8/6/2019 Bio EZ Cangahuala Et Al 09
1/18
Dear Author,
Here are the proofs of your article.
You can submit your correctionsonline, via e-mail or by fax.
For online submission please insert your corrections in the online correction form. Always
indicate the line number to which the correction refers.
You can also insert your corrections in the proof PDF andemail the annotated PDF.
For fax submission, please ensure that your corrections are clearly legible. Use a fine black
pen and write the correction in the margin, not too close to the edge of the page.
Remember to note thejournal title, article number, and your name when sending your
response via e-mail or fax.
Check the metadata sheet to make sure that the header information, especially author names
and the corresponding affiliations are correctly shown.
Check the questions that may have arisen during copy editing and insert your answers/corrections.
Check that the text is complete and that all figures, tables and their legends are included. Also
check the accuracy of special characters, equations, and electronic supplementary material if
applicable. If necessary refer to the Edited manuscript.
The publication of inaccurate data such as dosages and units can have serious consequences.
Please take particular care that all such details are correct.
Please do not make changes that involve only matters of style. We have generally introduced
forms that follow the journals style.
Substantial changes in content, e.g., new results, corrected values, title and authorship are notallowed without the approval of the responsible editor. In such a case, please contact the
Editorial Office and return his/her consent together with the proof.
If we do not receive your correctionswithin 48 hours, we will send you a reminder.
Your article will be published Online First approximately one week after receipt of your
corrected proofs. This is theofficial first publication citable with the DOI. Further changes
are, therefore, not possible.
The printed version will follow in a forthcoming issue.
Please note
After online publication, subscribers (personal/institutional) to this journal will have access to the
complete article via the DOI using the URL: http://dx.doi.org/[DOI].
If you would like to know when your article has been published online, take advantage of our free
alert service. For registration and further information go to: http://www.springerlink.com.
Due to the electronic nature of the procedure, the manuscript and the original figures will only be
returned to you on special request. When you return your corrections, please inform us if you would
like to have these documents returned.
http://www.springerlink.com/8/6/2019 Bio EZ Cangahuala Et Al 09
2/18
ELECTRONIC REPRINT ORDER FORM
After publication of your journal article, electronic (PDF) reprints may be purchased by arrangement with Springer andAries Systems Corporation.
The PDF file you will receive will be protected with a copyright system called DocuRights. Purchasing 50 reprints willenable you to redistribute the PDF file to up to 50 computers. You may distribute your allotted number of PDFs as youwish; for example, you may send it out via e-mail or post it to your website. You will be able to print five (5) copies of yourarticle from each one of the PDF reprints.
Please type or print carefully. Fill out each item completely.
1. Your name: __________________________________________________
Your e-mail address: __________________________________________________
Your phone number: __________________________________________________
Your fax number: __________________________________________________
2. Journal title (vol, iss, pp): __________________________________________________________________
3. Article title: __________________________________________________________________
4. Article author(s): __________________________________________________________________
5. How many PDF reprints do you want? __________________________________
6. Please refer to the pricing chart below to calculate the cost of your order.
Number of PDFreprints
Cost(in U.S. dollars)
50 $200100 $275150 $325200 $350
NOTE: Prices shown apply only to orders submitted by individual article authors or editors. Commercial orders must bedirected to the Publisher.
All orders must be prepaid. Payments must be made in one of the following forms: a check drawn on a U.S. bank an international money order Visa, MasterCard, or American Express (no other credit cards can be accepted)
PAYMENT (type or print carefully):
Amount of check enclosed: _________________ (payable to Aries Systems Corporation)
VISA __________________________________
MasterCard __________________________________
American Express __________________________________
Expiration date: _________________ Signature: _________________________________
Your PDF reprint file will be sent to the above e-mail address. If you have any questions about your order, or if you needtechnical support, please contact: [email protected]
For subscriptions and to see all of our other products and services, visit the Springer website at:http://www.springeronline.com
Print and send this form with paymentinformation to:
Aries Systems Corporation200 Sutton StreetNorth Andover, Massachusetts 01845Attn.: Electronic Reprints
OR Fax this to Aries at: 978-975-3811
http://www.springeronline.com/http://www.springeronline.com/8/6/2019 Bio EZ Cangahuala Et Al 09
3/18
Metadata of the article that will be visualized in OnlineFirst
Please note: Images will appear in color online but will be printed in black and white.
ArticleTitle Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca
sellowiana(Berg.) Burr.
Article Sub-Title
Article CopyRight Springer Science+Business Media B.V.
