racks were placed in a greenhouse for
germination Germination conditions included ambient light and
7000 relative humidity with average daily temperature 243 degC
(daytime temperature range 200-272degC) and average night
temperature 216degC (nighttime temperature range 206-239degC)
Tubes were watered at 2 hour intervals six times a day Germination
was recorded at weekly intervals 7 1421 and 28 days after planting
34
Table 5 Treatment Combinations for Experimental Layout of Randomized Complete Block-Water Birch
Treatment Number Stratification (Days) Separation Fraction
56 None
2 56 Floating
3 56 Sinking
4 21 None
5 21 Floating
6 21 Sinking
7 0 None
8 0 Floating
9 0 Sinking
10 0 None
DATA ANALYSIS
The seed refinement experiment was performed to determine
the mostadvantageous separation technique for use in the
germination studies with the percentage of filled seeds present in the
sinking fractions (percentage fill) and proportion of filled seeds
recovered from the total filled seeds available in the sample
(percentage recovery) as response variables and the preparation
protocols and seed sources as independent variables
The second experiment utilized the chosen seed refinement
method with levels of stratification seed separation fraction and seed
source as independent variables (or in the augmented factorial
treatment combination as the independent variable) with germination
percentage measured as the response variable Germination rate was
also recorded however the rapid germination between the time of
sowing and the first sampling (at 7 days) prevented meaningful
analysis of this da~
Data was analyzed by using categorical data modeling analysis
as found in the SAScopy statistical program The PROC CATMOD
procedure can perform analysis and giveanalysis of variance in the
36
general sense that it analyzes the response functions fits linear models
to functions of response frequencies and partitions the variation
among those functions into various sources (SAS Institute 1989)
CATMOD analyzes data that can be represented in a two-
dimensional contingency table with the rows corresponding to
populations or samples defined by one or more independent variables
and the columns corresponding to one or more dependent (response)
variables The frequencies in the table are assumed to follow a
product multinomial distribution with a simple random sample taken
for each population The probability for the response ofeach cell is
estimated and the vector (P) of these proportions is transformed into a
vector of functions F =F(P) If It denotes the vector of true
probabilities for the table then the functions of the true probabilities
F(It) are assumed to follow a linear model
I
where EA denotes asymptotic expectation X is the design matrix
containing fixed constants and Pis a vector ofparameters to be
37
estimated CA TMOD provides two estimation methods the
maximum-likelihood method and the weighted-least-squares method
which was used in this analysis (SAS Institute 1989)
Hypotheses about linear combinations of the parameters can be
tested these statistics are approximately distributed as chi-square for
sufficiently large sample sizes (SAS Institute 1989)
All of the response variables considered had a binomial type of
probability distribution (seed filled or not filled seed germinated or
not germinated) All treatments ofboth experiments were analyzed
using the PROC CATMOD procedure to examine the general model
as well as planned comparisons using contrast statements where ~
appropriate The PROC MEANS procedure was used to calculate
marginal percentages (main effect and interaction combinations)
along with standard errors Pairwise Z-tests were used to separate
percentages in those effects which were determined to be significant
by categorical modeling at the observed significance level laquo=005 this
method of percentage separation is analogous to Fishers LSD for
separating means
38
Categorical modeling of the seed refinement experiments used
two models one for the percentage of filled seeds attained in the
sinking and floating fractions and one for the percentage of filled
seeds recovered from those available in the baseline sample The
treatment structure for both of these seed refinement studies was a 5
X 2 X 4 factorial (preparation protocol by separation fraction by seed
source)
The model in CATMOD for the percentage of filled seeds
attained in the fractions is as follows
fill = source prep fraction sourceprep sourcefraction prepfraction sourceprepfraction
where fill is the response variable the number of filled seeds
source is the seed source prep is the LDS treatment protocol
(imbibition plus one of four drying times or no treatment) and
fraction is the separation fraction (floating or sinking)
The CATMOD model for the percentage of filled seed
recovered in the floating or sinking fractions is as follows
39
rec = source prep sourceprep
where rec is the response variable (number of filled seeds floating or
sinking) source is the seed source and prep is the IDS
treatment protocol
The treatment structure for the germination studies was a 3 X 3
factorial (stratification by separation) with an additional control (no
treatment) for 4 seed sources termed an augmented factorial design
by Lentner and Bishop (1986) The PROC CATMOD procedure
was used to analyze the data without the no treatment control as a
simple 3 X 3 X 4 factorial (stratification by separation by seed source)
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
LIST OF FIGURES
PageFigure
1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction 44
2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction 52
3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction 53
4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source 58
5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification 59
6 Alder Percentage Germination as Influenced by Imbibition and Seed Source 62
7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source 68
8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction 69
9 Birch Percentage Germination as Influenced by Stratification and Seed Source 70
10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification 71
XVI
Figure Page
11 Birch Percentage Germination as Influenced by Imbibition and Seed Source 75
12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 81
13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 83
INTRODUCTION
Birch (Betula) and alder (Alnus) are two genera of Betulaceae
trees found in riparian areas throughout New Mexico The presence
ofbirch and alder in riparian zones of New Mexico has been noted in
many early surveys of the region (Britton 1908 Sargent 1901 1905
Wooton and Standley 1915) Water birch (Betula ocddentalisHook
formerly B fontinalis Sarg) is found in the northern mountains of the
state (Martin and Hutchins 1980) New Mexico has two species of
alder Arizona alder (Alnus oblongifolia Torr) which is found in the
mountains of southwest New Mexico (Martin and Hutchins 1980
Vines 1960) and thinleaf alder (Alnus tenuifolia Nutt) designated by
Carter (1997) as Alnus incana ssp tenuifolia Nutt found in the
northern and western mountains (Martin and Hutchins 1980 Vines
1960) Until recently existence of these species has been of interest
mainly from a botanical standpoint However with increasing landshy
use in the western United States these trees may have a further
purpose in the revegetation of degraded riparian areas and as oasis
plants for those interested in native landscapes (Phillips 1995)
Successful revegetation of degraded areas is influenced by many
1
factors including the site conditions commonly encountered and the
chosen plant material Desirable plant material should be wellshy
adapted to the site have high survival and be economical to obtain or
produce
LITERATURE REVIEW
Revegetation and Reconstruction
Strategies for revegetation of disturbed lands are generally
divided into three categories restoration reclamation and
rehabilitation Restoration is the complete replication of the original
conditions species habitat and function of the area Reclamation
involves returning the area to a condition that is habitable by the
organisms that were originally present or organisms that approximate
the original inhabitants Rehabilitation involves returning the land to
a form and function which conforms to a prior land-use plan
including a stable ecological state that does not contribute
substantially to environmental deterioration and is consistent with
surrounding aesthetic values (Allen 1988 National Academy of
Sciences 1974) These three categories have been collectively termed
reconstruction by Allen (1988) Complete restoration is often not
practical as certain requisite intermediate conditions of varying
durations maybe necessary In the arid western United States
natural succession is slow and dependence on natural process risks
further site degradation (National Academy of Sciences 1974)
3
Reclamation and rehabilitation may be more workable concepts A
practical guiding philosophy would be the objective to create a stable
ecosystem that is compositionally and functionally similar to that
which existed prior to human disturbance with the realization that
such a goal is not completely attainable (Burton et al 1988)
Species Selection
It has long been the philosophy of those involved in
reconstruction efforts that the use ofnative and diverse species is
desirable rather than dependence on a few proven species (Daniel
et al 1979 Harker et al 1993 Nielson and Peterson 1973) The
rationale is that native species are better adapted to adverse site
conditions such as low moisture and high surface temperatures and
exposure (Nielson and Peterson 1973) Only native species survived
in a European study even though exotic species examined also
possessed characteristics which were well-adapted to the site (Herrera
et al 1993) Use of diverse native plant species can enhance
reconstruction efforts and sustain more diverse wildlife populations
(Harker et al 1993) Using plant material of local provenance (origin
of seed) to maximize survival is also important (Albers and Carpenter
4
1979 Burton et al 1988 Daniel et al 1979 Hobbs 1984) Species of
plants evolve within their habitat to site conditions including edaphic
topographic and climatic conditions such as temperature (Bewley and
Black 1994) photoperiod (Currie 1990) and growing season A plant
with origins in southern latitudes may not properly harden off for
winter in time to avoid early frost when grown in northern latitudes
with longer day1engths while a plant from northern latitudes may not
have optimal shoot growth in the shorter day1ength of southern areas
(Fowells 1965 Lane 1993)
PlantingMethods
Natural colonization processes can take anywhere from ten to
hundreds ofyears depending on site conditions (National Academy
of Sciences 1974) Planting methods used in reconstruction include
direct seeding wildling transplants and use ofbare-root or
containerized transplant material (Schubert et al 1970) Direct
seeding is often the least expensive planting method but success with
woody species is frequently limited Predation of seed germination
failure and adverse conditions for germinants can result in planting
failure (Fowells 1965 Haeussler et al 1995 Hibbs et al 1994
5
Monsen 1984 Pratt 1986) Wildling transplants may have poor
survival ifplanting is not timed properly and done carefully (Schubert
et al 1970) Use ofnursery grown seedlings bare-root or
containerized can improve survival rates relative to other
reconstruction efforts (Hobbs 1984) The ability to match stock type
(source physiological and morphological condition) to the site
known as the target seedling concept (Rose et al 1990) and greater
latitude in planting conditions (timing) can contribute to improved
transplant success of nursery stock relative to wildlings Combining
direct seeding ofnon-woody plants and nursery-grown seedlings can
be the most efficient and economical method of reconstruction when
costs ofproducing container stock can be kept low (Belcher 1982
Dunlap and Barnett 1984 Rose et al 1990) The success of
reconstruction efforts is heavily dependent on site conditions and the
quality of the plant material used (Monsen 1984) In tum quality of
plant material is dependent on well-developed germination and
culture protocols The economic feasibility of stock propagation for
reconstruction work is dependent on finding methods to efficiently
upgrade seed quality (proportion ofgerminable seeds) and optimize
6
germination capacity and seedling survival (Belcher 1982 Bonner
1984)
Birch and Alder Suitability in Reconstruction
Montane riparian vegetation zones are contained in areas where
the supply ofwater is constant (perennial) as well as areas with an
ephemeral (intermittent) water supply Riparian zones contain both
obligate and facultative riparian species Facultative riparian species
are also found in surrounding open spaces and in high cool nonshy
riparian locations (Dick-Peddie 1993) Riparian vegetation follows an
elevational gradient from the source to the mouth of the drainage
perpendicular to the zone of upland vegetation (Dick-Peddie 1993)
Other habitats where water may be caught but are not part of a true
drainage are termed pseudoriparian Pseudoriparian habitats include
gullies roadside ditches and the bottoms of talus slopes (Dick-Peddie
1993) Most of the obligate riparian species found in riparian and
pseudoriparian areas are adapted to flood conditions with the ability
to rapidly reproduce and colonize a devastated area Characteristics
ofobligate riparian species include prolific seed production efficient
7
seed dispersal fast growth short life-cycles and rapid attainment of
reproductive stage (Dick-Peddie 1993)
Birch and alder species are generally confined to montane
riparian zones (Elias 1980) Members ofboth genera have properties
indicative of obligate riparian species including fast growth prolific
seed production and short life-cycle these properties also make
members of these genera suitable candidates for use in reconstruction
efforts (Elias 1980) Birch and alder are known as pioneer species
which can successfully establish on denuded areas (Young and Young
1992) and which prefer mineral soil for germination and early growth
(Haeussler et al 1995 Schalin 1968) In addition most alder species
including thirlleaf alder and Arizona alder have the ability to fix
atmospheric nitrogen via a symbiotic relationship with root-nodule
forming species of Frankia actinomycetes (Bond 195519711976
Virtanen 1957) Many researchers believe the formation of a dynamic
rhizosphere of this type is critical to the rehabilitation of degraded
lands (Herrera et al 1993 Whitford 1988) Biological nitrogen
fixation in conjunction with the production of large amounts of litter
has been shown to help build up organic matter nitrogen and
8
improve soil structure in deficient soils such as glacial till (Bollen and
Lu 1968 Crocker and Major 1955 Tarrant and Trappe 1971)
Biological nitrogen fixation can also improve conditions for other
non-nitrogen fixing species (Tarrant 1961) and enhance species
diversity (Franklin and Pechanec 1968)
The use ofthese deciduous trees with the objective of improving
the site conditions (ie shade nutrients and organic matter) for other
species (Albers and Carpenter 1979) is a valuable strategy in the
reconstruction of disturbed areas such as mine spoils
Production ofStockP1ants
Efficient propagation ofnursery stock from seed requires
extensive knowledge of the germination requirements and cultural
methods needed for the particular species Little is known about the
propagation requirements for the two species used in this study
thinleaf alder and water birch This deficit is due in part to a lack of
demand for these species in the past Extensive work has been done
on the propagation of other species within the Alnus and Betula
genera specifically those species of commercial value to the timber
industry such as red alder (A rubra Bong) and paper birch (B
9
papyrifera Marsh) Information generated from propagation studies
on these species has elucidated some universal seed characteristics
and germination requirements for members ofBetulaceae Seeds aremiddot
characteristically very small and light and may have a winged
integument to aid in wind dispersal Average seed density for B
ocddentalis is about 2500 seeds per gram while A tenuifolia
averages about 1488 seeds per gram (Vines 1960) Seed quality and
germination capacity are often very low as it is difficult to separate
sound from empty seeds when size and weight are so low (Brinkman
1974 Schopmeyer 1974) Seed quality may vary considerably from
harvest to harvest (Bjorkbom et al 1965) Within species
germination requirements may differ with provenance (Fowler and
Dwight 1964 Wilcox 1968) or even within a provenance (Bjorkbom
et al 1965 Schopmeyer 1974) In some instances the requirements
for germination may be met but germination does not occur a
condition referred to as dormancy
Seed Dormanqr and Methods to Overcome It
Dormancy in seeds is defined as the condition where seeds will
not germinate even when environmental conditions (water
10
temperature and aeration) are permissive for germination (Bewley
and Black 1994 Hartmann et al 1997) This mechanism ensures that
germination does not take place in less than optimum conditions or at
