ORIGINAL PAPER In vitro propagation of four threatened Paphiopedilum species (Orchidaceae) Bo Long • Alex X. Niemiera • Zhi-ying Cheng • Chun-lin Long Received: 27 March 2009 / Accepted: 11 January 2010 / Published online: 2 February 2010 Ó Springer Science+Business Media B.V. 2010 Abstract The effects of seed maturity, media type, carbon source, and organic nutrient additives on seed germination, protocorm development, and plant growth of Paphiopedi- lum villosum var. densissimum Z. J. Liu et S. C. Chen were investigated. Micropropagation frequency was enhanced through the use of 200-day-old seed, Knudson C (KC) medium, and the presence of both glucose and coconut milk in the medium. The effects of various plant growth regula- tors on the frequency of shoot organogenesis in four Paphiopedilum species were also investigated. Explants of P. villosum var. densissimum and P. insigne (Lindl.) Stein incubated in the presence of 5 mg l -1 6-benzyladenine (BA) with 0.5 mg l -1 a-naphthalene acetic acid (NAA) and 0.2 mg l -1 BA with 0.1 mg l -1 NAA, respectively, showed a twofold increase in the frequency of shoot organogenesis. For explants of P. bellatulum (Rchb. f.) Stein and P. arme- niacum S. C. Chen et F. Y. Liu, the combination of 5.5 mg l -1 BA with 0.5 mg l -1 NAA and 4 mg l -1 BA with 0.1 mg l -1 NAA, respectively, resulted in the highest fre- quencies of shoot organogenesis. Keywords Micropropagation Á Orchid Á Seed germination Á Shoot proliferation Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid AC Activated charcoal BA 6-Benzyladenine IAA Indole-3-acetic acid IBA Indole-3-butyric acid KT Kinetin LH Lactalbumin hydrolysate NAA a-Naphthalene acetic acid TDZ 1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea Introduction Paphiopedilum spp. (Orchidaceae), commonly known as Lady’s slipper orchids, are often marketed as attractive and desirable container-grown plants. A few species are regarded as endangered or even extinct in the wild due to over-collection from natural areas and large-scale illegal trade. Paphiopedilum spp. have been designated as endangered by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES; CITES Appendices 2008). In a natural setting, Paphio- pedilum seed germinates relatively slowly due to the absence of an endosperm. In P. armeniacum, the interval between seed germination and tiller production is about 4 years (Liu et al. 2006), although some orchid hybrids and tropical orchids initiate tiller production within 6 months B. Long Á Z. Cheng Á C. Long (&) Kunming Institute of Botany, Chinese Academy of Sciences, 650204 Kunming, China e-mail: [email protected]; [email protected]B. Long e-mail: [email protected]B. Long Graduate School, Chinese Academy of Sciences, 100039 Beijing, China A. X. Niemiera Department of Horticulture, Virginia Polytechnic and State University, Blacksburg, VA 24061, USA e-mail: [email protected]C. Long College of Life and Environmental Sciences, Minzu University of China, 100081 Beijing, China 123 Plant Cell Tiss Organ Cult (2010) 101:151–162 DOI 10.1007/s11240-010-9672-1
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ORIGINAL PAPER
In vitro propagation of four threatened Paphiopedilum species(Orchidaceae)
Bo Long • Alex X. Niemiera • Zhi-ying Cheng •
Chun-lin Long
Received: 27 March 2009 / Accepted: 11 January 2010 / Published online: 2 February 2010
� Springer Science+Business Media B.V. 2010
Abstract The effects of seed maturity, media type, carbon
source, and organic nutrient additives on seed germination,
protocorm development, and plant growth of Paphiopedi-
lum villosum var. densissimum Z. J. Liu et S. C. Chen were
investigated. Micropropagation frequency was enhanced
through the use of 200-day-old seed, Knudson C (KC)
medium, and the presence of both glucose and coconut milk
in the medium. The effects of various plant growth regula-
tors on the frequency of shoot organogenesis in four
Paphiopedilum species were also investigated. Explants of
P. villosum var. densissimum and P. insigne (Lindl.) Stein
incubated in the presence of 5 mg l-1 6-benzyladenine (BA)
with 0.5 mg l-1 a-naphthalene acetic acid (NAA) and
0.2 mg l-1 BA with 0.1 mg l-1 NAA, respectively, showed
a twofold increase in the frequency of shoot organogenesis.