(This will be the copyright line in the final PDF)
Journal Name Plant Growth Regulation
Corresponding Author Family Name Guerra
Particle
Given Name Miguel Pedro
Suffix
Division Graduate Program in Plant Genetic Resources, Plant Developmental
Physiology and Genetics Laboratory
Organization Federal University of Santa Catarina
Address 88040-900, Florianpolis, SC, Brazil
Email [email protected]
Author Family Name Cangahuala-Inocente
Particle
Given Name Gabriela Claudia
Suffix
Division Graduate Program in Plant Genetic Resources, Plant Developmental
Physiology and Genetics Laboratory
Organization Federal University of Santa Catarina
Address 88040-900, Florianpolis, SC, Brazil
Email [email protected]
Author Family Name Silveira
Particle
Given Name Vanildo
Suffix
Division Biotechnology Laboratory
Organization CBB, State University of North Fluminense (UENF)
Address Av. Alberto Lamego, 2000, 28013-600, Campos dos Goytacazes, Rio de
Janeiro, Brazil
Email [email protected]
Author Family Name Caprestano
Particle
Given Name Clarissa Alves
Suffix
Division Graduate Program in Plant Genetic Resources, Plant Developmental
Physiology and Genetics Laboratory
Organization Federal University of Santa Catarina
Address 88040-900, Florianpolis, SC, Brazil
8/6/2019 Bio EZ Cangahuala Et Al 09
4/18
Author Family Name Ducroquet
Particle
Given Name Jean Pierre Henry Joseph
Suffix
Division
Organization Epagri, So Joaquim Experimental StationAddress 88600-000, So Joaquim, SC, Brazil
Email [email protected]
Author Family Name Floh
Particle
Given Name E. I. S.
Suffix
Division Laboratory of Plant Cell Biology, Department of Botany
Organization IB-University of So Paulo
Address 05422-970, So Paulo, SP, Brazil
Email [email protected]
Schedule
Received 10 December 2008
Revised
Accepted 9 June 2009
Abstract Acca sellowiana(Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a
domestication and breeding program. Biotechnological tools have been used to assist in this program. The
establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and
fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the
general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis.
Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines
(PAs), IAA and ABA, in different stages ofA. sellowianazygotic embryogenesis. Starch is the predominant
storage compound during zygotic embryo development. Increased synthesis of amino acids in thecotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed
increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages.
IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing
basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results
here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this
species as well as in other woody angiosperms.
Keywords (separated by '-') Embryo development -Feijoa sellowiana - IAA - Polyamine - Starch - Storage protein
Footnote Information
8/6/2019 Bio EZ Cangahuala Et Al 09
5/18
Author Query Form
Please ensure you fill out your response to the queries raised below
and return this form along with your corrections
Dear Author,
During the preparation of your manuscript for typesetting, some questions have arisen. These are listed below. Please check yourtypeset proof carefully and mark any corrections in the margin of the proof or compile them as a separate list. This form shouldthen be returned with your marked proof/list of corrections [email protected]
Disk useIn some instances we may be unable to process the electronic file of your article and/or artwork. In that case we have, for
efficiency reasons, proceeded by using the hard copy of your manuscript. If this is the case the reasons are indicated below:
Disk damaged Incompatible file format LaTeX file for non-LaTeX journal
Virus infected Discrepancies between electronic file and (peer-reviewed, therefore definitive) hard copy
Other: .................................................................................................................................................................................
We have proceeded as follows:
Manuscript scanned Manuscript keyed in Artwork scanned
Files only partly used (parts processed differently: .....)
Bibliography
If discrepancies were noted between the literature list and the text references, the following may apply:
The references listed below were noted in the text but appear to be missing from your literature list. Please complete the list orremove the references from the text.
Uncited references: This section comprises references that occur in the reference list but not in the body of the text.Please position each reference in the text or delete it. Any reference not dealt with will be retained in this section.
Queries and/or remarks
Section/paragraph Details required Authors response
Front matter Please check and confirm the inserted
citation of Fig. 7 is correct. If not, please
suggest an alternate citation.
Body matter Please check section headings are
correctly identified.
Back matter Umbreit et al. (1964) has been changed to
Umbreit and Burris (1960) so that this
citation matches the list.
Bassumer et al. 2007 has been changed to
Bassuner et al. 2007 so that this citation
matches the list.
Please update reference Umbreit and
Burris (1960).
Bassumer et al. 2007 has been changed to
Bassuner et al. 2007 so that this citation
matches the list.