the wrong time (Bewley and Black 1994 Thompson 1971) For
example in some species seeds of southern provenance require
longer stratifications (Fowler and Dwight 1964) probably to prevent
germination in areas where there are intermittent periods ofwarm
weather followed by frost Seed dormancy results from a
combination ofgenetic and environmental conditions and it is not
always possible to predict the dormancy of a particular species from
characteristics of other species within the genus (Schopmeyer 1974)
There are different systems for classifying dormancy but the
condition may be divided into four basic types exogenous
endogenous double or combinational and secondary (Hartmann et
al 1997) The seed dormancy exhibited by birch and alder falls under
the category of endogenous dormancy a dormancy imposed by
embryonic factors This includes morphological dormancy (an
underdeveloped embryo) and physiological dormancy ofvarying
degrees (non-deep intermediate and deep) Non-deep physiological
11
dormancy is characterized by the need for after-ripening or exposure
to red light (photodormancy) Intermediate physiological dormancy
is characterized by the need for moderate periods of cold stratification
(up to 56 days) Deep physiological dormancy requires long periods
of cold stratification more than 56 days (Hartmann et al 1997)
Seeds ofboth Alnus and Betula exhibit varying degrees of
dormancy in most cases broken by coolmoist stratification andor
germination under red light (Brinkman 1974 Dirr and Heuser 1987
Schopmeyer 1974 Young and Young 1992) In some species of these
genera chemical treatments such as potassium nitrate have been
effective to overcome dormancy (Bradbeer 1988 Hartmann et al
1997 Young et al 1984) Many birch species are known to possess a
phytochrome light detection system which prevents germination
when seeds are buried too deep to allow seedling survival after
germination (Bewley and Black 1994 Black and Wareing 1955
Bradbeer 1988) Where the phytochrome detection mechanism is
present exposure to red light during germination is required for
breaking dormancy Most species of birch and alder have seeds that
ripen in late summer or early fall fall germination would result in
12
seedling loss over the winter so an after-ripening or stratification
requirement decreases the possibility of fall germination Joseph
(1929) found non-stratified birch seeds had a higher temperature
requirement for germination The current theory is that stratification
causes phase changes in membrane fluidity and triggers membraneshy
related signal transduction pathways activating enzymes and
hormones thus allowing dormancy release (Bewley and Black 1994
Ross and Bradbeer 1971)
Leaching of certain chemical inhibitors from seeds can also
break dormancy it maybe that this is part of the mechanism by
which photo dormancy is broken by moist stratification as only small
amounts of moisture are needed (Brad beer 1988) Research indicates
that the testa and pericarp of the seeds are involved in dormancy not
because they contain the inhibitor but because they prevent leaching
of the inhibitor (Villiers and Wareing 1964 Webb and Wareing
1972) Ru40lf (1950) found that cold-soaking might in some cases be
an acceptable substitute for stratification in some conifer species this
might be due to the leaching mechanism
13
The role ofpotassium nitrate in breaking dormancy has not
been clarified but there is speculation that the nitrogen supplied or
the oxygenating properties of the nitrate are involved (Brad beer
1988) Biswas et al (1972) found that the chemical treatment
enhanced the effect of stratification but did not necessarily replace it
Hilton (1985) found the germination-stimulating properties ofnitrate
depend on the presence of light nitrate in the presence of red light is
believed to be a cofactor to the phytochrome system which is involved
in the synthesis ofgibberellins that promote germination (Hilhorst et
al 1986)
Germination Requirements
General requirements for germination include moisture
favorable temperatures adeq-qate gas exchange and for some species I)
light In the presence of these conditions the quiescent seed can
imbibe water causing the seed to swell and the seed coat to split or
break Enzymatic activity within the seed accelerates increasing
respiration and use of stored energy resulting in the commencement
of growth processes within the seed (Bewley and Black 1994
14
Pretreatment requirements for germination of alder seed are
quite variable both between and within species For many species of
alder cold stratification periods of60-180 days are recommended
(Dirr and Heuser 1987) In one study ofthinleaf alder prechilling
(stratification) did not improve germination percentage while in
European speckled alder 180 days of stratification did improve
percentage germination (Young and Young 1992) Several other
treatments including light freezing and potassium nitrate
independently and with stratification have been shown to enhance
germination ofalders In red alder stratification was not necessary
when seed was germinated in light (Kenady 1978 Radwan and
DeBell 1981) Evidence of a phytochrome-regulated dormancy was shy
subsequently found in this species (Bormann 1983) Several general
horticultural texts recommend a pretreatment with 0200 potassium
nitrate (wv) to enhance stratification effects (Hartmann et al 1997
Young and Young 1992) In one study stratification followed by
freezing of seed for 3 days at -20degC enhanced germination (Schalin
1968)
16
Water Birch
Birch species are widely distributed in the northern hemisphere
found further north than alders can grow in various habitats and are
tolerant of a wide range of soils and moisture levels but are sensitive
to drought (Ashburner 1993 deJong 1993) Birch species are thought
to be more resistant to drought than alder species (McVean 1956) B
ocddentaJis Hook occurs as a shrub or small tree along streams or in
moist canyons and occasionally in dryer sites of the mountain West
( at elevations of 1500-2700 meters (Foxx and Hoard 1995 Vines
1960) It is known in the vernacular as water birch red birch and
black birch A small tree it is not used for lumber but can be used as
firewood posts browse by livestock or wildlife and sometimes as a
landscape tree (BrenzeI1995 Elias 1980 Preston 1968 Vines 1960)
Germination requirements for species of Betula generally
include stratification or red light treatment (Brinkman 1974)
indicating the presence ofphytochrome far-red inhibition (Bevington
1986 Bevington and Hoyle 1981 Schopmeyer 1974) Occasionally
both red light and stratification are recommended to improve
germination rate (Dirr and Heuser 1987) Potassium nitrate 02
17
pretreatment is recommended for birch species by Hartmann et al
(1997) Seeds of this species are considered to have a fairly shallow
dormancy (Lane 1993)
Seed Quality Improvements
Methods to upgrade seed quality (separate viable from nonshy
viable seeds) have been developed for different species Conventional
seed separation techniques are based on density such as air column or
liquidseparation or by size and shape such as with screens
Separation ofviable and non-viable seeds is extremely problematic
with very light winged seeds like those of alder and birch Air
separation techniques may not be practical for winged light-weight
seed Flotation techniques often employ lighter-than-water solvents
but some of these substances may have adverse effects on seed
viability (Barnett 1971 McLemore 1965) Widescale use of some
solvents is not considered desirable because of health and safety
concerns
A method of seed refinementupgrade originally developed in
Sweden by Milan Simak called the LDS method (Incubation
Drying Separation) shows promise for separating live and dead seeds
18
(cited in Bonner 1984 Downie and Wang 1992 Simak 1983
Sweeney et al 1991) Seeds are imbibed for several hours then
incubated at cool temperatures (15~or several hours in 100
relative humidity Seeds are then dried for several hours at 35
relative humidity at cool temperatures (timing and relative humidity
must be adjusted for the particular species) During the drying
dead seeds will lose most of the water previously imbibed while live
seeds should retain most of their imbibed water This differential
moisture content would make separation by flotation and other
density separation methods potentially feasible Similar methods of
conditioning have been shown to improve seed quality in lettuce
tomato and onion (Hill et al 1989) It has also been shown that
drying of stratified seeds for storage or for separation from
stratification medium need not result in loss of viability (Danielson
and Tanaka 1978 Schopmeyer 1974)
19
OBJECTIVES OF THIS STUDY
The purpose of this study is to determine the effectiveness of the
LDS seed refinement technique and othi separation procedures in
increasing the percentage of live seeds in a seed lot and to develop
germination strategies for water birch and thinleaf alder investigating
the use of stratification Secondly this study will examine the within-
species variability of different seed lots in their response to LDS and
stratification treatments
METHODS AND MATERIALS
Sources
Alder strobiles were collected in October and November of
1998 in Catron County New Mexico near the towns of Luna and
Reserve in the Cottonwood Canyon Campground and in the Head of
the Ditch Campground and in Taos County New Mexico in the
Red River Canyon near the Molycorp molybdenum mine Table 1
shows the seed source elevations and locations Strobiles were kept
cool and allowed to dry for several weeks Seeds were separated from
the opening strobiles by rubbing on a coarse screen
Birch strobiles were collected in October and N overrtber of 1998
in Taos County in the Red River Canyon near the Mo1ycorp
molybdenum mine (Table 1) Strobiles were kept cool and allowed to
dry for several weeks allowing the release of seeds from the bracts
In addition commercial seed sources ofbirch and alder were
purchased in the summer of 1999 (collected in the fall of 1998) The
seed lots collected in 1998 (Table 1) were used in the seed refinement
study providing four seed lots for that study For the final seed
refinement-germination study the two Red River Canyon seed lots of
21
Table 1 Seed Source Locations and Elevations
Species Source Lot Baseline Description Elevation Latitude Notes No Fill (meters) Longitude
Thinleaf Alder Luna NA 234 Head ofthe Ditch CG 2134 N 33deg49 W 108deg59
t+
Reserve NA 268 Cottonwood Canyon 1829 N 33deg37 W 108deg55
t+
RRC-l 98108 08 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
RRC-2 98109 09 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 537 W of Poncha Springs CO 2438 N 38deg31 W 106deg05
I
Water Birch RRC-3 98104 69 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Moly-1 98106 39 Molycorp Tailings Rd 2469 N 36deg41 W 105deg29
t+
Moly-2 98107 52 Molycorp Low Dump 2469 N 36deg41 W 105deg29
t+
Mo1y-3 98105 56 Molycorp Front Dump 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 297 W ofPoncha Springs CO
t used in Experiment 1--Seed Refinement I used in Experiment 2--Germination Requirements sectpooled for use in Experiment 2-Germination Requirements
2377 N 38deg31 W 106deg05
I
alder and the Red River Canyon and Moly 3 seed lots ofbirch were )
pooled due to limited amounts of seed The seed lots collected in
1998 and the 1998 purchased seed lots from Chaffee County
Colorado provided four seed lots of each species for that study
All seed sources were evaluated for percentage of filled seeds by
means ofdissection performed under a dissecting microscope at 30X
magnification (Berry and Torrey 1985) Alder species baseline
percentage of filled seeds was estimated using 25 samples of 100 seeds
pooled into one percentage response for each seed source Birch
species baseline percentage of filled seeds was determined using 15
samples of 50 seeds pooled into one percentage response for each seed
source Baseline percentage fill (Table 1) is the estimate of the
percentage of filled seed in the entire seed collection for each source
Separation Media
Ethanol and water were not particularly effective in separation
ofthinleaf alder seeds either using IDS methods or when separating
dry seed It was necessary to choose a fluid with a lower specific
gravity than ethanol (SG=O 79) in order to separate filled and empty
seeds with very low densities Falleri and Pacella (1997) found that
23
low-density London plane tree (Platanus x acerifolia [Aid Willd)
seeds could not be separated using water as the separation medium
due to the very small density differences between sound and empty
seeds and chose petroleum ether as a separation medium Petroleum
ether was chosen for the separation of thinleaf alder seeds because of
its low specific gravity (SG middot060) its relative stability low
reactivity and rating as a slight health risk Contact with skin may
cause dryness and irritation but no chronic systematic effects have
been reported with industrial use (Mallinckrodt Baker Inc 1997a)
As observed previously for thinleaf alder seeds the simple
specific gravity method using water was not effective for separating
water birch seeds In preliminary studies ethanol and petroleum
ether were found to be effective in separation of dry water birch seeds
and petroleum ether ethanol and water were somewhat effective in
separation of water birch seeds treated by the LDS method but
ethanol was chosen as the separation medium because of its lower
cost greater effectiveness and availability
Denatured ethanol is actually rated a greater health risk than
petroleum ether because ingestion is more likely to result in death or
24
permanent damage and prolonged skin contact may affect the
nervous system and other organ systems of the body Ethanol also
has a higher reactivity rating Gloves goggles and lab coat (personal
protective equipment) proper ventilation avoidance of ingestion and
proper fire safety measures should prevent problems with use of either
solvent (Mallinckrodt Baker Inc 1997a 1997b)
Seed Refinement
Thinleaf Alder
Separation treatments examined includeddensity separation of
dry seed samples in petroleum ettter (the control) and imbibed seed ~
samples treated with the IDS method at 0 1 18 and 24 hour drying
times followed by density separation in petroleum ether (Table 2)
Seeds were imbibed for 24 hours by submersion in a 10-gallon glass
aquarium filled with distilled water and equipped with an aeration
pump and filter Seeds were packaged in filter paper then the
packages were enclosed in wire cages (purchased tea balls were used
for this purpose) weighted with marbles to keep them submerged At
the end of the imbibition period seeds were removed from the cages
thoroughly blotted and placed on clean filter paper The drying
25
incubation was performed in a closed chamber with a constant
humidity obtained by the use ofCaC12middot6H20 salt in a saturated
solution prepared by adding SOOOg CaClzmiddot6HzO to 30 liters of
distilled water (Slavik 1974 Young 1967) Imbibed seeds were placed
on filter paper and suspended on a screen above the calcium chloride
solution Humidity was monitored using an hygrometer and held
steady at 50 in the presence of the wet seeds and filter paper
Table 2 Alder Preparation Protocols for S~d Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) 0 0
2
3
24
24
0
1 )
4 24 18
5 24 24
At the end of the appropriate drying incubation the seeds were
placed in petroleum ether and briefly and vigorously stirred to
separate seeds adhering to one another Floating seeds were removed
from the surface of the petroleum ether by means of a small net
andor a spatula placed on clean moistened filter paper and placed in
26
a labeled plastic bag to await counting The sinking seeds were
strained through the net and packaged in a similar manner Five
repetitions were performed for each of the five treatments using 100
seeds per repetition Percentage of filled seeds contained in each
fraction was determined by means of dissection tests performed on the
floating and sinking fractions using a scalpel and a dissecting
microscope with 30X magnification
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that particular repetition
of filled seeds in the sinking fraction X 100=percentage recovery
of filled seeds in the sinking fraction + of filled seeds in the floating fraction
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product of percentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
27
Water Birch
Separation treatments included density separation of dry seed in
95 ethanol (the control) and seed samples treated with the IDS
method at 005 1 and 2 hour drying times followed by density
separation in 95 ethanol (Table 3) Seeds were imbibed for 12 hours
by submersion in a 10-gallon glass aquarium filled with distilled water
and equipped with an aeration pump and filter Seeds were packaged
in filter paper then the packages were enclosed in wire cages
(purchased tea balls were used for this purpose) weighted with
marbles to keep them submerged At the end of the imbibition
period seeds were removed from the cages thoroughly blotted and
placed on clean filter paper The drying incubation was performed in
a closed chamber with a constant humidity obtained by the use of
CaCI2middot6H20 salt in a saturated solution prepared as described in the
previous section (Slavik 1974 Young 1967) Imbibed seeds were