For explants of P. bellatulum (Rchb. f.) Stein and P. arme-
niacum S. C. Chen et F. Y. Liu, the combination of
5.5 mg l-1 BA with 0.5 mg l-1 NAA and 4 mg l-1 BA with
0.1 mg l-1 NAA, respectively, resulted in the highest fre-
80 0 c 0 c 0 c 0.12 c 0.14 c 0.74 c 0.9 c 0.71 c 31.33 a 13.33 b
Data are given as the mean in vitro germination frequency of seed of P. villosum var. densissimum collected from fruit harvested between 120
and 300 days after pollination (DAP). Each mean is based on microscopic observation. Means followed by different lower-case letters are
significantly different at P \ 0.05 level by the LSD testa Duration of time between placement of medium (plating) and germination
154 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
The effect of PGRs on callus formation
Few seed-derived protocorms induced callus (data not
shown; Fig. 4c, d). All leaf pieces taken from 1.5-year-old
plantlets (cultured in containers; Fig. 4e) as callus induction
material turned brown and ultimately died. The combination
of 0.5 mg l-1 NAA with 2.0 mg l-1 BA resulted in leaves
that remained green (no browning, no callus) during the
Medium
Fre
quen
cy o
f ger
min
atio
n %
¡r/ p
roto
corm
siz
e m
m2
0
10
20
30
40
50
60
70Frequency of germination after 80days following inoculationPromcorm size after 100days following inoculation
C
A
B
C
ab ab ab
1/4MS KC VW 1/2MS
Fig. 1 Effect of medium composition on seed germination and
protocorm size of in vitro Paphiopedilum villosum var. dentissimum.Mean in vitro seed germination and protocorm size of P. villosum var.
densissimum in relation to composition of the medium. Data were
recorded 3 months following culture initiation. Each mean is based on
a visual observation. a Data were square-root transformed prior to
analysis of variance (ANOVA) and converted to the original scale for
demonstration in the figure. b Means with different letters within a
column are significantly different at P \ 0.05 level by the LSD test.
Bars: Standard errors. KC Knudson medium, VW Vacin and Went
medium, MS Murashige and Skoog medium (1/4, 1/2 quarter- and
half-strength, respectively). Details on the various media are given in
the text
Carbon sourceSucrose Glucose Maltose Mannitol
Freq
uenc
y of
ger
mia
ntio
n (
%)
/ Lea
f len
gth
(mm
)
0
5
10
15
20
25
Frequency of germinationLeaf lengthLeaf width
A
B
C
D
aba
ab
b
a a
ab
b
/ Lea
f wid
th (
mm
)
Fig. 2 Effect of carbon source
on mean in vitro seed
germination and leaf size of in
vitro Paphiopedilum villosumvar. dentissimum. Data were
recorded 3 months following
culture initiation. Each mean is
based on a visual observation. aData were expressed as
germination frequency; leaf
length was square-root and
reciprocal square-root
transformed prior to ANOVA
and converted to the original
scale for demonstration in the
figure. b Means with different
letters within a column are
significantly different at the
P \ 0.05 level by the LSD test.
Bars: Standard errors
Plant Cell Tiss Organ Cult (2010) 101:151–162 155
123
0
5
10
15
20
25
Frequency of germinationLeaf lengthLeaf width
Potato Apple Chayote LH Coconut
Organic nutrient additives
Fre
quen
cy o
f ger
min
atio
n (
%)
/ Le
af le
ngth
(m
m)
B
B
C C
A
a
a
a
b
a
a aab
bab
/ Le
af w
idth
(m
m)
Fig. 3 Effect of organic
nutrient additives on mean seed
germination and leaf size of
P. villosum var. dentissimum in
vitro. Data were recorded
3 months following culture
initiation. each mean is based on
a visual observation. Means
with different letters within a
column are significantly
different at the P \ 0.05 level
by the LSD test. LHLactalbumin hydrolysate. Bars:
Standard errors
Fig. 4 In vitro propagation process of Paphiopedilum spp. aGermination of 300-DAP (day after pollination) seed of P. villosumvar. densissimum: swollen embryo and ruptured testa (Bar: 0.1 mm).
b Protocorm growth (Bar: 1 mm), c Callus mass developed from a
protocorm of P. bellatulum. d Shoot cluster formed from callus of P.bellatulum. e 1.5-year-old P. insigne in vitro seedling with roots. f–I
Shoot multiplication from seedlings of P. villosum var. densissimum(f), P. insigne (g), P. bellatulum (h), and P. armeniacum (I).