Journal: 10725
Article: 9393
mailto:[email protected]:[email protected]:[email protected]8/6/2019 Bio EZ Cangahuala Et Al 09
6/18
UNCO
RREC
TED PR
OOF
O RI G I N A L P A P E R1
2 Dynamics of biochemical and morphophysiological changes
3 during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.
4 Gabriela Claudia Cangahuala-Inocente Vanildo Silveira
5 Clarissa Alves Caprestano Jean Pierre Henry Joseph Ducroquet
6 E. I. S. Floh Miguel Pedro Guerra
7 Received: 10 December 2008 / Accepted: 9 June 20098 Springer Science+Business Media B.V. 2009
9 Abstract Acca sellowiana (Berg.) Burr. is a native
10 Myrtaceae from southern Brazil and Uruguay, now the11 subject of a domestication and breeding program. Bio-
12 technological tools have been used to assist in this pro-
13 gram. The establishment of a reliable protocol of somatic
14 embryogenesis has been pursued, with a view to capturing
15 and fixing genetic gains. The rationale behind this work
16 relies on the fact that deepening comprehension of the
17 general metabolism of zygotic embryogenesis may cer-
18 tainly improve the protocol for somatic embryogenesis.
19 Thus, in the present work we studied the accumulation of
20 protein, total sugars, starch, amino acids, polyamines
21 (PAs), IAA and ABA, in different stages of A. sellowiana
22zygotic embryogenesis. Starch is the predominant storage
23compound during zygotic embryo development. Increased24synthesis of amino acids in the cotyledonary stage, mainly
25of asparagine, was observed throughout development.
26Total free PAs showed increased synthesis, whereas total
27conjugated PAs were mainly observed in the early devel-
28opmental stages. IAA decreased and ABA increased with
29the progression from early to late embryogenesis. Besides
30providing basic information on the morphophysiological
31and biochemical changes of zygotic embryogenesis, the
32results here obtained may provide adequate strategies
33towards the modulation of somatic embryogenesis in this
34species as well as in other woody angiosperms.
35
36Keywords Embryo development Feijoa sellowiana
37IAA Polyamine Starch Storage protein
38
39Introduction
40Acca sellowiana (Berg.) Burr. is a native fruit species from
41southern Brazil and northern Uruguay. In New Zealand,
42Australia, the USA and even certain countries in Europe, it
43has been commercially cultivated since the beginning of
44the twentieth century (Thorp and Bieleski 2002). Recently,
45efforts have been made in Brazil to launch a domestication
46program with this species.
47Conventional techniques for the vegetative propagation
48of A. sellowiana based on cuttings and grafting, are diffi-
49cult due to the negative effects of phenolic compounds (Dal
50Vesco and Guerra 2001). Micropropagation techniques
51have been employed to overcome such problems, with
52studies being focused on the establishment of reliable
53protocols for somatic embryogenesis (Canhoto and Cruz
541996; Dal Vesco and Guerra 2001; Stefanello et al. 2005;
A1 G. C. Cangahuala-Inocente C. A. Caprestano
A2 M. P. Guerra (&)
A3 Graduate Program in Plant Genetic Resources,
A4 Plant Developmental Physiology and Genetics Laboratory,
A5 Federal University of Santa Catarina, Florianopolis,
A6 SC 88040-900, Brazil
A7 e-mail: [email protected]
A8 G. C. Cangahuala-Inocente
A9 e-mail: [email protected]
A10 V. Silveira
A11 Biotechnology Laboratory, CBB, State University of North
A12 Fluminense (UENF), Av. Alberto Lamego, 2000, Campos dosA13 Goytacazes, Rio de Janeiro 28013-600, Brazil
A14 e-mail: [email protected]
A15 J. P. H. J. Ducroquet
A16 Epagri, Sao Joaquim Experimental Station, Sao Joaquim, SC
A17 88600-000, Brazil
A18 e-mail: [email protected]
A19 E. I. S. Floh
A20 Laboratory of Plant Cell Biology, Department of Botany,
A21 IB-University of Sao Paulo, Sao Paulo, SP 05422-970, Brazil
A22 e-mail: [email protected]
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
Plant Growth Regul
DOI 10.1007/s10725-009-9393-9
8/6/2019 Bio EZ Cangahuala Et Al 09
7/18
UNCO
RREC
TED PR
OOF
55 Cangahuala-Inocente et al. 2007). One of the main limi-
56 tations of these protocols is associated with the low rate of
57 somatic embryo conversion to plantlets. These constraints
58 are mainly a result of the lack of knowledge on the phys-
59 iological and biochemical changes that occur during
60 development and maturation of the zygotic embryo.