placed on filter paper and suspended on a screen above the calcium
chloride solution Humidity was monitored using an hygrometer and
held steady at 50 in the presence of the wet seeds and filter paper
28
Table 3 Birch Preparation Protocols for Seed Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) o o
2 12 o
3 12 05
4 12 1
5 12 2
At the end of the appropriate drying incubation the seeds were
placed in 9500 ethanol and briefly and vigorously stirred to separate
seeds adhering to one another Floating seeds were removed from the
surface of the ethanol by means of a small net andor a spatula
placed on clean moistened filter paper and placed in a labeled plastic
bag to await counting The sinking seeds were strained through the
net and packaged in a similar manner Three repetitions were
performed for each of the five treatments using 50 seeds per
repetition Percentage of filled seeds contained in each fraction was
determined by means of dissection tests performed on the floating and
sinking fractions using a scalpel and a dissecting microscope with
30X magnification
29
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that repetition (as given in the previous equation)
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product ofpercentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
Germination Requirements
Thinleaf Alder
Separations were performed using the separation method
chosen from the seed refinement study alder preparation protocol 4
24-hour imbibition followed by I8-hour drying time and separation in
petroleum ether as described in the seed refinement experiment
(Table 2) Unseparated imbibed seeds and seeds from both the
floating and sinking fractions were subsequently treated with 028
and 56 days of stratification In addition an unseparated nonshy
stratified control of dry seeds was tested for germination Seeds for
stratification treatments were placed in layers ofpaper towel
30
moistened with 25 m1 of distilled water and placed in sealed zip-lock
plastic bags The bags were placed in a cooler at I-5degC (average
temperature 50degC) for periods of 28 or 56 days Initiation of
stratification treatments was staggered so that all treatments came out
ofstratification at the same time
Following stratification the seeds were sown in Ray-Leach
Super Cells (Steuwe amp Sons Inc Corvalis OR) containing a 2 1 1
ratio ofpeatmossperlitevermiculite (vvv) with OsmocoteR 14-14-10
slow release fertilizer at a rate of 4007 gm3bull Five seeds were sown
per tube Treatments were distributed in a randomized complete
block design consisting of4 blocks (locations on the greenhouse
bench) with each block containing the 10 treatment combinations for
each of four seed lots (Table 4) Each repetition contained 20 tubes
repetitions were placed in random order four repetitions to a rack ten
racks to each block Each repetition for each treatment contained 100
seeds therefore 100 seeds were used for each seed source by
treatment by block combination pooled to one measurement for the
response variable germination percentage Racks were placed in a
greenhouse for germination Germination conditions included
31
ambient light and 70 relative humidity with average daily
temperature 243degC (daytime temperature range 200-272degC) and
average night temperature 216degC (nighttime temperature range 206shy
239degC) Tubes were watered at 2 hour intervals six times a day
Germination was recorded at weekly intervals 7 1421 and 28 days
after planting
Table 4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder
Treatment Number Stratification (Days) Separation Fraction
1 56 None
2 56 Floating
3 56 Sinking
4 28 None
5 28 Floating
6 28 Sinking
7 0 None
8 0 Floating
9 0 Sinking
blocks Qocations on the greenhouse bench) with each block
containing the 10 treatment combinations for each of four seed lots
(Table 5) Each repetition contained 20 tubes repetitions were placed
in random order four repetitions to a rack ten racks to each block
Each repetition for each treatment contained 100 seeds therefore 100
seeds were used for each seed source by treatment by block
combination pooled to one measurement for the response variable
germination percentage Racks were placed in a greenhouse for
germination Germination conditions included ambient light and
7000 relative humidity with average daily temperature 243 degC
(daytime temperature range 200-272degC) and average night
temperature 216degC (nighttime temperature range 206-239degC)
Tubes were watered at 2 hour intervals six times a day Germination
was recorded at weekly intervals 7 1421 and 28 days after planting
34
Table 5 Treatment Combinations for Experimental Layout of Randomized Complete Block-Water Birch
Treatment Number Stratification (Days) Separation Fraction
56 None
2 56 Floating
3 56 Sinking
4 21 None
5 21 Floating
6 21 Sinking
7 0 None
8 0 Floating
9 0 Sinking
10 0 None
DATA ANALYSIS
The seed refinement experiment was performed to determine
the mostadvantageous separation technique for use in the
germination studies with the percentage of filled seeds present in the
sinking fractions (percentage fill) and proportion of filled seeds
recovered from the total filled seeds available in the sample
(percentage recovery) as response variables and the preparation
protocols and seed sources as independent variables
The second experiment utilized the chosen seed refinement
method with levels of stratification seed separation fraction and seed
source as independent variables (or in the augmented factorial
treatment combination as the independent variable) with germination
percentage measured as the response variable Germination rate was
also recorded however the rapid germination between the time of
sowing and the first sampling (at 7 days) prevented meaningful
analysis of this da~
Data was analyzed by using categorical data modeling analysis
as found in the SAScopy statistical program The PROC CATMOD
procedure can perform analysis and giveanalysis of variance in the
36
general sense that it analyzes the response functions fits linear models
to functions of response frequencies and partitions the variation
among those functions into various sources (SAS Institute 1989)
CATMOD analyzes data that can be represented in a two-
dimensional contingency table with the rows corresponding to
populations or samples defined by one or more independent variables
and the columns corresponding to one or more dependent (response)
variables The frequencies in the table are assumed to follow a
product multinomial distribution with a simple random sample taken
for each population The probability for the response ofeach cell is
estimated and the vector (P) of these proportions is transformed into a
vector of functions F =F(P) If It denotes the vector of true
probabilities for the table then the functions of the true probabilities
F(It) are assumed to follow a linear model
I
where EA denotes asymptotic expectation X is the design matrix
containing fixed constants and Pis a vector ofparameters to be
37
estimated CA TMOD provides two estimation methods the
maximum-likelihood method and the weighted-least-squares method
which was used in this analysis (SAS Institute 1989)
Hypotheses about linear combinations of the parameters can be
tested these statistics are approximately distributed as chi-square for
sufficiently large sample sizes (SAS Institute 1989)
All of the response variables considered had a binomial type of
probability distribution (seed filled or not filled seed germinated or
not germinated) All treatments ofboth experiments were analyzed
using the PROC CATMOD procedure to examine the general model
as well as planned comparisons using contrast statements where ~
appropriate The PROC MEANS procedure was used to calculate
marginal percentages (main effect and interaction combinations)
along with standard errors Pairwise Z-tests were used to separate
percentages in those effects which were determined to be significant
by categorical modeling at the observed significance level laquo=005 this
method of percentage separation is analogous to Fishers LSD for
separating means
38
Categorical modeling of the seed refinement experiments used
two models one for the percentage of filled seeds attained in the
sinking and floating fractions and one for the percentage of filled
seeds recovered from those available in the baseline sample The
treatment structure for both of these seed refinement studies was a 5
X 2 X 4 factorial (preparation protocol by separation fraction by seed
source)
The model in CATMOD for the percentage of filled seeds
attained in the fractions is as follows
fill = source prep fraction sourceprep sourcefraction prepfraction sourceprepfraction
where fill is the response variable the number of filled seeds
source is the seed source prep is the LDS treatment protocol
(imbibition plus one of four drying times or no treatment) and
fraction is the separation fraction (floating or sinking)
The CATMOD model for the percentage of filled seed
recovered in the floating or sinking fractions is as follows
39
rec = source prep sourceprep
where rec is the response variable (number of filled seeds floating or
sinking) source is the seed source and prep is the IDS
treatment protocol
The treatment structure for the germination studies was a 3 X 3
factorial (stratification by separation) with an additional control (no
treatment) for 4 seed sources termed an augmented factorial design
by Lentner and Bishop (1986) The PROC CATMOD procedure
was used to analyze the data without the no treatment control as a
simple 3 X 3 X 4 factorial (stratification by separation by seed source)
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245
Figure Page
11 Birch Percentage Germination as Influenced by Imbibition and Seed Source 75
12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 81
13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol 83
INTRODUCTION
Birch (Betula) and alder (Alnus) are two genera of Betulaceae
trees found in riparian areas throughout New Mexico The presence
ofbirch and alder in riparian zones of New Mexico has been noted in
many early surveys of the region (Britton 1908 Sargent 1901 1905
Wooton and Standley 1915) Water birch (Betula ocddentalisHook
formerly B fontinalis Sarg) is found in the northern mountains of the
state (Martin and Hutchins 1980) New Mexico has two species of
alder Arizona alder (Alnus oblongifolia Torr) which is found in the
mountains of southwest New Mexico (Martin and Hutchins 1980
Vines 1960) and thinleaf alder (Alnus tenuifolia Nutt) designated by
Carter (1997) as Alnus incana ssp tenuifolia Nutt found in the
northern and western mountains (Martin and Hutchins 1980 Vines
1960) Until recently existence of these species has been of interest
mainly from a botanical standpoint However with increasing landshy
use in the western United States these trees may have a further
purpose in the revegetation of degraded riparian areas and as oasis
plants for those interested in native landscapes (Phillips 1995)
Successful revegetation of degraded areas is influenced by many
1
factors including the site conditions commonly encountered and the
chosen plant material Desirable plant material should be wellshy
adapted to the site have high survival and be economical to obtain or
produce
LITERATURE REVIEW
Revegetation and Reconstruction
Strategies for revegetation of disturbed lands are generally
divided into three categories restoration reclamation and
rehabilitation Restoration is the complete replication of the original
conditions species habitat and function of the area Reclamation
involves returning the area to a condition that is habitable by the
organisms that were originally present or organisms that approximate
the original inhabitants Rehabilitation involves returning the land to
a form and function which conforms to a prior land-use plan
including a stable ecological state that does not contribute
substantially to environmental deterioration and is consistent with
surrounding aesthetic values (Allen 1988 National Academy of
Sciences 1974) These three categories have been collectively termed
reconstruction by Allen (1988) Complete restoration is often not
practical as certain requisite intermediate conditions of varying
durations maybe necessary In the arid western United States
natural succession is slow and dependence on natural process risks
further site degradation (National Academy of Sciences 1974)
3
Reclamation and rehabilitation may be more workable concepts A
practical guiding philosophy would be the objective to create a stable
ecosystem that is compositionally and functionally similar to that
which existed prior to human disturbance with the realization that
such a goal is not completely attainable (Burton et al 1988)
Species Selection
It has long been the philosophy of those involved in
reconstruction efforts that the use ofnative and diverse species is
desirable rather than dependence on a few proven species (Daniel
et al 1979 Harker et al 1993 Nielson and Peterson 1973) The
rationale is that native species are better adapted to adverse site
conditions such as low moisture and high surface temperatures and
exposure (Nielson and Peterson 1973) Only native species survived
in a European study even though exotic species examined also
possessed characteristics which were well-adapted to the site (Herrera
et al 1993) Use of diverse native plant species can enhance
reconstruction efforts and sustain more diverse wildlife populations
(Harker et al 1993) Using plant material of local provenance (origin
of seed) to maximize survival is also important (Albers and Carpenter
4
1979 Burton et al 1988 Daniel et al 1979 Hobbs 1984) Species of
plants evolve within their habitat to site conditions including edaphic
topographic and climatic conditions such as temperature (Bewley and
Black 1994) photoperiod (Currie 1990) and growing season A plant
with origins in southern latitudes may not properly harden off for
winter in time to avoid early frost when grown in northern latitudes
with longer day1engths while a plant from northern latitudes may not
have optimal shoot growth in the shorter day1ength of southern areas
(Fowells 1965 Lane 1993)
PlantingMethods
Natural colonization processes can take anywhere from ten to
hundreds ofyears depending on site conditions (National Academy
of Sciences 1974) Planting methods used in reconstruction include
direct seeding wildling transplants and use ofbare-root or
containerized transplant material (Schubert et al 1970) Direct
seeding is often the least expensive planting method but success with
woody species is frequently limited Predation of seed germination
failure and adverse conditions for germinants can result in planting
failure (Fowells 1965 Haeussler et al 1995 Hibbs et al 1994
5
Monsen 1984 Pratt 1986) Wildling transplants may have poor
survival ifplanting is not timed properly and done carefully (Schubert
et al 1970) Use ofnursery grown seedlings bare-root or
containerized can improve survival rates relative to other
reconstruction efforts (Hobbs 1984) The ability to match stock type
(source physiological and morphological condition) to the site
known as the target seedling concept (Rose et al 1990) and greater
latitude in planting conditions (timing) can contribute to improved
transplant success of nursery stock relative to wildlings Combining
direct seeding ofnon-woody plants and nursery-grown seedlings can
be the most efficient and economical method of reconstruction when
costs ofproducing container stock can be kept low (Belcher 1982
Dunlap and Barnett 1984 Rose et al 1990) The success of
reconstruction efforts is heavily dependent on site conditions and the
quality of the plant material used (Monsen 1984) In tum quality of
plant material is dependent on well-developed germination and
culture protocols The economic feasibility of stock propagation for
reconstruction work is dependent on finding methods to efficiently
upgrade seed quality (proportion ofgerminable seeds) and optimize
6
germination capacity and seedling survival (Belcher 1982 Bonner
1984)
Birch and Alder Suitability in Reconstruction
Montane riparian vegetation zones are contained in areas where
the supply ofwater is constant (perennial) as well as areas with an
ephemeral (intermittent) water supply Riparian zones contain both
obligate and facultative riparian species Facultative riparian species
are also found in surrounding open spaces and in high cool nonshy
riparian locations (Dick-Peddie 1993) Riparian vegetation follows an
elevational gradient from the source to the mouth of the drainage
perpendicular to the zone of upland vegetation (Dick-Peddie 1993)
Other habitats where water may be caught but are not part of a true
drainage are termed pseudoriparian Pseudoriparian habitats include
gullies roadside ditches and the bottoms of talus slopes (Dick-Peddie
1993) Most of the obligate riparian species found in riparian and
pseudoriparian areas are adapted to flood conditions with the ability
to rapidly reproduce and colonize a devastated area Characteristics
ofobligate riparian species include prolific seed production efficient
7
seed dispersal fast growth short life-cycles and rapid attainment of
reproductive stage (Dick-Peddie 1993)
Birch and alder species are generally confined to montane
riparian zones (Elias 1980) Members ofboth genera have properties