j P. insigne root tubercles. k–m Transplanted seedlings of P. villosumvar. densissimum (k), P. insigne (l), and P. bellatulum (m) in a pot
(Bar: 1 cm)
156 Plant Cell Tiss Organ Cult (2010) 101:151–162
123
3-month culture period, but after subculture, the leaves
gradually turned brown (Table 2). A number of PGR com-
binations reduced the amount of browning, such as in the
case of P. insigne treated with 2,4-D (at 2.0, 4.0, and
8.0 mg l-1) in combination with KT (0.1 mg l-1) and TDZ
(0.02, 0.05 mg l-1; Table 2). Explant size also affected the
browning phenomenon; 1.5-cm2 leaf pieces had less
browning than all other sizes, and 2-cm2 pieces showed the
most browning (data not shown).
The effect of PGRs on shoot multiplication
The length and number of shoots of the four Paphiopedi-
lum spp. was influenced by the concentrations and com-
binations of cytokinins and auxins contained in the medium
(Table 3; Fig. 4f-I). In a 3-month period, three new
plantlets were produced (per three plants) by: (1) P.
villosum var. densissimum for the combinations of
3.0 mg l-1 BA with 1.0 mg l-1 NAA, 5.0 mg l-1 BA with
0.5 mg l-1 NAA, and 6.0 mg l -1 BA with 0.1 mg l-1
NAA; (2) P. insigne for the combinations of 0.2 mg l-1
BA with 0.1 mg l-1 NAA and 1.0 mg l-1 BA with
0.5 mg l-1 NAA. For P. bellatulum, the combination
5.5 mg l-1BA with 0.5 mg l-1 NAA induced maximum
shoot organogenesis (0.33; one new plantlet per three
plants) and produced the longest shoots (0.6 cm); this same
result was obtained with the 1.0 mg l-1 BA and 0.5 mg l-1
NAA treatment. Organogenesis occurred im all PGR
combinations for P. armeniacum; for this species, shoot
number was highest in the 4.0 mg l-1 BA and 0.1 mg l-1
NAA treatment (Table 3).
After 2 months, plantlets with three or more 3- to 6-cm-
long roots and four to five leaves were transferred to pots
with a peat moss substrate (Fig. 4k–m). Although the
survival frequency of these greenhouse-grown rooted
plants was about 60%, the seedlings grew very slowly.
Discussion
Seed germination and protocorm growth in vitro
Seed maturity significantly affected the germination fre-
quency of P. villosum var. densissimum (Table 1). The
germination frequency of 200-DAP seeds was highest on
the basal medium (inoculation); seed coat formation started
about 200 DAP. Seeds\200-DAP turned brown soon after
inoculation, indicating that the embryo was seemingly too
underdeveloped to absorb nutrients from the medium. In
contrast, the frequency of germination for 300-DAP seed
was lower than that of 200-DAP seed, likely because the
seed coat had already formed (microscopic observation),
possibly attenuating nutrient uptake. A similar effect of
seed maturity on the germination of Paphiopedilum seed
has been shown by Nagashima (1982) and Ding et al.
(2004). Ren and Wang (1987) found that if a P. godefroyae
(Godef.) Stein embryo had 10–40 cells at 100 ± 5 DAP,
then suspensor and endosperm nuclei degenerated at
120 ± 5 DAP; however, at 200 ± 10 DAP, the globular
embryo of mature seed had 120–140 cells, and there was a
single cell layer in the seed coat. Lee (1998) reported
optimal seed germination of P. delenatii Guillanmin at 150
DAP (68%), with the germination frequency decreasing
Table 2 Effect of cytokinins in combination with auxins on brown-
ing of leaf fragments
Cytokinins (mg l-1) Auxin (mg l-1) Browninga (%)
2,4-D NAA
KT
0.1 1 0 100
0.1 2 0 66.7
0.1 4 0 57.1
0.1 8 0 42.9
TDZ
0.02 1 0 28.6
0.02 2 0 71.4
0.05 4 0 33.3
0.05 8 0 71.4
0.1 1 0 85.7
0.1 2 0 100
0.5 4 0 100
0.5 8 0 100
BA
1 0 0.1 100
2 0 0.1 100
4 0 0.1 100
8 0 0.1 100
1 0 0.5 100
2 0 0.5 0
4 0 0.5 100
8 0 0.5 100
1 0 1 100
2 0 1 100
4 0 1 100
8 0 1 100
KT Kinetin, TDZ 1-phenyl-3-(1, 2, 3-thiadiazol-5-yl)-urea, BA 6-ben-