61 Zygotic embryogenesis is a complex and highly orga-
62 nized process that plays a central role in the life cycle of63 higher plants. It can usually be divided into two main steps,
64 namely, (1) an initial morphogenetic phase which is char-
65 acterized by cell division and the onset of cell differenti-
66 ation, followed by (2) a maturation phase associated with
67 the accumulation of major storage products and preparation
68 for seed desiccation, dormancy and germination (Mord-
69 horst et al. 1997; Sallandrouze et al. 2002).
70 Both the synthesis and accumulation of storage com-
71 pounds play a central role in zygotic embryogenesis
72 (Merkle et al. 1995). Storage proteins are the source of
73 amino acids for seed germination (Misra et al. 1993). A
74 special class of storage compounds, the LEA proteins, play75 an important role in seed dehydration (Wise and Tunnac-
76 liffe 2004). Apart from proteins, carbon is stored in lipids
77 and starch. The levels of protein, lipids, and starch vary
78 between angiosperms species (Dam et al. 2009). Soluble
79 sugars, such as glucose and sucrose, are involved with the
80 regulation of developmental processes occurring from
81 embryo development to seed maturation (Gibson 2005).
82 Amino acids are important in nitrogen metabolism and
83 protein synthesis, as well as in the transition from hetero-
84 trophy to autotrophy (Ortiz-Lopez et al. 2000).
85 The polyamines, aliphatic amines with a positive charge
86 in neutral pH, play a basic role in cell proliferation and
87 differentiation (Bouchereau et al. 1999; Puga-Hermida
88 et al. 2003; Baron and Stasolla 2008), and in protein syn-
89 thesis and responses to water stress in plants (Bais and
90 Havishankar 2002; Kusano et al. 2007). Taken together,
91 these substances are reliable biochemical markers of
92 zygotic and somatic embryo quality.
93 From a physiological point of view, auxins are involved
94 in cell division and expansion, and differentiation of the
95 vascular system (Liu et al. 1993). These hormones are also
96 associated with regulation of the embryonic patterns of
97 histodifferentation (Fischer-Iglesias and Nauhaus 2001;
98 Bassuner et al. 2007). ABA is another hormone which plays
99 a major role in the embryo, by preventing precocious ger-
100 mination (Kermode 1995) and promoting the accumulation
101 of storage compounds (Cailloux et al. 1996), and thus,
102 embryo maturation (Black1991). In somatic embryogenesis
103 ABA reduces the frequency of embryo malformation (Eti-
104 enne et al. 1993) and the occurrence of secondary or
105 repetitive embryogenesis (Nuutila et al. 1991).
106 The present work aimed to study the dynamics of biochemi-
107 cal and physiological changes during the development
108ofA. sellowiana zygotic embryos, such as the levels of total
109proteins, amino acids, carbohydrates, polyamines, IAA and
110ABA. The accumulation patterns of certain of these sub-
111stances in specific stages of seed development were also
112assayed.
113Materials and methods
114Plant material
115Biological material was compiled together with the A. sel-
116lowiana germplasm collection at the Epagri Sao Joaquim
117Experimental Station (28180 Latitude S, 49560 Longitude
118W), Santa Catarina, in the south of Brazil, from November
1192004 to March 2005. About 3,800 flowers were emascu-
120lated followed by manual pollination. Ovules from non-
121pollinated flowers were considered as the zero-time limit.
122Fruits were collected after 21 and 30 days, then every
12315 days until reaching fruit maturation which occurred124after 120 days (Fig. 1). The fruits were transported in
125plastic boxes with dry ice. Using a stereoscope (Olympus
126SZH10) the seeds were extracted from the fruits, and stored
127at -20C.
128Histological analysis
129Samples were fixed for 24 h in a 0.2 M phosphate buffer
130(pH 7.3) containing 2.5% paraformaldehyde. After fixation,
131these were dehydrated in a graded ethanol series and
132embedded in historesin (Leica), as described by Arnold
133et al. (1975). 5 lm thick sections were sliced with a rotary
134microtome (Slee Technik Cut 4055) and fixed onto slides
135by heating. The sections were stained with 0.5% toluidine
136blue O (TBO) (C.I. 52040) in a 0.2 M phosphate buffer
137(pH 6.8) for 1 min (OBrien et al. 1965). Photographs were
138taken with a standard Olympus BX 40 microscope and
139Olympus SZH10 stereoscope.
140Soluble proteins
141For each collecting date 300 mg of seeds were extracted.
142The seeds were stored in eppendorff tubes and stored at
143-20C until the processing. Total soluble protein extraction
144was performed according to Cangahuala-Inocente et al.