indicative of obligate riparian species including fast growth prolific
seed production and short life-cycle these properties also make
members of these genera suitable candidates for use in reconstruction
efforts (Elias 1980) Birch and alder are known as pioneer species
which can successfully establish on denuded areas (Young and Young
1992) and which prefer mineral soil for germination and early growth
(Haeussler et al 1995 Schalin 1968) In addition most alder species
including thirlleaf alder and Arizona alder have the ability to fix
atmospheric nitrogen via a symbiotic relationship with root-nodule
forming species of Frankia actinomycetes (Bond 195519711976
Virtanen 1957) Many researchers believe the formation of a dynamic
rhizosphere of this type is critical to the rehabilitation of degraded
lands (Herrera et al 1993 Whitford 1988) Biological nitrogen
fixation in conjunction with the production of large amounts of litter
has been shown to help build up organic matter nitrogen and
8
improve soil structure in deficient soils such as glacial till (Bollen and
Lu 1968 Crocker and Major 1955 Tarrant and Trappe 1971)
Biological nitrogen fixation can also improve conditions for other
non-nitrogen fixing species (Tarrant 1961) and enhance species
diversity (Franklin and Pechanec 1968)
The use ofthese deciduous trees with the objective of improving
the site conditions (ie shade nutrients and organic matter) for other
species (Albers and Carpenter 1979) is a valuable strategy in the
reconstruction of disturbed areas such as mine spoils
Production ofStockP1ants
Efficient propagation ofnursery stock from seed requires
extensive knowledge of the germination requirements and cultural
methods needed for the particular species Little is known about the
propagation requirements for the two species used in this study
thinleaf alder and water birch This deficit is due in part to a lack of
demand for these species in the past Extensive work has been done
on the propagation of other species within the Alnus and Betula
genera specifically those species of commercial value to the timber
industry such as red alder (A rubra Bong) and paper birch (B
9
papyrifera Marsh) Information generated from propagation studies
on these species has elucidated some universal seed characteristics
and germination requirements for members ofBetulaceae Seeds aremiddot
characteristically very small and light and may have a winged
integument to aid in wind dispersal Average seed density for B
ocddentalis is about 2500 seeds per gram while A tenuifolia
averages about 1488 seeds per gram (Vines 1960) Seed quality and
germination capacity are often very low as it is difficult to separate
sound from empty seeds when size and weight are so low (Brinkman
1974 Schopmeyer 1974) Seed quality may vary considerably from
harvest to harvest (Bjorkbom et al 1965) Within species
germination requirements may differ with provenance (Fowler and
Dwight 1964 Wilcox 1968) or even within a provenance (Bjorkbom
et al 1965 Schopmeyer 1974) In some instances the requirements
for germination may be met but germination does not occur a
condition referred to as dormancy
Seed Dormanqr and Methods to Overcome It
Dormancy in seeds is defined as the condition where seeds will
not germinate even when environmental conditions (water
10
temperature and aeration) are permissive for germination (Bewley
and Black 1994 Hartmann et al 1997) This mechanism ensures that
germination does not take place in less than optimum conditions or at
the wrong time (Bewley and Black 1994 Thompson 1971) For
example in some species seeds of southern provenance require
longer stratifications (Fowler and Dwight 1964) probably to prevent
germination in areas where there are intermittent periods ofwarm
weather followed by frost Seed dormancy results from a
combination ofgenetic and environmental conditions and it is not
always possible to predict the dormancy of a particular species from
characteristics of other species within the genus (Schopmeyer 1974)
There are different systems for classifying dormancy but the
condition may be divided into four basic types exogenous
endogenous double or combinational and secondary (Hartmann et
al 1997) The seed dormancy exhibited by birch and alder falls under
the category of endogenous dormancy a dormancy imposed by
embryonic factors This includes morphological dormancy (an
underdeveloped embryo) and physiological dormancy ofvarying
degrees (non-deep intermediate and deep) Non-deep physiological
11
dormancy is characterized by the need for after-ripening or exposure
to red light (photodormancy) Intermediate physiological dormancy
is characterized by the need for moderate periods of cold stratification
(up to 56 days) Deep physiological dormancy requires long periods
of cold stratification more than 56 days (Hartmann et al 1997)
Seeds ofboth Alnus and Betula exhibit varying degrees of
dormancy in most cases broken by coolmoist stratification andor
germination under red light (Brinkman 1974 Dirr and Heuser 1987
Schopmeyer 1974 Young and Young 1992) In some species of these
genera chemical treatments such as potassium nitrate have been
effective to overcome dormancy (Bradbeer 1988 Hartmann et al
1997 Young et al 1984) Many birch species are known to possess a
phytochrome light detection system which prevents germination
when seeds are buried too deep to allow seedling survival after
germination (Bewley and Black 1994 Black and Wareing 1955
Bradbeer 1988) Where the phytochrome detection mechanism is
present exposure to red light during germination is required for
breaking dormancy Most species of birch and alder have seeds that
ripen in late summer or early fall fall germination would result in
12
seedling loss over the winter so an after-ripening or stratification
requirement decreases the possibility of fall germination Joseph
(1929) found non-stratified birch seeds had a higher temperature
requirement for germination The current theory is that stratification
causes phase changes in membrane fluidity and triggers membraneshy
related signal transduction pathways activating enzymes and
hormones thus allowing dormancy release (Bewley and Black 1994
Ross and Bradbeer 1971)
Leaching of certain chemical inhibitors from seeds can also
break dormancy it maybe that this is part of the mechanism by
which photo dormancy is broken by moist stratification as only small
amounts of moisture are needed (Brad beer 1988) Research indicates
that the testa and pericarp of the seeds are involved in dormancy not
because they contain the inhibitor but because they prevent leaching
of the inhibitor (Villiers and Wareing 1964 Webb and Wareing
1972) Ru40lf (1950) found that cold-soaking might in some cases be
an acceptable substitute for stratification in some conifer species this
might be due to the leaching mechanism
13
The role ofpotassium nitrate in breaking dormancy has not
been clarified but there is speculation that the nitrogen supplied or
the oxygenating properties of the nitrate are involved (Brad beer
1988) Biswas et al (1972) found that the chemical treatment
enhanced the effect of stratification but did not necessarily replace it
Hilton (1985) found the germination-stimulating properties ofnitrate
depend on the presence of light nitrate in the presence of red light is
believed to be a cofactor to the phytochrome system which is involved
in the synthesis ofgibberellins that promote germination (Hilhorst et
al 1986)
Germination Requirements
General requirements for germination include moisture
favorable temperatures adeq-qate gas exchange and for some species I)
light In the presence of these conditions the quiescent seed can
imbibe water causing the seed to swell and the seed coat to split or
break Enzymatic activity within the seed accelerates increasing
respiration and use of stored energy resulting in the commencement
of growth processes within the seed (Bewley and Black 1994
14
Pretreatment requirements for germination of alder seed are
quite variable both between and within species For many species of
alder cold stratification periods of60-180 days are recommended
(Dirr and Heuser 1987) In one study ofthinleaf alder prechilling
(stratification) did not improve germination percentage while in
European speckled alder 180 days of stratification did improve
percentage germination (Young and Young 1992) Several other
treatments including light freezing and potassium nitrate
independently and with stratification have been shown to enhance
germination ofalders In red alder stratification was not necessary
when seed was germinated in light (Kenady 1978 Radwan and
DeBell 1981) Evidence of a phytochrome-regulated dormancy was shy
subsequently found in this species (Bormann 1983) Several general
horticultural texts recommend a pretreatment with 0200 potassium
nitrate (wv) to enhance stratification effects (Hartmann et al 1997
Young and Young 1992) In one study stratification followed by
freezing of seed for 3 days at -20degC enhanced germination (Schalin
1968)
16
Water Birch
Birch species are widely distributed in the northern hemisphere
found further north than alders can grow in various habitats and are
tolerant of a wide range of soils and moisture levels but are sensitive
to drought (Ashburner 1993 deJong 1993) Birch species are thought
to be more resistant to drought than alder species (McVean 1956) B
ocddentaJis Hook occurs as a shrub or small tree along streams or in
moist canyons and occasionally in dryer sites of the mountain West
( at elevations of 1500-2700 meters (Foxx and Hoard 1995 Vines
1960) It is known in the vernacular as water birch red birch and
black birch A small tree it is not used for lumber but can be used as
firewood posts browse by livestock or wildlife and sometimes as a
landscape tree (BrenzeI1995 Elias 1980 Preston 1968 Vines 1960)
Germination requirements for species of Betula generally
include stratification or red light treatment (Brinkman 1974)
indicating the presence ofphytochrome far-red inhibition (Bevington
1986 Bevington and Hoyle 1981 Schopmeyer 1974) Occasionally
both red light and stratification are recommended to improve
germination rate (Dirr and Heuser 1987) Potassium nitrate 02
17
pretreatment is recommended for birch species by Hartmann et al
(1997) Seeds of this species are considered to have a fairly shallow
dormancy (Lane 1993)
Seed Quality Improvements
Methods to upgrade seed quality (separate viable from nonshy
viable seeds) have been developed for different species Conventional
seed separation techniques are based on density such as air column or
liquidseparation or by size and shape such as with screens
Separation ofviable and non-viable seeds is extremely problematic
with very light winged seeds like those of alder and birch Air
separation techniques may not be practical for winged light-weight
seed Flotation techniques often employ lighter-than-water solvents
but some of these substances may have adverse effects on seed
viability (Barnett 1971 McLemore 1965) Widescale use of some
solvents is not considered desirable because of health and safety
concerns
A method of seed refinementupgrade originally developed in
Sweden by Milan Simak called the LDS method (Incubation
Drying Separation) shows promise for separating live and dead seeds
18
(cited in Bonner 1984 Downie and Wang 1992 Simak 1983
Sweeney et al 1991) Seeds are imbibed for several hours then
incubated at cool temperatures (15~or several hours in 100
relative humidity Seeds are then dried for several hours at 35
relative humidity at cool temperatures (timing and relative humidity
must be adjusted for the particular species) During the drying
dead seeds will lose most of the water previously imbibed while live
seeds should retain most of their imbibed water This differential
moisture content would make separation by flotation and other
density separation methods potentially feasible Similar methods of
conditioning have been shown to improve seed quality in lettuce
tomato and onion (Hill et al 1989) It has also been shown that
drying of stratified seeds for storage or for separation from
stratification medium need not result in loss of viability (Danielson
and Tanaka 1978 Schopmeyer 1974)
19
OBJECTIVES OF THIS STUDY
The purpose of this study is to determine the effectiveness of the
LDS seed refinement technique and othi separation procedures in
increasing the percentage of live seeds in a seed lot and to develop
germination strategies for water birch and thinleaf alder investigating
the use of stratification Secondly this study will examine the within-
species variability of different seed lots in their response to LDS and
stratification treatments
METHODS AND MATERIALS
Sources
Alder strobiles were collected in October and November of
1998 in Catron County New Mexico near the towns of Luna and
Reserve in the Cottonwood Canyon Campground and in the Head of
the Ditch Campground and in Taos County New Mexico in the
Red River Canyon near the Molycorp molybdenum mine Table 1
shows the seed source elevations and locations Strobiles were kept
cool and allowed to dry for several weeks Seeds were separated from
the opening strobiles by rubbing on a coarse screen
Birch strobiles were collected in October and N overrtber of 1998
in Taos County in the Red River Canyon near the Mo1ycorp
molybdenum mine (Table 1) Strobiles were kept cool and allowed to
dry for several weeks allowing the release of seeds from the bracts
In addition commercial seed sources ofbirch and alder were
purchased in the summer of 1999 (collected in the fall of 1998) The
seed lots collected in 1998 (Table 1) were used in the seed refinement
study providing four seed lots for that study For the final seed
refinement-germination study the two Red River Canyon seed lots of
21
Table 1 Seed Source Locations and Elevations
Species Source Lot Baseline Description Elevation Latitude Notes No Fill (meters) Longitude
Thinleaf Alder Luna NA 234 Head ofthe Ditch CG 2134 N 33deg49 W 108deg59
t+
Reserve NA 268 Cottonwood Canyon 1829 N 33deg37 W 108deg55
t+
RRC-l 98108 08 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
RRC-2 98109 09 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 537 W of Poncha Springs CO 2438 N 38deg31 W 106deg05
I
Water Birch RRC-3 98104 69 Red River Canyon 2469 N 36deg41 W 105deg29
t+sect
Moly-1 98106 39 Molycorp Tailings Rd 2469 N 36deg41 W 105deg29
t+
Moly-2 98107 52 Molycorp Low Dump 2469 N 36deg41 W 105deg29
t+
Mo1y-3 98105 56 Molycorp Front Dump 2469 N 36deg41 W 105deg29
t+sect
Chaffee NA 297 W ofPoncha Springs CO
t used in Experiment 1--Seed Refinement I used in Experiment 2--Germination Requirements sectpooled for use in Experiment 2-Germination Requirements
2377 N 38deg31 W 106deg05
I
alder and the Red River Canyon and Moly 3 seed lots ofbirch were )
pooled due to limited amounts of seed The seed lots collected in
1998 and the 1998 purchased seed lots from Chaffee County
Colorado provided four seed lots of each species for that study
All seed sources were evaluated for percentage of filled seeds by
means ofdissection performed under a dissecting microscope at 30X
magnification (Berry and Torrey 1985) Alder species baseline
percentage of filled seeds was estimated using 25 samples of 100 seeds
pooled into one percentage response for each seed source Birch
species baseline percentage of filled seeds was determined using 15
samples of 50 seeds pooled into one percentage response for each seed
source Baseline percentage fill (Table 1) is the estimate of the
percentage of filled seed in the entire seed collection for each source
Separation Media
Ethanol and water were not particularly effective in separation
ofthinleaf alder seeds either using IDS methods or when separating
dry seed It was necessary to choose a fluid with a lower specific
gravity than ethanol (SG=O 79) in order to separate filled and empty
seeds with very low densities Falleri and Pacella (1997) found that
23
low-density London plane tree (Platanus x acerifolia [Aid Willd)
seeds could not be separated using water as the separation medium
due to the very small density differences between sound and empty
seeds and chose petroleum ether as a separation medium Petroleum
ether was chosen for the separation of thinleaf alder seeds because of
its low specific gravity (SG middot060) its relative stability low
reactivity and rating as a slight health risk Contact with skin may
cause dryness and irritation but no chronic systematic effects have
been reported with industrial use (Mallinckrodt Baker Inc 1997a)
As observed previously for thinleaf alder seeds the simple
specific gravity method using water was not effective for separating
water birch seeds In preliminary studies ethanol and petroleum
ether were found to be effective in separation of dry water birch seeds
and petroleum ether ethanol and water were somewhat effective in
separation of water birch seeds treated by the LDS method but
ethanol was chosen as the separation medium because of its lower
cost greater effectiveness and availability
Denatured ethanol is actually rated a greater health risk than
petroleum ether because ingestion is more likely to result in death or
24
permanent damage and prolonged skin contact may affect the
nervous system and other organ systems of the body Ethanol also
has a higher reactivity rating Gloves goggles and lab coat (personal
protective equipment) proper ventilation avoidance of ingestion and