145(2009). Three repetition of samples (300 mg fresh weight-
146FW each) were macerated at 4C with 1 ml of extraction
147buffer (pH 7.0) containing 50 mM sodium phosphate
148dibasic, 0.2 M b-mercaptoethanol, 17.3 mM sodium dode-
149cyl sulfate (SDS) and 1 mM phenylmethylsulfonyl fluoride
150(PMSF), to then be centrifuged to 4C by 20 min at
1518,000 rpm. The supernatant containing total soluble pro-
152teins was removed and the pellet stored at -20C. Soluble
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
8/18
UNCO
RREC
TED PR
OOF
153 proteins were sedimented at 0C by adding two volumes of
154 100% ethanol into the supernatant and then centrifuging
155 4C by 20 min at 10,000 rpm. The sedimented proteins
156 were solubilized in 50 mM sodium phosphate dibasic (pH
157 7.0). Protein content was determined by the Bradford (1976)
158 method, using bovine serum albumin as standard.
159 Total sugar and starch
160 The extraction of total soluble sugars was performed161 according to Shannon (1968). The pellet from the protein
162 extraction was macerated using 2 ml methanolchloro-
163 formwater (MCW) (12:5:3), centrifuged for 10 min at
164 2,000 rpm. The supernatant was recovered and the pellet
165 was re-extracted using 2 ml MCW. One part chloroform and
166 1.5 part water were added for each four parts of the super-
167 natant, followed by centrifuging for 10 min at 2,000 rpm,
168 from which two phases were obtained. The upper aqueous
169 phase was removed for dosage using anthrone at 0.2%, in
170 accordance with Umbreit and Burris (1960).
171 The extraction and determination of starch levels were
172 based on the procedures of McCready et al. (1950). The
173 pellets used in the total soluble sugar extraction were
174 ground with 1 ml of 30% perchloric acid and centrifuged
175 for 15 min at 10,000 rpm. The supernatant containing
176 starch was removed and the pellets were re-extracted
177 twofold. The supernatants were combined and the pellets
178 eliminated. For dosage was using anthrone at 0.2%. The
179 sugar and starch concentrations were calculated using
180 glucose as standard. The absorbance was read in UVVIS
181 UV-1203 spectrophotometer (Shimadzu) at 620 nm.
182Amino acid
183Amino acid determination was carried out according to
184Santa-Catarina et al. (2006). Three biological samples
185(200 mg FW) of each developmental stage were ground,
186individually, in 6 ml of 80% (v/v) ethanol and evaporated
187in a speed vac centrifuge concentrator (ThermoSavant,
188Milford, USA). Samples were re-suspended in 2 ml of
189MilliQ type water and centrifuged at 20,000g for 10 min.
190The supernatant was filtered through a 20 lm membrane.191Amino acids were derivatizated with o-phthaldialdehyde
192(OPA) and identified by HPLC using a C-18 reverse phase
193column (SUPELCOSILTM, 5 lm particle size, L9 I.D.
19425 cm 9 4.6 mm). The gradient was developed by mixing
195increasing proportions of 65% methanol with a buffer
196solution (50 mM sodium acetate, 50 mM sodium phos-
197phate, 20 ml l-1 methanol, 20 ml l-1 tetrahydrofuran and
198pH 8.1 adjusted with acetic acid). The gradient of 65%
199methanol was programmed to 20% over the first 32 min,
200from 20 to 100% between 32 and 71 min, and 100%
201between 71 and 80 min, at a 1 ml min-1 flow and 40C.
202Fluorescence excitation and emission wavelengths of 250
203and 480 nm, respectively, were used for amino acid
204detection. Peak areas and retention times were measured by
205comparison with known quantities of standard amino acids.
206Polyamines
207Putrecine (Put), Spermidine (Spd) and Spermine (Spm)
208were defined according to Silveira et al. (2004a). Three
209biological samples (300 mg FW) of each developmental
Fig. 1 Development of the seed
of A. sellowiana after
pollination directed. 0 DAP:
floral button at the stage
balloon, 21 DAP: formation of
the zygotic embryo with
successive cell divisions
causing a pro-embryo, 30 DAP:
zygotic embryo at the globular
stage, 45 DAP: zygotic embryo
at the heart stage, 60 DAP:
zygotic embryo at the torpedo
stage with endosperm still
liquid, 75 DAP: zygotic embryo
at the cotyledonary stage with
presence of endosperm, 90, 105
and 120 DAP: zygotic embryo
at the cotyledonary stage
without presence of endosperm.
Bar= 1 mm
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
9/18
UNCO
RREC
TED PR
OOF
210 stage were ground, individually, in 3 ml of 5% (v/v) per-
211 chloric acid. After 1 h, extracted samples were centrifuged
212 for 20 min at 15,000g and 0C. In each case, the super-
213 natant containing free polyamines was removed and the
214 pellets re-extracted. Supernatants were then combined and
215 the pellets eliminated. Free polyamines were determined
216 directly from the supernatant. Conjugated polyamines were
217 extracted by hydrolyzing 200 ll of supernatant with 200 ll218 of 12 N HCl for 18 h at 110C. The samples were dried
219 under nitrogen. The conjugated polyamines were solubi-
220 lized in 200 ll of 5% Perchloric acid.
221 Free and conjugated polyamines were derivatizated by
222 dansyl chloride made up in acetone at a concentration of
223 5 mg ml-1. A 40 ll aliquot of the sample was added to
224 100 ll of dansyl chloride, 20 ll of 0.05 mM diaminohep-
225 tane (internal standard) and 50 ll of saturated sodium
226 carbonate. In sequence, the samples were incubated in the
227 dark for 50 min at 70C. The toluene phase was collected
228 and dried under nitrogen. Finally, dansylated polyamines
229 were solubilized in 200 ll of acetonitrile.230 Twenty ll of the dansylated polyamines were separated
231 by reverse phase HPLC in a C-18 reverse phase column
232 (Shimadzu SHIM-PACK CLC-ODS, 5 lm particle size,
233 L9 I.D. 25 cm 9 4.6 mm). The gradient was developed
234 by mixing increasing proportions of absolute acetonitrile to
235 10% acetonitrile in water (pH 3.5). The gradient of abso-
236 lute acetonitrile was programmed at 65% over the first
237 10 min, from 65 to 100% between 10 and 13 min and at
238 100% between 13 and 21 min. The flow was 1 ml min at
239 40C. The fluorescence detector was set at 340 nm (exci-
240 tation) and 510 nm (emission). A mixture of putrescine
241 (Put), spermidine (Spd) and spermine (Spm) was used as
242 standard.
243 IAA and ABA determination
244 IAA and ABA contents were determined according to
245 Santa-Catarina et al. (2006). Three biological samples
246 (1,000 mg FW) of each developmental stage were ground,
247 individually, in a 5 ml extraction buffer (80% ethanol ?
248 1% polyvinylpyrrolidone-40). [3H]IAA and [3H]ABA were
249 added to the samples as internal radioactive standards. After
250 90 min of incubation, samples were centrifuged during
251 15 min at 15,500g, and 4C. Supernatants were concen-
252 trated in a speed vac centrifuge concentrator (Ther-
253 moSavant, Milford, USA) at 45C, until reaching 20% of
254 the initial volume (1 ml). Volumes were then raised (Mil-
255 liQ water type) to 3 ml and the pH adjusted to 2.5 using
256 HCl (1 N). The samples were partitioned twice by using
257 ethyl ether as an organic solvent. Organic layers were
258 combined and the aqueous residue eliminated by freezing
259 the base of the tubes during 5 s in liquid nitrogen, with
260 transference of the organic phase to a clean tube. The
261organic phase containing IAA and ABA was dried in a
262speed vac at 45C, dissolved in 200 ll of 100% methanol,
263transferred to micro tubes and after stored at -80C until
264analysis. Aliquots (40 ll) of stored samples were analyzed
265by reverse phase HPLC, by using a C-18 reverse phase
266column (Shimadzu SHIM-PACK CLC-ODS, 5 lm particle
267size, L9 I.D. 25 cm 9 4.6 mm). The gradient was devel-
268oped by mixing increasing proportions of absolute methanol269to 10% methanol plus 0.5% acetic acid in water. The IAA
270content was defined by using a fluorescence detector at
271280 nm (excitation) and 350 nm (emission) and the ABA
272content by means of a UVVIS detector at 254 nm. A
273mixture of IAA and ABA was used as a standard. Fractions
274containing IAA and ABA were collected and analyzed in
275the Packard Tri-Carb liquid scintillation counter to estimate
276losses and data validation.
277Data analysis
278Data were analyzed by ANOVA (P\ 0.05) followed by279the Student-Newman-Keuls (SNK) test, using Statistica
280version 7.0 software. In the event of ANOVA and SNK
281tests not being used, the mean of three replicates and
282standard error were then applied to analyze data.
283Results and discussion
284Formation of zygotic embryos
285 A. sellowiana ovules are anatropous, developing asyn-
286chronously (Fig. 2a) with axillary placenta. The nucellus
287presents layers of cells with prominent nuclei (Fig. 2b).