proper fire safety measures should prevent problems with use of either
solvent (Mallinckrodt Baker Inc 1997a 1997b)
Seed Refinement
Thinleaf Alder
Separation treatments examined includeddensity separation of
dry seed samples in petroleum ettter (the control) and imbibed seed ~
samples treated with the IDS method at 0 1 18 and 24 hour drying
times followed by density separation in petroleum ether (Table 2)
Seeds were imbibed for 24 hours by submersion in a 10-gallon glass
aquarium filled with distilled water and equipped with an aeration
pump and filter Seeds were packaged in filter paper then the
packages were enclosed in wire cages (purchased tea balls were used
for this purpose) weighted with marbles to keep them submerged At
the end of the imbibition period seeds were removed from the cages
thoroughly blotted and placed on clean filter paper The drying
25
incubation was performed in a closed chamber with a constant
humidity obtained by the use ofCaC12middot6H20 salt in a saturated
solution prepared by adding SOOOg CaClzmiddot6HzO to 30 liters of
distilled water (Slavik 1974 Young 1967) Imbibed seeds were placed
on filter paper and suspended on a screen above the calcium chloride
solution Humidity was monitored using an hygrometer and held
steady at 50 in the presence of the wet seeds and filter paper
Table 2 Alder Preparation Protocols for S~d Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) 0 0
2
3
24
24
0
1 )
4 24 18
5 24 24
At the end of the appropriate drying incubation the seeds were
placed in petroleum ether and briefly and vigorously stirred to
separate seeds adhering to one another Floating seeds were removed
from the surface of the petroleum ether by means of a small net
andor a spatula placed on clean moistened filter paper and placed in
26
a labeled plastic bag to await counting The sinking seeds were
strained through the net and packaged in a similar manner Five
repetitions were performed for each of the five treatments using 100
seeds per repetition Percentage of filled seeds contained in each
fraction was determined by means of dissection tests performed on the
floating and sinking fractions using a scalpel and a dissecting
microscope with 30X magnification
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that particular repetition
of filled seeds in the sinking fraction X 100=percentage recovery
of filled seeds in the sinking fraction + of filled seeds in the floating fraction
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product of percentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
27
Water Birch
Separation treatments included density separation of dry seed in
95 ethanol (the control) and seed samples treated with the IDS
method at 005 1 and 2 hour drying times followed by density
separation in 95 ethanol (Table 3) Seeds were imbibed for 12 hours
by submersion in a 10-gallon glass aquarium filled with distilled water
and equipped with an aeration pump and filter Seeds were packaged
in filter paper then the packages were enclosed in wire cages
(purchased tea balls were used for this purpose) weighted with
marbles to keep them submerged At the end of the imbibition
period seeds were removed from the cages thoroughly blotted and
placed on clean filter paper The drying incubation was performed in
a closed chamber with a constant humidity obtained by the use of
CaCI2middot6H20 salt in a saturated solution prepared as described in the
previous section (Slavik 1974 Young 1967) Imbibed seeds were
placed on filter paper and suspended on a screen above the calcium
chloride solution Humidity was monitored using an hygrometer and
held steady at 50 in the presence of the wet seeds and filter paper
28
Table 3 Birch Preparation Protocols for Seed Refinement
Preparation Protocol Imbibition Time (Hours) Drying Time (Hours)
1- (Control) o o
2 12 o
3 12 05
4 12 1
5 12 2
At the end of the appropriate drying incubation the seeds were
placed in 9500 ethanol and briefly and vigorously stirred to separate
seeds adhering to one another Floating seeds were removed from the
surface of the ethanol by means of a small net andor a spatula
placed on clean moistened filter paper and placed in a labeled plastic
bag to await counting The sinking seeds were strained through the
net and packaged in a similar manner Three repetitions were
performed for each of the five treatments using 50 seeds per
repetition Percentage of filled seeds contained in each fraction was
determined by means of dissection tests performed on the floating and
sinking fractions using a scalpel and a dissecting microscope with
30X magnification
29
In addition the percentage recovery of filled seeds from the
sinking fraction was calculated based on the total number of filled
seeds present in that repetition (as given in the previous equation)
The most effective drying-incubation time combination was chosen
for use in the germination testing portion of the study on the basis of
the largest product ofpercentage of filled seeds in the sinking fraction
multiplied by percentage recovery of filled seed from the sinking
fraction
Germination Requirements
Thinleaf Alder
Separations were performed using the separation method
chosen from the seed refinement study alder preparation protocol 4
24-hour imbibition followed by I8-hour drying time and separation in
petroleum ether as described in the seed refinement experiment
(Table 2) Unseparated imbibed seeds and seeds from both the
floating and sinking fractions were subsequently treated with 028
and 56 days of stratification In addition an unseparated nonshy
stratified control of dry seeds was tested for germination Seeds for
stratification treatments were placed in layers ofpaper towel
30
moistened with 25 m1 of distilled water and placed in sealed zip-lock
plastic bags The bags were placed in a cooler at I-5degC (average
temperature 50degC) for periods of 28 or 56 days Initiation of
stratification treatments was staggered so that all treatments came out
ofstratification at the same time
Following stratification the seeds were sown in Ray-Leach
Super Cells (Steuwe amp Sons Inc Corvalis OR) containing a 2 1 1
ratio ofpeatmossperlitevermiculite (vvv) with OsmocoteR 14-14-10
slow release fertilizer at a rate of 4007 gm3bull Five seeds were sown
per tube Treatments were distributed in a randomized complete
block design consisting of4 blocks (locations on the greenhouse
bench) with each block containing the 10 treatment combinations for
each of four seed lots (Table 4) Each repetition contained 20 tubes
repetitions were placed in random order four repetitions to a rack ten
racks to each block Each repetition for each treatment contained 100
seeds therefore 100 seeds were used for each seed source by
treatment by block combination pooled to one measurement for the
response variable germination percentage Racks were placed in a
greenhouse for germination Germination conditions included
31
ambient light and 70 relative humidity with average daily
temperature 243degC (daytime temperature range 200-272degC) and
average night temperature 216degC (nighttime temperature range 206shy
239degC) Tubes were watered at 2 hour intervals six times a day
Germination was recorded at weekly intervals 7 1421 and 28 days
after planting
Table 4 Treatment Combinations for Experimental Layout of Randomized Complete Block--Thinleaf Alder
Treatment Number Stratification (Days) Separation Fraction
1 56 None
2 56 Floating
3 56 Sinking
4 28 None
5 28 Floating
6 28 Sinking
7 0 None
8 0 Floating
9 0 Sinking
blocks Qocations on the greenhouse bench) with each block
containing the 10 treatment combinations for each of four seed lots
(Table 5) Each repetition contained 20 tubes repetitions were placed
in random order four repetitions to a rack ten racks to each block
Each repetition for each treatment contained 100 seeds therefore 100
seeds were used for each seed source by treatment by block
combination pooled to one measurement for the response variable
germination percentage Racks were placed in a greenhouse for
germination Germination conditions included ambient light and
7000 relative humidity with average daily temperature 243 degC
(daytime temperature range 200-272degC) and average night
temperature 216degC (nighttime temperature range 206-239degC)
Tubes were watered at 2 hour intervals six times a day Germination
was recorded at weekly intervals 7 1421 and 28 days after planting
34
Table 5 Treatment Combinations for Experimental Layout of Randomized Complete Block-Water Birch
Treatment Number Stratification (Days) Separation Fraction
56 None
2 56 Floating
3 56 Sinking
4 21 None
5 21 Floating
6 21 Sinking
7 0 None
8 0 Floating
9 0 Sinking
10 0 None
DATA ANALYSIS
The seed refinement experiment was performed to determine
the mostadvantageous separation technique for use in the
germination studies with the percentage of filled seeds present in the
sinking fractions (percentage fill) and proportion of filled seeds
recovered from the total filled seeds available in the sample
(percentage recovery) as response variables and the preparation
protocols and seed sources as independent variables
The second experiment utilized the chosen seed refinement
method with levels of stratification seed separation fraction and seed
source as independent variables (or in the augmented factorial
treatment combination as the independent variable) with germination
percentage measured as the response variable Germination rate was
also recorded however the rapid germination between the time of
sowing and the first sampling (at 7 days) prevented meaningful
analysis of this da~
Data was analyzed by using categorical data modeling analysis
as found in the SAScopy statistical program The PROC CATMOD
procedure can perform analysis and giveanalysis of variance in the
36
general sense that it analyzes the response functions fits linear models
to functions of response frequencies and partitions the variation
among those functions into various sources (SAS Institute 1989)
CATMOD analyzes data that can be represented in a two-
dimensional contingency table with the rows corresponding to
populations or samples defined by one or more independent variables
and the columns corresponding to one or more dependent (response)
variables The frequencies in the table are assumed to follow a
product multinomial distribution with a simple random sample taken
for each population The probability for the response ofeach cell is
estimated and the vector (P) of these proportions is transformed into a
vector of functions F =F(P) If It denotes the vector of true
probabilities for the table then the functions of the true probabilities
F(It) are assumed to follow a linear model
I
where EA denotes asymptotic expectation X is the design matrix
containing fixed constants and Pis a vector ofparameters to be
37
estimated CA TMOD provides two estimation methods the
maximum-likelihood method and the weighted-least-squares method
which was used in this analysis (SAS Institute 1989)
Hypotheses about linear combinations of the parameters can be
tested these statistics are approximately distributed as chi-square for
sufficiently large sample sizes (SAS Institute 1989)
All of the response variables considered had a binomial type of
probability distribution (seed filled or not filled seed germinated or
not germinated) All treatments ofboth experiments were analyzed
using the PROC CATMOD procedure to examine the general model
as well as planned comparisons using contrast statements where ~
appropriate The PROC MEANS procedure was used to calculate
marginal percentages (main effect and interaction combinations)
along with standard errors Pairwise Z-tests were used to separate
percentages in those effects which were determined to be significant
by categorical modeling at the observed significance level laquo=005 this
method of percentage separation is analogous to Fishers LSD for
separating means
38
Categorical modeling of the seed refinement experiments used
two models one for the percentage of filled seeds attained in the
sinking and floating fractions and one for the percentage of filled
seeds recovered from those available in the baseline sample The
treatment structure for both of these seed refinement studies was a 5
X 2 X 4 factorial (preparation protocol by separation fraction by seed
source)
The model in CATMOD for the percentage of filled seeds
attained in the fractions is as follows
fill = source prep fraction sourceprep sourcefraction prepfraction sourceprepfraction
where fill is the response variable the number of filled seeds
source is the seed source prep is the LDS treatment protocol
(imbibition plus one of four drying times or no treatment) and
fraction is the separation fraction (floating or sinking)
The CATMOD model for the percentage of filled seed
recovered in the floating or sinking fractions is as follows
39
rec = source prep sourceprep
where rec is the response variable (number of filled seeds floating or
sinking) source is the seed source and prep is the IDS
treatment protocol
The treatment structure for the germination studies was a 3 X 3
factorial (stratification by separation) with an additional control (no
treatment) for 4 seed sources termed an augmented factorial design
by Lentner and Bishop (1986) The PROC CATMOD procedure
was used to analyze the data without the no treatment control as a
simple 3 X 3 X 4 factorial (stratification by separation by seed source)
where germ is the response variable strat is the level of
stratification sep is the IDS fraction and source is the seed
source
40
The augmented factorial design (3 X 3 factorial plus a noshy
treatment control for 4 seed sources) was also analyzed by PROe
CATMOD in order to evaluate the effect of imbibition The
treatment structure for the germination studies in this analysis was a
lOX 4 factorial (treatment by seed source) with the model in
CATMOD as follows
germ = treat source treatsource
where germ is the response variable (germinated or not) treat is
the treatment combination (LDS fraction plus level ofstratification)
and source is the seed source
RESULTS
Seed Refinement
Thinleaf Alder Fill Enhancement
Preparation protocol seed source and the separation fraction
had significant (alpha=O05) effect on the percentage fill (Table 6)
The effect of separation fraction was influenced by both source and
preparation protocol
Table 6 Analysis ofVariance Table for Thinleaf Alder Percentage of Filled Seeds as Influenced by Preparation Protocol Separation Fraction and Seed Source-Factorial
Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 11663 00000
Seed Source 3 17367 00000
Preparation Protocol 4 4490 00000
Separation Fraction 1 8829 00000
SourcePrep 12 541 09427
SourceFraction 3 971 00212
PrepFraction 4 986 00429
SourcePrepFraction 11 714 07878
Protocols 1 4 and 5 the control and 24 hour imbibition
followed by either 18 or 24 hours drying respectively all had greater
than 8000 filled seed in the sinking fraction (Table 2 Figure 1)
Twenty-four hour imbibition alone or in conjunction with 1 hour of
drying both had lower percentages of filled seeds in the sinking
fraction (less than 3500) Protocol 4 the 24-hour imbibition followed
by 18 hours of drying and density separation in petroleum ether was
chosen as the separation method for the germination requirements
study
The proportion of filled seed in the sinking and floating
fractions was also influenced by seed source Percentage of filled
seeds in the sinking fraction ranged from 444 for the Red River
Canyon 1 source to over 86 for the Luna source (Table 7)
Percentage of filled seeds in the floating fraction ranged from less than
1 to just over 1200 while the baseline percentage of filled seeds in
the seed sources ranged from less than 100 to over 26 The
separation process improved percentage fill in the sinking fraction
compared to the percentage fill in the floating fraction by about sevenshy
43
bull bull bull bull bull
100~------------------------------------------~
80
60El ~
~ ~ 40s ~ 1-4 0 ~
20
0
used to represent the percentage (protocols are described in Table 2)
44
_ Floating Fraction -0- Sinking Fraction
1- (010) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 1 Alder Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent +- one Standard Error Bars which are not visible are smaller than the symbol
Table 7 Thlnleaf Alder Percentage ofFilled Seeds in the Fractions as Influenced by Source and Compared to Baseline Fill Uninfluenced
by Preparation Protocol
Seed Baseline Fill- SE Fill- SE n Source Fill Sinking Fraction Floating Fraction
Luna 234 8634c 180 1265d 072 4000
Reserve 268 4644b 139 631c 070 4000
RRC-1 08 444a 131 O44a 014 4000
RRC-2 09 909a 328 062a 0)5
Percentages followed by the same letter are not significandy different at laquo=0 5
fold for the Luna and Reserve seed sources ten-fold for the Red River
Canyon 1 source and almost fifteen-fold for the Red River Canyon
2 source Separation improved the percentage of filled seeds in the
sinking fraction compared to the unseparated seed source by almost
four-fold for the Luna source almost two-fold for the Reserve source
almost six-fold for the Red River Canyon 1 source and ten-fold for
the Red River Canyon 2 source
Floating separation fractions had a much lower percentage of
filled seeds (464) than sinking fractions (471100) (Table 8)
Percentage of filled seeds was consistently low in the floating fraction