288Members of the order Myrtales, which includes the Myrt-
289aceae, have a crassinucellate ovule, the micropyle being
290formed by external and internal teguments, with two layers
291of cells, ephemeral or absent antipodal cells and a nuclear-
292type endosperm (Tobe and Raven 1983).
293Twenty one days after pollination (DAP), the first cel-
294lular division, leading to the formation of the pro-embryo
295(Fig. 2c) with embryonal and suspensor cells (Fig. 2d),
296was noted. According to Dodeman et al. (1997), several
297structural alterations occur in the pro-embryo, mostly
298associated with vacuole organization, nucleus location, the
299presence of organelles and cell wall changes. After 30 DAP
300the apical cells of the pro-embryo originated a spherical
301mass of cells, corresponding to the globular stage (Fig. 2e, f).
302The endosperm of the nuclear position was constituted of
303a cell layer with conspicuous nuclei (Fig. 2e). In Arabid-
304opsis thaliana the embryo in the globular stage stems
305from a sequence of divisions of embryonal cells, the
306embryo soon acquiring a heart shape (Mansfield and Bri-
307arty 1991).
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
10/18
UNCO
RREC
TED PR
OOF
308 Forty five DAP, histological sections at heart stage
309 embryos showed a well-defined proto-dermal layer
310 (Fig. 3a, b). Sixty days after pollination, the embryo was in
311 the torpedo-stage with an extended apicalbasal axis
312 (data not shown). According to Goldberg et al. (1994),
313 dramatic changes occur in the transition from globular to
314 torpedo-stage embryos. Among other features, cotyledons
315 arise from two lateral domains, this resulting in bilateral
316 symmetry.
317 The early cotyledonary stage was observed at 75 DAP
318 (Fig. 3c) showing rudimentary cotyledonary leaves sur-
319 rounded by the endosperm. At 90 DAP, the endosperm
320 present in seed had almost been consumed (Fig. 3d). In
321 addition, at this stage cells in the apical (Fig. 3e) and basal
322 (Fig. 3f) meristems were observed at the end of the
323 hypocotylsradicle axis. Starting from 105 to 120 days,
324 fruit development was concluded with maturation. Con-
325 comitantly, embryos showed a thickened cotyledons with
326 storage compounds (see Fig. 1).
327Protein and carbohydrates
328Total protein levels during embryo development ranged
329from 0.24 to 0.39 mg g FW-1 (Fig. 4). Storage proteins
330provide free amino acids to be used for early embryonal
331stages until reaching plantlet autotrophy (Prewein et al.
3322006). During zygotic embryogenesis of Ocotea catharin-
333ensis, proteins were only detected at high concentrations in
334the late developmental stages, peaking at the mature
335embryo stage (Santa-Catarina et al. 2006). In Araucaria
336angustifolia zygotic embryogenesis, the embryonic axis
337protein content increased until the cotyledonary stage, with
338further stabilization in the mature seed (Silveira et al. 2008).
339Total soluble sugars ranged from 1.45 to 2.23 mg g
340FW-1 during zygotic embryo development (Fig. 4). Starch
341content remained quite stable during embryo development,
342the highest value (24.5 mg g FW-1) being observed at 21
343DAP (Fig. 4). This phase is coincident with fertilization and
344zygote formation. Similar results were reported by Pescador
Fig. 2 Histological sections of
the process of zygotic
embryogenesis ofA. sellowiana.
a Ovule of the immature fruit 0
DAP, stained with AT-O, b the
opening of the micropyle detail
and the presence of the
teguments protection,
c formation of the pro-embryo
in the immature fruit, d cells in
the suspensor and pro-embryo,
e immature fruit at 30 DAP,
observing the zygotic embryo in
globular stage, f details of the
zygotic embryo in globular
stage and the presence of the
outer tegument of protection.
Bar= 10 lM. Mi micropyle,
teg tegument, ca chalaza, nu
nucellus, fu funicular, ex-teg
outer tegument, in-teg internal
tegument, en endosperm, ze
zygotic embryo, sus suspensor,
pro-en pro-embryo, glo globular
stage
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
11/18
UNCO
RREC
TED PR
OOF
345 et al. (2008) with this same species. Starch and sugar con-
346 tents play a major role in seed development through the
347 supply of compounds to be consumed during transition
348 from seed heterotrophy to plantlet autotrophy (Merkle et al.
349 1995). In Quercus robur, starch accumulated gradually in
350 the late embryonal developmental stages (Prewein et al.