but varied with the preparation protocol in the sinking fraction
(Figure I)
45
Table 8 Thinleaf Alder Percentage ofFilled Seeds as Influenced by Separation Fraction
Percentages followed by the same letter are not significantly different at laquo=005
Thinleaf Alder Recovery
Seed source and preparation protocol both influenced the
percentage of filled seeds recovered (Table 9) In contrast to the
percentage of filled seeds in the sinking fraction (Figure 1) the
percentage of seeds recovered was improved by 24 hours imbibition
alone or with one hour drying at 5000 humidity (Table 10) These two
treatments had in excess of 80 recovery whereas the other three
separation treatments all averaged less than 6700 recovery
Table 9 Analysis ofVariance Table for Thinleaf Alder Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 694 00084
Seed Source 3 11055 00000
Preparation Protocol 4 2371 00001
SourcePrep 12 1715 01439
Percentage recovery as influenced by source varied from
approximately 3200 for the Red River Canyon 2 collection to over
88 for the Reserve seed source (Table 11) The Red River Canyon
47
1and Luna sources both had percentage recoveries slightly greater
than 50
Table 10 Thlnleaf Alder Percentage ofFilled Seeds Recovered in the Sinking Fraction as Influenced by
Preparation Protocol
Protocol (SoakDry) Recovery SE n
1- 010 6447a 290 273
2 - 240 8094b 236 278
3 - 241 8225b 230 276
4-2418 6667a 312 228
5 -2424 6041 a 312 245
Percentages followed by the same letter are not significantly different at cx=005
Table 11 Thlnleaf Alder Percentage of Filled Seeds Recovered in the Sinking Fraction as Influenced by Seed Source
Seed Source Recovery SE n
Luna 5392b 206 586
Reserve 8852c 123 671
RRC-l 5238ab 1090 21
RRC-2 3182a 993 22
Percentages followed by the same letter are not significantly differerit at a=005
48
Water Birch Fill Enhancement
The preparation protocol and the separation fraction influenced
the percentage of filled seed in the fractions Seed source did not
impact the percentage of filled seed in the fractions The effect of
separation fraction on percentage of filled seed in the fractions was
influenced by both seed source and preparation protocol
independently (Table 12)
Table 12 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds as Influenced by Preparation Protocol Separation
Fraction and Seed Source--Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 47402 00000
Seed Source 3 603 01103
Preparation Protocol 4 956 00486
Separation Fraction 1 8190 00000
SourcePrep 12 1204 04423
SourceFraction 3 2223 00001
PrepFraction 4 1304 00111
SourcePrepFraction 12 1453 02680
Overall the sinking fraction had higher percentage of filled seed
than the floating fraction (Table 13) The influence ofpreparation
protocol on the percentage of filled seed in the two fractions is
illustrated in Figure 2 All four of the LDS treatments reduced the
percentage of filled seeds in the sinking fraction relative to the nonshy
imbibed control treatment (Table 3 Figure 2) The percentage of
filled seeds in the sinking fraction ranged from near 900 to 12 for the
imbibed treatments whereas the percentage in the control treatment
was over three times these amounts The floating fractions had
consistently low percentage of filled seeds while the sinking fraction
treated by protocol 1 (separation without imbibition) had a higher
percentage of filled seeds than the sinking fractions treated by the
other protocols (all with imbibition) The control treatment (protocol
1) was chosen as the separation protocol for the germination
requirements study
The influence ofseed source on the percentage of filled seed in
the two fractions is illustrated in Figure 3 The Moly 2 and Moly 3
seed sources had much higher percentage of filled seeds in the sinking
50
Table 13 Water Birch Percentage ofFilled Seeds as Influenced by Separation Fraction
Separation Fraction Fill SB n
Floating Fraction 177a 030 1867
Sinking Fraction 1165b 095 1133
Percentages followed by the same letter are not significantly different at ct=O05
fraction than the Red River Canyon 3 and the Moly 1 sources All
of the floating fractions had a low percentage of filled seeds
bull bull bull
50--------------------------------------------
40
30S It ltU
$ 20I ltU ~ ltU
tl-i 10
0
-e- Floating Fraction -0- Sinking Fraction
t-----
1- (00) 2- (120) 3- (1205) 4- (1211) 5- (1212)
Preparation Protocol
Figure 2 Birch Percentage Fill as Influenced by Preparation Protocol and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage (protocols are described in Table 3)
52
bull bull
60-------------------------------------------~
50
~ ~
~ 5 t
p
40
30
20
10
o
__ Floating Fraction -0- Sinking Fraction
RRC3 Molyl Moly2 Moly3
Seed Source
Figure 3 Birch Percentage Fill as Influenced by Seed Source and Separation Fraction Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
53
Water Birch Recovery
Preparation protocol and seed source did not influence the
percentage of sound seeds recovered in the sinking fraction (Table 14)
Percentage of filled seeds recovered for the various protocols ranged
from 64-91 Percentage of filled seeds recovered in the sinking
fractions for the 4 seed sources ranged from 70-89
Table 14 Analysis ofVariance Table for Water Birch Percentage ofFilled Seeds Recovered in the Sinking and Floating Fractions as Influenced by Preparation Protocol
Table 16 Thinleaf Alder Percentage Germination as Influenced by Source--Factorial Analysis
Source Percentage Germination SB n
Luna 2011b 067 3600
Reserve 1914b 066 3600
RRCpoo1 075a 014 3600
Chaffee 3283c 078 3600
Percentages followed by the same letter are not significantly different at =005
56
Table 17 Thlnleaf Alder Percentage Gennination as Influenced by Separation-Factorial Analysis
Separation Fraction Percentage Germination SE n
No Separation 1960b 057 4800
Floating Fraction 292a 024 4800
Sinking Fraction 32l0c 067 4800
Percentages followed by the same letter are not significantly different at a==005
unseparated controls however regardless of separation fraction or
stratification treatment overall germination was quite low ( lt 100) in
this seed source (Table 16)
The influence of stratification was variable across all four seed
sources and the separation fractions evaluated (Figure 5) For
example only in the sinking fraction of the Chaffee source did
germination continue to increase with increasing stratification
duration (Figure 5d) In several combinations of separation protocol
and seed source the mid-level (28 days) of stratification had the
highest germination In the case of the sinking fraction of the Reserve
source this level actually reduced the germination response (Figure )
5b) Overall the low germinating fractions (Red River Canyon and
57
60
___ Luna 50
t 400-a
~
5 E 30 d v 00 ~ 20 t v U M V
10p
0
-0- Reserve -T shy RRC
~ -v- Chaffee
Q
~
Nosep Floating Sinking
Separation Fraction
Figure 4 Alder Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
58
M-------------______~ ~-------------------~
21 21
Stratification Period (Days) Stratification Period (Days)
Figure 5a Luna Source Figure Sb Reserve Source
M-------------------~ ~-------------------~
212S
Stratification Period (Days) Stratification Period (Days)
Figure 5c RRC Source Figure 5d Chaffee Source I
-- No Separation --0- Floating Fraction -- Sinking Fraction
Figure 5 Alder Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
59
the floating fractions of the other three seed sources) were slightly
impacted by stratification duration
In the analysis of the augmented factorial stratification and
separation fraction treatments were grouped to consider the effect of
imbibition (Table 18) Treatment 10 (the non-imbibed control) was
compared to the imbibed O-day stratification treatments (789) using
planned comparisons by contrast to determine ifpre-soaking of the
seeds had an effect Seed sources were also compared using planned
comparisons by contrast (Table 19) Overall pre-soaking (imbibing)
did not intluence germination however individual sources varied in
- response to this procedure (Tables 18 and 19 Figure 6) Pre-soaking
did not influence the percentage germination of the Red River
Canyon pool or the Chaffee seed sources (Figure 6) However preshy
soaking increased percentage germination in the Luna seed source
and decreased the percentage germination in the Reserve seed source
In order to detect differences in seed source in response to
treatment the Luna source and the Reserve source from the same
general geographical area were compared to each other and to the
Red River Canyon source and the Chaffee source The Luna and
60
Table 18 Analysis ofVariance Table for Thinleaf Alder Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source OF Chi-Square Observed Significance
Level
Intercept 1 123226 00000
Treatment 9 28267 00000
Seed Source 3 41066 00000
TreatSource 27 19476 00000
Table 19 Thinleaf Alder Analysis of Contrasts-Augmented Factorial
Contrast OF Chi-Square Observed Significance
Level
Treatment 10 vs 7+8+9t 1 230 01292
Treatment 10 vs 3 1549 00014 7+8+9Seed Sourcet
Luna + Reserve vs RRCsect 1 14117 00000
Luna + Reserve vs Chaffeesect 1 20161 00000
Luna vs Reservesect 022 06368
tOegrees of freedom by Treatment tDegrees of freedom from Source Main Effect sectOegrees offreedom by Source
61
40
35
30Q00tl CIS
25o~ d 20 Q) t)I)
S Q 15Q)
~ Q)
~ 10
5
0
_ Non-Imbibed _Imbibed
Luna Reserve RRC Chaffee
Seed Source
Figure 6 Alder Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent + - one Standard Error
62
Reserve sources were not different from each other but these sources
differed from the Red River Canyon source and the Chaffee source
(Table 19)
It was noted that the Red River Canyon seed source had
uniformly poor germination which might affect the interpretation of
the analysis (Figures 4 Sc) The analysis was repeated deleting this
source (Table 20) With the elimination of the Red River Canyon
seed source stratification effect becomes significant but the
differences are not dramatic (Table 21) There was no interaction
between stratification and separation or stratification and source the
other effects and interactions are similar to those detected in the
analysis using all sources
r
Table 20 Thinleaf Alder Analysis ofVariance--Factorial Analysis without Red River Canyon Seed Source
Source DF Chi-Square Observed Significance
Level
Intercept 1 150244 00000
Stratification 2 936 00093
Separation Fraction 2 81026 00000
Strat Sep 4 918 00568
Seed Source 2 18498 00000
Strat Source 4 752 01108
Sep Source 4 10657 00000
Strat Sep Source 8 2225 00045
Table 21 Thinleaf Alder Percentage Germination as Influenced by Stratification without Red River Canyon Seed Source-Factorial
Analysis
Stratification Period Percentage Germination SE n
oDays 2167a 069 3600
28 Days 260Ob 073 3600
56 Days 2442b 072 3600
Percentages followed by the same letter are not significantly different at laquo=005
64
Water Birch
Total germination of imbibed water birch seed was influenced
by stratification separation fraction and seed source and by all
interactions of these three treatments (Table 22) Increasing
stratification length improved germination (Table 23) Seed in the
sinking fraction regardless of seed source or stratification had the
greatest total germination (Table 24 Figure 7) While stratification
regardless of duration improved germination overall seeds in the
sinking fraction responded best to the 56-day stratification treatment
(Table 23 Figure 8) The Chaffee seed source had the greatest total
germination followed by the Moly 2 and the Red River Canyon
seed sources while the Moly 1 source had a low germination
percentage (Table 25) Improvement in germination of the sinking
fraction relative to the unseparated control ranged from two-fold for
the Chaffee source to over ten-fold for the Moly 2 source (Figure 7)
The response of individual seed sources to stratification varied
considerably (Figure 9) The response to stratification was also not
consistent across seed sources and separation fractions (Figure 10)
The lack ofconsistent stratification effects is most apparent in the
65
Table 22 Water Birch Percentage Germination Analysis of Variance Table-Factorial Analysis
Source DF Chi-Square Observed Significance
Level
Intercept 1 74900 00000
Stratification 2 4503 00000
Separation Fraction 2 7719 00000
Strat Sep 4 2266 00001
Seed Source 2 2671 00000
Strat Source 4 9154 00000
Sep Source 4 7030 00000
Strat Source 8 13885 00000
Table 23 Water Birch Percentage Germination as Influenced by Stratification-Factorial Analysis
Stratification Period Percentage Germination SE n
oDays 1108a 045 4800
21 Days 1363b 050 4800
56 Days 1623c 053 4800
Percentages followed by the same letter are not significantly different at laquo=005
66
1
Table 24 Water Birch Percentage Germination as Influenced by Separation-Factorial Analysis
----------------~-------
Separation Fraction Percentage Gennination SE ----------------shy
n
No Separation 669b 057 4800
Floating Fraction 123a 024 4800
Sinking Fraction 3302c 067 4800
Percentages followed by the same letter are not significantly different at laquo=005
Table 25 Water Birch Percentage Gennination as Influenced by Seed Source-Factorial Analysis
Source Percentage Germination SE n
Moly-1 475a 034 3600
Moly-2 1503c 056 3600
RRCpool 1295b 053 3600
Chaffee 1855d 061 3600
Percentages followed by the same letter are not significantly different at laquo=005
67
---
50
40
I= 0
0 d 30
~ Q)
d 20
s ~ I=
~ 10 Q)
~
0
-e-- Moly 1 -0- Moly2 -T RRC -ry- Chaffee Atj
Itt ~
Iffjl
-shy-- J-~I
Nosep Floating
Separation Fraction
Sinking
Figure 7 Birch Percentage Germination as Influenced by Separation Fraction and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
68
40
50~--------------------------------------------~
-- shy _--i ____ Y- --- -shy
y---- ---shy___ No Separation
-0- Floating Fraction - - Sinking Fraction
bullbullbull _-0 0------()---shyo
o 21 56
Stratification Period (Days)
Figure 8 Birch Percentage Germination as Influenced by Stratification and Separation Fraction Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
69
r
35
30
25 0=
Q
5 ~
20 e ti Cl 15 ~ ~ = 10 ~ v ~
5
0
-+- Moly 1 -0- Moly2 -- RRC -V- Chaffee
o 21
Stratification Period (Days)
Figure 9 Birch Percentage Germination as Influenced by Stratification and Seed Source Error bars represent one + - Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
70
56
bullbull bull
80
Q 0 60 c
5 ~ D 40
t 20
~ p
J
8O-----------------~
0-- -0--0---shy
2
Stratification Period (Days)
Figure lOc RRC Source
t=
21 56
Stratification Period (Days)
Figure lOa Moly 1 Source
100--------------- ~
I
2
Stratification Period (Days)
Figure lOb Moly 2 Source
100--------------
Stratification Period (Days)
Figure lOd Chaffee Source
- No Separation -0- Floating Fraction -I- Sinking Fraction
Figure 10 Birch Percentage Germination as Influenced by Separation Fraction Seed Source and Stratification Error bars represent + - one Standard Error Bars which are not visible are smaller than the symbol used to represent the percentage
71
variable responses in the sinking fraction among seed sources In the
Moly 1 source 21 days ofstratification had no impact while 56 days
had a 7-fold improvement in germination (Figure lOa) However in
the Moly 2 source 21 days of stratification yielded the highest
germination and continued stratification reduced total germination
(Figure lOb) A similar but less pronounced trend was seen in the
germination ofseeds in the sinking fraction of the Red River Canyon
source (Figure IDe) where 21 days of stratification increased
germination of the sinking fraction but 56 days ofstratification
reduced germination below the level ofno stratification
Stratification regardless of duration reduced germination in seeds
from the sinking fraction of the Chaffee source however the decrease
was not linear with stratification length (Figure lOd) The floating
fraction and unseparated seed were relatively unaffected by
stratification treatment
Treatment (combination of stratification and separation
fraction) and source were considered in the analysis of the augmented
factorial in order to evaluate the effect ofpre-soaking by the use of
planned contrasts (Tables 26 27) Pre-soaking (the contrast of
72
Table 26 Analysis ofVariance Table for Water Birch Percentage Germination as Influenced by Treatment
Combination and Seed Source-Augmented Factorial
Source DF Chi-Square Observed Significance
Level
Intercept 1 88622 00000
Treatment 9 101947 00000
Seed Source 3 2994 00000
TreatSource 27 34727 00000
Table 27 Water Birch Analysis of Contrasts--Augmented Factorial
Contrast DF Chi-Square Observed Significance
Level
Treatment 10 vs 7+ 8+9t 1 1777 00000
Treatment 10 3 3280 00000 vs 7+8+9 Seed Sourcet
tDegrees of freedom by Treatment tDegrees offreedom from Seed Source Main Effect
73
Treatment 10 with Treatments 7 8 and 9) influenced germination
regardless of source but the seed sources varied in their response to
irnbibition (the contrast of Treatment 10 with the average of
Treatments 7 8 and 9seed source) Imbibition improved
germination for the Red River Canyon and Chaffee seed sources
Germination of the Moly 1 source was reduced by imbibition while
Moly 2 imbibed seeds had no germination (Figure 11)
35
30
c 250-Q
Cd
-~ 20 Q)
d ~ 15Cd c ~ Q) 10p
5
0
I Non-Imbibed _Imbibed
Moly 1 Moly 2 RRC Chaffee
Seed Source
Figure 11 Birch Percentage Germination as Influenced by Imbibition and Seed Source Error bars represent one + -Standard Error
75
DISCUSSION
Seed Refinement
Traditionally seed refinement has been thought of as enhancing
the number ofpotentiaily viable seeds (filled seeds) in a seed lot
Previously published studies have used total germination as the
measure of seed refinement efficacy In this study the number of
filled seeds in the sinking fraction was used The LDS treatments
imposed did not improve the number of filled seeds in the sinking
fraction in comparison with ordinary gravity separation for either of
the species evaluated in this study In two of the alder LDS
treatment levels 24-hour soak with either no drying time or one hour
of drying time actually reduced the percentage of filled seeds in the
sinking fraction The two remaining alder ID S treatments had
considerably longer drying times and resulted in percentages of filled
seeds in the sinking fraction similar to those of the non-imbibed
control treated by gravity separation The influence of drying time on
the efficacy of the LDS treatment has been seen in other species
(Faileri and Pacella 1997 Sweeney et al 1991) In a study ofLondon
plane tree researchers found that as drying time increased from 75
76
I
hours to 24 hours observed germination percentage was greater than
control (Falleri and Pacella 1997) At drying times less than 75
hours observed germination was comparable to unseparated controls
In the same study only seed receiving 24 hours of drying as part of an
IDS treatment had greater germination than non-treated seed
separated in petroleum ether
The response of the alder seed to IDS indicates there may be
potential for IDS as a seed refinement tool using longer imbibition
and drying times The difference in times from the I-hour to the 18shy
hour drying is considerable and corresponds to a significant difference
in the percentage of filled seeds in the sinking fraction The shorter
drying times may have been of insufficient duration to allow the
unfilled seed to lose sufficient moisture and hence these seeds ended
up in the sinking fraction In contrast the 18- and 24-hour drying
times may have allowed the imbibed unfilled seeds to lose the
majority of the water imbibed and resulted in percentages of filled
seeds in the sinking fraction similar to those seen in the non-imbibed
controls
77
The percentage of filled seeds in the sinking fraction in response
to the ID S treatments used in the water birch experiment indicates
that drying times may have been too short to allow the empty seeds to
lose sufficient moisture This would result in an increase in the
percentage ofempty seeds in the sinking fraction The seeds ofwater
birch are similar to those of thinleaf alder both are borne in strobiles
and have winged integuments almost entirely surrounding the seed
The alder integument is rather leathery while the birch integument
appears thinner Water birch seeds are smaller and rounder as
opposed to the flattened shape of alder seeds (alder seeds averaged
1469gram birch seeds 12S4gram) The seeds may also differ in
their ability to lose water following imbibition The assumption was
made that the birch seeds being smaller with larger integuments
relative to the size of the seed would lose imbibed water at a faster
rate This may not have been the case as indicated by the higher
percentage of empty seeds in the sinking fraction
While all thinleaf alder sources had improved percentages of
filled seeds in the sinking fractions there appear to be differences
between sources in response to seed refinement This difference was
78
detailed studies examining source differences in the rate of moisture
loss would be beneficial
The above discussion focuses primarily on reducing the number
of empty or non-viable seeds in a seed lot During seed refinement
some viable seed is also lost in the floating fraction (Downie and
Wang 1992 Falleri and Pacella 1997 Sweeney et al 1991) In cases
where there is more than adequate seed supply the loss of viable seed
in the floating fraction is not a problem In those cases where the
amount of available viable seed is limited and losses of viable seeds
needs to be minimized other criteria can be used to determine the
most effective seed refinement technique Such was the case in this
study
The percentage of filled seeds recovered in the sinking fraction
provides a measure ofhow efficient the refinement technique is at
reducing the number of filled (potentially viable) seeds lost in the
floating fraction In the current study involving alder those protocols
with low percentages of filled seeds in the sinking fraction had a high
percentage of filled seeds recovered (Figure 12) In the case of alder
the high recovery of filled seeds was inversely related to the LDS
80
r
0
~ S Il Q) u M Q)
~
50
40
30
20
-e- Fill -0- Recovery
1- (00) 2- (240) 3- (241) 4- (2418) 5- (24124)
Preparation Protocol
Figure 12 Alder Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (Protocols described in Table 2)
81
~ 0 u ~ M 0
S ~ I+-lt
90
80
70
60
treatments ability to remove non-viable seed A similar trend was
observed in another study in an attempt to upgrade germinated
cabbage seeds using density gradients As percentage recovery
increased the percentage of germinated seeds decreased because of
the increased recovery of non-germinated seeds (Taylor and Kenny
1985) In the case ofwater birch separation technique did not
impact the percentage of filled seeds recovered but there were
differencesin the percentage of filled seeds in the sinking fraction
(Figure 13) The technique employed to determine which seed
refinement protocol to use in the germination studies was to multiply
the percentage of filled seeds in the sinking fraction by the percentage
of filled seeds recovered This value addresses both the protocols
ability to remove non-viable seeds as well as its ability to reduce the J
loss of potentially viable seeds
Depending on a growers constraints either greenhouse space
or seed supply the evaluation of a seed refinement technique could be
based on one of three criteria discussed above percentage of filled
seeds in the sinking fraction percentage of filled seeds recovered or
the product generated by multiplying these two values as was done in
82
-
120~------------------------------------------~
___ Fill
-0- Recovery
20
J O~------~----~------~------~------~----~
1- (010) 2- (120) 3- (1205) 4- (121) 5- (1212)
Preparation Protocol
Figure 13 Birch Percentage Fill and Percentage Recovery of the Sinking Fraction as Influenced by Preparation Protocol Error bars represent + - one Standard Error (protocols described in Table 3)
83
this case In cases where seed supply is a greater constraint selection
of seed refinement technique may be based solely on the percentage
of filled seeds recovered This seed refinement technique may not be
as efficient in removing unfilled seeds but loss of filled seeds would
be minimized In the case where growing space is the greater
constraint the percentage of filled seeds in the sinking fraction would
be the criteria used for seed refinement technique selection Ifboth
greenhouse space and seed supply are limited then the product of the
two may be used to determine the appropriate protocol The use of
this information in conjunction with spreadsheet-based seed sowing
programs allows nursery managers to select the best seed refinement
technique for their nursery (Harrington and Glass 1997 Wenny
1993)
The particular separation medium found to be most effective
will vary with species Large and dense seeds may often be effectively
separated using water as the medium (Simak 1983) This is known as
the specific gravity method of separation when used on untreated
seeds In very small seeds where the density gradient between
empty dead and filled live seeds is not great water may not be
84
effective and it is more advantageous to adjust the specific gravity of
the separation medium rather than trying to make fine adjustments in
the density gradient of the seeds to be separated (Downie and Wang
1992)
Germination Requirements
Thinleaf Alder
The IDS separation process significantly improved alder
percentage germination with greater improvement in the better
quality seedlots Simak (1983) achieved an enhancement of lodgepole
pine seeds in which the sinking fraction had almost 7000 germination
at 7 days following sowing compared to 13 for the control and over
90 germination at 21 days post-sowing compared to 6800 for the
control Falleri and Pacella (1997) improved the germination of
London plane tree to 86) with LDS compared to 4800 for the
control
As was the case for total numbers of filled seeds seed sources
varied considerably in the observed germination There was no
distinct latitudinal gradient observed in the data The extremely low
observed germination of the Red River Canyon seed source has been
85
observed in previous studies (Dreesen and Harrington 1998)
Provenance variation in seed properties and germination is not
uncommon and has been reported for a wide range of other woody
species (Young and Young 1992 Baskin and Baskin 1998)
Germination percentage differences in source were noted in a study of
seaside alder (A maritima [Marsh] Nutt) (Schrader and Graves
2000) In the latter study non-stratified seeds varied in germination
percentage from less than 20 for seeds from the Georgia and
Delmarva peninsula sources to more than 40 for the Oklahoma
source Stratified seeds of the Oklahoma source had a 55
germination while the Georgia source had just over 31 germination
and the Delmarva source had nearly 15 germination Schrader and
Graves also noted that germination varied among half-sibling groups
within each source popUlation Stratified groups within the
Oklahoma seed source had germination percentages ranging from 38shy
82 within the Georgia seed source ranging from 12-58 and
within the Delmarva source ranging from 4-29 Non-stratified seeds
from one group in the Delmarva source had germination percentage
less than 1 (Schrader and Graves 2000)
86
There was an interaction between separation and source
Sources with a moderately low percentage ofviable seeds responded
more effectively to the IDS separation method than the source with
a very low percentage fill or the sources with a moderate percentage
of filled seeds Germination for the Luna source (1400 germination in
the non-separated fraction) and the Red River Canyon source (0500
germination in the non-separated fraction) can both be improved
three-fold by separation However a three-fold improvement of a low
germination percentage still results in a low germination percentage
Donald (1985) found that the ID S technique could improve the
viability of a seed source of Pinus eDiottiwhich had a reasonably
high germination capacity but that the technique might not be
valuable for seed stock of very low viability because it cannot
separate normal live seeds from live seeds which have abnormal
germination
Stratification appears to be advantageous for many species of
alder but the influence of stratification was not detectable when all
seed sources were analyzed When analyzed without the very low
viability seed source (Red River Canyon) stratification does affect
87
germination but the actual difference in percentage germination is
not impressive Longer stratification period (56 days) does not appear
to confer any advantage In seaside alder response to stratification is
also source-specific One Oklahoma seaside alder source had
optimum germination with six weeks of stratification and longer
periods tended to reduce germination percentage (Schrader and
Graves 2000) Germination in the other two sources of seaside alder
was not improved by six weeks of stratification This suggests that
stratification and source interactions may be a feature of Alnus
species
Interaction between stratification and separation fraction
among sources can best be explained by the fact that the floating
fractions of all sources and all fractions of the Red River Canyon
source had very little viable seed and therefore very little response to
stratification In contrast the non-separated and sinking fractions of
the other three sources which contained greater amounts ofviable
seed responded to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
in comparison to the sinking fractions
88
There is a three-way interaction between stratification
separation and source The floating fractions of all sources tended to
have a low germination percentage which was not significantly
affected by stratification as was the case with the non-separated and
sinking fraction of the very low viability Red River Canyon source
While the sinking fractions and non-separated samples of the Luna
source and the non-separated Chaffee seed sources responded best to
28 days of stratification the non-separated Reserve seed source
showed no response to stratification The sinking fraction of the
Reserve source responded negatively to 28 days of stratification and
germination at 56 days of stratification was similar to the germination
of the non-stratified sinking fraction Only the sinking fraction of the
Chaffee source responded in linear fashion to increasing stratification
With the elimination of the Red River Canyon source these
interactions are still present (Figure 6)
Pre-soaking of alder seeds had variable effects on germination
depending on seed source Imbibition improved germination for the
Luna source decreased it for the Reserve source and had no effect on
the Red River Canyon or Chaffee sources Pre-soaking with aeration
89
was found to improve ge~nation of red alder and speckled alder
(Berry and Torrey 1985)
Water Birch
Water birch percentage germination was significantly improved
by the density separation in ethanol Falleri and Pacella (1997) found
that density separation of London plane tree seeds in petroleum ether
improved germination to 6000 compared to 4800 for the unseparated
control
Source influenced the effectiveness of separation Seed sources
with moderately rather than extremely low germination percentage
(Moly 2 and Red River Canyon) had an encouraging ten-fold
improvement The Moly 1 source which had an even lower nonshy
separated germination percentage had a five-fold improvement while
the Chaffee source with a 17 germination in the non-separated
portion had a two-fold improvement We can again refer to the study
by Donald (1985) and see that in the case ofwater birch seeds the
moderately low germination percentages of the non-separated seeds
percentage with the longest stratification period showing the most
improvement but the actual gain in percentage was rather low at the
cost ofseveral weeks of time The four seed sources responded
differently to stratification The Moly 2 and Red River Canyon
sources had the greatest germination at 21 days of stratification while
the Moly 1 source had an increase in germination only at 56 days
The Chaffee source responded negatively to stratification especially
at 21 days Paper birch also responds to stratification (Bevington and
Hoyle 1981) with an optimum chilling period of 2 to 3 weeks This
response varied between the New Hampshire and Alaska seed sources
studied with the germination of the New Hampshire source
decreasing somewhat with longer periods of stratification but the
Alaska source maintaining a high percentage with longer stratification
periods
Interaction between stratification and separation fraction can
best be explained by the fact that the floating fractions had very little
viable seed and therefore very little response to stratification while
the non-separated and sinking fractions which contained viable seed
91
had a response to stratification This would also explain the
somewhat muted response of the non-separated seeds to stratification
Source was a significant factor in birch germination percentage
with the Moly 1 source showing very poor germination and the
other sources ranging from 13-1900 Germination of distinct paper
birch sources differed in response to different treatments (Bevington
1986) and germination of separate populations of seaside alder varied
by source (Schrader and Graves 2000)
Pre-soaking was beneficial for the Red River Canyon and
Chaffee seed sources but not the Moly 1 or Moly 2 sources This
difference may be due more to the poor quality of the seed sources
than to different responses to pre-soaking Overall pre-soaking
germination was more than twice that of the non-treated control
General Observations
Stratification increases the germination percentage ofwater
birch and thinleaf alder but does not appear to afford great
improvements in view of the time required Thinleaf alder benefitted
most from a 28-day stratification while water birch responded best to
92
the longer 56-day stratification There are considerable differences in
response to stratification among seed sources for both species
The seed refinement process significantly improves germination
percentage for both thinleaf alder and water birch The LDS method
appears to be an effective tool for seed refinement in thinleaf alder
seed collections where there is a percentage of filled seeds high
enough to make the process practical and the same could be said for
the specific gravity methodmiddotof separation for water birch seeds using
ethanol as a separation medium The techniques could be further
refined for each species In the case ofthinleaf alder an optimum
drying period between 1 hour and 18 hours could be identified using
differences in moisture content to pinpoint the best drying time An
LDS treatment for water birch using drying periods longer than 2
hours might be more effective for seed refinement than the specific
gravity method
Seed source and quality of the seed lot would also determine
how effectively the method works Fine adjustments in method could
make the process for the respective species and seed lot more
effective Preliminary evaluations of a particular seed collection for
93
percentage of filled seeds moisture content and general response to
separation medium might point to the most effective manner of
dealing with that seed source Whether the process is worthwhile in
particular cases would depend on the value of the seed collection in
relation to its potential for improvement and the previously
mentioned constraints of seed supply and growing space
LITERATURE CITED
Albers Daniel J and Stanley B Carpenter 1979 Influence of site environmental conditions mulching and herbaceous ground cover on survival growth and water relations ofEuropean alder seedlings planted on surface mine spoil In Proceedings of the Symposium on Surface Mining Hydrology Sedimentology andReclamation ed Stanley B Carpenter 23-32 Lexington Univ ofKentucky College ofEngineering Press
Allen Edith B 1988 Ecological approaches in theory and practice To what degree is reconstruction possible The Reconstruction ofDisturbedAridLands -An Ecological Approach Academy for the Advancement of Science Sel Symposium 109 ed Edith B Allen 1-4 257-261 Boulder Westview Press
Ashburner K B 1993 Birches in the wild their habitats and ecology In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 19-28 Surrey International Dendrology Society
Barnett James P 1971 Flotation in ethanol reduces storability of southern pine seeds Forest Science 17(1)50-51
Baskin C C and J M Baskin 1998 Within-species variations in seed dormancy Chapter 8 of Seeds Ecology Biogeography andEvolution ofDonnancyand Gennination San Diego CA Academic Press
Belcher E W 1982 You reap what you sow In Proceedings ofthe Southem Containerized Forest Tree Seedling Conference USDA FS GTR-SO-37 eds R W Guilan and James P Barnett 25-28 New Orleans Southern Forest Experiment Station
95
Berry Alison Mand John G Torrey 1985 Seed Germination seedling inoculation and establishment of Alnus spp in containers in greenhouse trials Plant and Soi187161-173
Bevington John M and Merrill C Hoyle 1981 Phytochrome action during prechilling induced germination of Betula papyrifera Marsh Plant Physiol 67705-710
Bevington John M 1986 Geographic differences in the seed germination ofpaper birch (Betula papyrifera) American Journal ofBotany 73(4)564-573
Bewley J Derek and Michael Black 1994 Seeds Physiology of Development and Germination 2nd ed New York Plenum Pub Corp pp 213-287
Biswas P K P A Bonamy and K B Paul 1972 Germination promotion of loblolly pine and baldcypress seeds by stratification and chemical treatments PhysiolPlant 2771-76
Bjorkbom John C D A Marquis and F E Cunningham 1965 The variability ofpaper birch seed production dispersal and germination USFS Research Paper NE-41 Washington GPO
Black M and P F Wareing 1955 Growth studies in woody species VII Photoperiodic control of germination in Betula pubescens Ehrh Physiol Planta 8300-316
Bollen W B and K C Lu 1968 Nitrogen Transformation in soils beneath red alder and conifers In Biology ofAlder eds J M Trappe et aI 141-148 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
96
Bond G 1955 An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus Myrica and Hippophae Joum ofExper Botany6303-311
---------- 1971 Root nodule formation in non-leguminous angiosperms In Biological Nitrogen Fixation in Natural and Agricultural Habitats Proceedings ofthe Technical Meetings on Biological Nitrogen Fixation ofthe Intemational Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and EG Mulder 317-324 The Hague Martinus Nijhoff Plant and SoilSpecial Volume 1971
---------- 1976 Evidence for fixation of nitrogen by root nodules of alder(Alnus) under field conditions New Phytologist 55147-153
Bonner FT 1984 New forests from better seeds The role of seed physiology In Seedling Physiology and Reforestation Success eds Mary L Duryea and Gregory N Brown 37-60 Dordrecht Martinus NijhoffDrW Junk Pub
Bormann Bernard T 1983 Ecological implications of phytochrome-mediated seed germination in red alder Forest Science 29734-738
Bradbeer JW 1988 Seed Dormancy and Germination London Chapman amp Hall pp 39-131
Brenzel Kathleen N (ed) 1995 Sunset Westem Garden Book Menlo Park Sunset Publishing Corporation pp 183-184
Brinkman Kenneth A 1974 Betula L Birch In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 252-257 Washington GPO
Britton Nathaniel Lord 1908 North American Trees New York Henry Holt amp Co pp246-264
97
Burton Philip J Kenneth R Robertson Louis R Iverson and Paul G Risser 1988 Use of resource partitioning and disturbance regimes in the design and management of restored prairies In The Reconstruction ofDisturbedAnaLands - An Ecological Approach ed E B Allen 46-88 BoulderWestview Press
Carter Jack L 1997 Trees and Shrubs ofNew Mexico Boulder Mimbres Publishing pp 400-402
Crocker Robert L and Jack Major 1955 Soil development in relation to vegetation and surface age at Glacier Bay Alaska Journal ofEcology43427-448
Daniel Theodore John A Helms and Frederick S Baker 1979 Principles ofSilviculture 2nd ed New York McGraw Hill Book Co pp373-376
Danielson H Rodger and Yasuomi Tanaka 1978 Drying and storing stratified ponderosa pine and douglas-fir seeds Forest Science 24(1)11-16
deJong PC 1993 An introduction to Betula its morphology evolution classification and distribution with a survey of recent work In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 7-18 Surrey International Dendrology Society
Dick-Peddie William A 1993 Riparian vegetation Chapter 9 of New Mexico Vegetation Pas Present andFuture Albuquerque UNM Press
Dirr Michael A and Charles W Heuser 1987 The Reference Manual ofWoody Plant Propagation From Seed to Tissue Culture Athens GA Varsity Press ppII-2290-9196-97
98
Donald D G M 1985 The separation of full dead seed from live seed in Pinus elliottii In Proceedings ofthe Intemational Symposium on Nursery Management Practices for the Southem Pines Montgomery AL August 4-9 1985 ed David B South 83-88 Auburn AL Auburn University
Downie Bruce and Ben S P Wang 1992 Upgrading germinability and vigour ofjack pine lodgepole pine and white spruce by the IDS technique Can J For Res 22(8)1124-1131
Dreesen D R and J T Harrington 1998 Propagation of native plants for restoration projects in the southwestern U S shyPreliminary investigations In Proceedings ofthe Westem Forest and Conservation Nursery Association Meeting Boise ID August 19-21 1997 ed T D Landis pp 77-88
Dunlap J R and J P Barnett 1984 Manipulating loblolly pine (Pinus taeda L) seed germination with simulated moisture and temperature stress In Seedling Physiology andReforestation Success eds Duryea Mary L and Gregory N Brown 61-74 Dordrecht Martinus NijhoffDrWJunk Pub
Elias Thomas S 1980 The Complete Trees ofNorth Amenca-Field Guide andNatural History New York Outdoor LifeNature Books Van Nostrand Reinhold Co pp 385-412
Falled Elisabetta and Rosetta Pacella 1997 Applying the IDS method to remove empty seeds in Platanus x acemolia Can J For Res 271311-1315
Fowler D P and T W DWight 1964 Provenance differences in the stratification requirements of white pine Can Joum ofBotany 42669-675
Fowells H A 1965 Silvics ofForest Trees ofthe United States USDA-Forest Service Agricultural Handbook 271 compo H A Fowells 1-4 82-88 92-109 Washington GPO
99
Foxx Teralene S and Dorothy Hoard 1995 Flowering Plants ofthe Southwestern Woodlands Los Alamos Otowi Crossing Press pp26-27
Franklin JerryF andAnnaA Pechanec 1968 Comparison of vegetation in adjacentalder conifer and mixed alder-conifer communities In Biology ofAlder eds J M Trappe et al 37shy44 PortlandUSDA FS Pacific Northwest Forest arid Range Experiment Station
Haeussler Sybille J C Tappeiner II and B J Greber 1995 Germination survival and early growth of red alder seedlings in the ~entral Coast Range of Oregon Canadian Journal of Forest Research 25(10) 1639-1651
Harker Donald Sherri Evans Marc Evans and Kay Harker 1993 Landscape Restoration Handbook Boca Raton Lewis Publications pp 19-2465-71
Harrington J T and P A Glass 1997 Determining the number of seeds to sow per cell An application of the geometric distributions Tree Planters Notes 828-34
Hartmann Hudson T Dale E Kester Fred T Davies Jr and Robert L Geneve 1997 Plant Propagation Principles and Practices 6th ed Upper Saddle River NJ Simon amp Schuster pp194-215 671673-674
Herrera M A C P Salamanca and J M Barea 1993 Inoculation ofwoody legumes with selected arbuscular mycorrhizal fungi and Rhizobia to recover desertified Mediterranean ecosystems AppliedandEnv Micro 59129-133
Hibbs David E Dean S DeBell and Robert F Tarrant eds 1994 The Biology andManagement ofRedAlder Corvallis Oregon State University Press
100
Hilhorst H WM A Smitt and C M Karssen~ 1986 Gibberellin-biosynthesis and sensitivity mediated stimulation of seed germination of Sisymbrium oflicinale by red light and nitrate Physiol Plant 67285-290
Hill HI A G Taylor and T -G Min 1989 Density separation of imbibed and primed vegetable seeds J Amer Soc Hort Sci 114(4)661-665
Hilton Janet R 1985 The influence of light and potassium nitrate on the dormancy and germination of A vena fatua L (wild oat) seed stored buried under natural conditions JExp Botany 36974-979
Hobbs S D 1984 The influence of species and stocktype selection on stand establishment An ecophysiological perspective In Seedling Physiology andReforestation Success eds Mary L Duryea and Gregory N Brown 179-224 Dordrecht Martinus NijhoffDrWJunk Pub
Johnson Frederic D 1968 Taxonomy and distribution of Northwestern alders In Biology ofAlder eds J M Trappe et aI 9-22 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Joseph Hilda C 1929 Germination and vitality ofbirch seeds Bot Gazette 87 127-151
Kenady Reid M 1978 Regeneration of red alder In Utilization and Management ofAlder compiled by D G Briggs D S DeBell and W A Atkinson 183-191 USDA Forest Service GTR-PNW-70 Portland Pacific Northwest Forest and Range Experiment Station
101
-
Lane C G 1993 Propagation of the genus Betula In Betula Proceedings ofthe IDS Betula Symposium Sussex England October 1992 ed David Hunt 51-60 Surrey International Dendrology Society
Lentner M and T Bishop 1986 Experimental Design and Analysis Blacksburg VA Valley Book Company p 173
Mallinckrodt Baker Inc 1997a Material Safety Data Sheet-Petroleum Ether MSDS Number P1696 Phillipsburg NJ Mallinckrodt Baker Inc
Mallinckrodt Baker Inc 1997b Material Safety Data Sheet-Proprietary Solvent III-I Anhydrous (Denatured Ethanol) MSDSNumberP6735 Phillipsburg NJ Mallinckrodt Baker Inc
Martin W C and C R Hutchins 1980 A Flora ofNew Mexico Vaduz J Cramer AR Gantner Verlag K G pp510-514
Mayer A M and A Poljakoff-Mayber 1989 The Germination of Seeds 4th ed Oxford Pergamon Press pp 71-99
McLemore B F 1965 Pentane flotation for separating full and empty longleaf pine seeds Forest Science 11(2)242-243
McVean D N 1956 Ecology ofAlnusglutinosa (L) Gaertn III Seedling establishment JEcol44195-218
Monsen Stephen B 1984 Use of shrubs on mine spoils In The Challenge ofProducing Native Plants for the Intermountain Area Proc Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT-168 26-31 Ogden Intermountain Forest and Range Experimental Station
102
National Academy ofSciences (NAS) 1974 Rehabilitation Potential ofWestem Coal Lands eds Thadis W Box Richard F Hadley andM Gordon Wolman 11-1549-7273-94 Cambridge Ballinger Pub Co for the N A S (for the Ford Foundation)
Nielson Rex F and HB Peterson 1973 Establishing vegetation on mine tailings waste In Ecology andReclamation ofDevastated Land vol II eds Russell J Hutnik and Grant Davis 103-115 New York Gordon amp Breach
Phillips Judith 1995 Plants for Natural Gardens Santa Fe Museum ofNew Mexico Press ppl08-109
Pratt Carl R 1986 Environmental factors affecting seed germination ofgray birch (Betula popuJifoJia) collected from abandoned anthracite coal mine spoils in northeast Pennsylvania Ann Appl BioI 108649-658
Preston Richard J Jr 1968 RockyMountain Trees A Handbook of the Native Species New York Dover Publications Inc pp 120shy123124-129134-135
Radwan M A andD S DeBell 1981 Germination of red alder seed USDA Forest Service Res Note PNW-370 Portland Pacific Northwest Forest and Range Experiment Station
Rose R W C Carlson and P Morgan 1990 The target seedling concept In Target Seedling Symposium Proceedings Combined Meeting ofthe Westem Forest Nursery Associations August 13-17 1990 Roseburg Oregon eds Robin Rose et al General Technical Report RM-200 Ft Collins CO USDA Forest Service Rocky Mountain Forest and Range Experiment Station
103
Ross JD and J W Bradbeer 1971 Studies in seed dormancy V The content of endogenous gibberellins in seeds of Corylus avellana L Planta 100288-302
Rudolf PaulO 1950 Cold soaking-a short cut substitution for stratification Joum ForeSl1y4831-32
SAS Institute Inc 1989 SASSTATUsers Guide Version 6 Fourth Edition Volume 1 Cary NC SAS Institute Inc
Sargent Charles S 1901 New or little known North American trees m Bot Gazette 31(4)217-240
---------- 1905 ManuaJ ofthe Trees ofNorth America New York Houghton Mifflin (reprint 1965 New York Dover Pub) pp 205-206218-220223-226
Schalin TImari 1968 Germination analysis of grey alder (Alnus incana) and black alder (Alnus glutinosa) seeds In Biology of Alder eds J M Trappe et aI 107-114 Portland USDA FS Pacific Northwest Forest and Range Experiment Station
Schopmeyer C S 1974 AlnusB Ehrh In Seeds ofWoody Plants in the United States USDA Agricultural Handbook 450 ed C S Schopmeyer 19-40 126-135 140-152206-211 Washington GPO
Schrader James A and William R Graves 2000 Seed germination and seedling growth ofAlnus maritima from its three disjunct populations J Amer Soc Hort Sd 125(1)128-134
Schubert Gilbert H L J Heidman and M M Larson 1970 ArtificiaJ Reforestation Practices for the Southwest USDA Agricultural Handbook 370 Washington GPO
104
Simak Milan 1983 A new method for improvement of the quality of Pinus contorta seeds In Lodgepole pine regeneration and management ed Mayo Murray 39-41 USDA For Servo Gen shyTech Rep PNW-157
Slavik Bohdan 1974 Water exchange between plant and atmosphere Chapter 5 of Methods ofStudying Plant Water Relations Prague Academia Publishing House of the Czechoslovak Academy ofSciences
Sweeney J D Y A EI-Kassaby D W Taylor D G W Edwards and G E Miller 1991 Applying the IDS method to remove seeds infested with the seed chalcid Megastigmus spermotrophus Wachtl in douglas-fir Pseudotsuga menziesii (Mirb) Franco New Forests 5327-334
Taylor A G and T J Kenny 1985 Improvement of germinated seed quality by density separation J Amer Soc Hort Sci 110(3)347-349
Tarrant Robert F 1961 Stand development and soil fertility in a douglas-fir - red alder plantation Forest Science 7238-246
Tarrant Robert F and James M Trappe 1971 The role of Alnus in improving the forest environment In Biological Nitrogen Fixation in Natural andAgricultural Habitats Proceedings of the technical meetings on biological nitrogen fixation ofthe Ind Biological Programme (Section PP-N) Prague and Wageningen 1970 eds T A Lie and E G Mulder 335-348 The Hague Martinus Nijhoff Plant and Soil Special Volume 1971
Thompson P A 1971 Research into seed dormancy and germination Comb Proc Inter Plant Prop Soc 21211-228
Villiers T A and P F Wareing 1964 Dormancy in fruits of Fraxinus excelsior L Joum Exp Botany 15(44)359-367
105
Vines Robert A 1960 Trees Shrubs and Woody Vines ofthe Southwest Austin University of Texas Press pp139-142
Virtanen Artturi 1 1957 Investigations on nitrogen fixation by the alder II Associated culture of spruce and inoculated alder without combined nitrogen Physio1 P1anta 10164-169
Webb DP and PF Wareing 1972 Seed dormancy in Acer Endogenous germination inhibitors and dormancy in Acer pseudop1atanusL P1anta 104115-125
WennyDL 1993 Calculating filled and empty cells based on number of seeds sown per cell A microcomputer application Tree Planters Notes 4449-52
Whitford W 1988 Decomposition and nutrient cycling in disturbed arid ecosystems In The Reconstruction ofDisturbedArid Lands -An Ecological Approach ed E B Allen 136-16l Boulder Westview Press
Wilcox James R 1968 Sweetgum seed stratification requirements related to winter climate at seed source Forest Science 1416-19
Wooton E O and Paul C Standley 1915 Flora ofNew Mexico Washington GPO pp 163-164
Young James A Jerry D Bundy and Raymond A Evans 1984 Germination of seeds of wildland plants In The Challenge of Producing Native Plants for the Intermountain Area Proceedings ofthe Intermountain Nurserymans Assoc 1983 Conference Las Vegas Nevada USDA Forest Service GTR-INT 168 1-5 Ogden Intermountain Forest and Range Experimental Station
106
Young James A and Cheryl G Young 1986 Collecting Processing and Germinating Seeds ofWildland Plants Portland Timber Press pp 59-65 84-85
Young James A and Cheryl G Young 1992 Seeds ofWoody Plants in North America Portland Dioscorides Press pp 26-28 55-59
Young J F 1967 Humidity control in the laboratory using salt solutions - a review J Applied Chem 17241-245