351 2006). Studies using an Arabidopsis mutant showed the role
352of sugars in the regulation of genetic expression, prolifer-
353ation and cell death, in seedling-growth, leaf expansion and
354senescence, and in seed development (Smeekens 2000;
355Gibson 2005; Rolland et al. 2006).
356Histochemical analysis of mature A. sellowiana zygotic
357embryos (120 DAP) revealed a low starch-content and a
358high content of protein and lipidic bodies in cotyledon cells
Fig. 3 Histological sections of
the process of zygotic
embryogenesis ofA. sellowiana.
a Immature fruit at 45 DAP
looking up the zygotic embryo
in stage cordiforme, b zygotic
embryo in detail the heart stage
and the presence of protoderm,
c longitudinal section of the
zygotic embryo at the initial
cotyledonary stage to 75 DAP,
d longitudinal section the
zygotic embryo at the
cotyledonary stage at 90 DAP,
e details of the root meristem,
f detail of the apical meristem.
Note that the cells of the leaves
cotyledonary are somewhat
thickened. Bar= 10 lM. ex-teg
outer tegument, he heart stage,
protoderprotoderm, cot
cotyledon, procam procambium,
ap-me apical meristem, ra-me
radicle meristem, cap root cap
cbcbcbcabcabcabc ab
a
a
aaab
bcab
bcdcddcd
cd
cddcdcd
bc
b
a
0,0
0,5
1,0
1,5
2,0
2,5
3,0
0 21 30 45 60 75 90 105 120
Days after pollination
Concentration(mg/gFW)
0
5
10
15
20
25
30
Concentration(mg/gFW)
Protein Sugar StarchFig. 4 Mean concentration of
total protein, starch and totalsugars (mg/g) of fresh weight
(FW) for the formation of the
zygotic embryo ofA. sellowiana
(n = 3). Values followed by
different letters, inside of
parameters indicating
significant differences
according to the SNK test
(P\ 0.05)
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
12/18
UNCO
RREC
TED PR
OOF
359 (data not shown). It is commonly accepted that the main
360 storage compounds during seed development are proteins,
361 carbohydrates and lipids, and that the ratio of this com-
362 position is different in each species (Bewley and Black
363 1994). Thus, Caesalpinia peltophoroides seed cotyledons
364 presented about 50% of lipids, 32% of soluble carbohy-
365 drates, 7.7% of starch and 6.8% of soluble proteins (Corte
366 et al. 2006). On the other hand, the predominant reserve367 was starch in seed cotyledons of Anthyrium andraeaman,
368 whereas endosperm reserves were mainly constituted of
369 starch and proteins (Matsumoto et al. 1998).
370 Amino acids
371 Amino acids are important primary assimilation products
372 in nitrogen metabolism (Ortiz-Lopez et al. 2000). In
373 addition to the synthesis of proteins, amino acids directly
374 or indirectly control various aspects of plant growth and
375 development (Coruzzi and Last 2000).
376 In the present work, amino acid content was enhanced377 after 45 days from the pollination period, when the embryos
378 had already reached the heart stage (Table 1). At this stage,
379 amino acids may be used for the synthesis of specific pro-
380 teins associated with histodifferentiation, since this is the
381 moment when the embryo begins to establish its radial
382 symmetry, vascularization and formation of the cotyledons.
383 The total amino acid content peaked at 75 DAP
384 (Table 1), a period in which the zygotic embryo was in the
385 cotyledonary stage. These results confirm those obtained
386 during embryonic development of A. angustifolia (Astarita
387 et al. 2003a), Pinus taeda (Silveira et al. 2004b) and
388 O. catharinensis (Santa-Catarina et al. 2006).
389 A decrease in amino acid levels was observed from 90 to
390 105 DAP (Table 1). This could be ascribed to their con-
391 sumption for the synthesis of various, mainly reserve,
392 proteins. According to Weber et al. (1997) the transport of
393 amino acid to the cotyledons can be initially passive,
394 although an additional system of active absorption can be
395 established in late developmental stages, when large
396 amounts of proteins are stored to ensure seedling devel-
397 opment. A decrease in total amino acid levels in the mature
398 embryo was also observed in O. catharinensis (Santa-
399 Catarina et al. 2006).
400 Asparagine was the prevalent amino acid, with a peak
401 (Table 1) in the late embryonal stages (7590 DAP), when
402 the cotyledons were fully developed, fully suggesting its
403 involvement in the mobilization of embryo reserves. This
404 result confirms that reported by Feirer (1995) and Santa-
405 Catarina et al. (2006) during seed development of
406 P. strobus and. O. catharinensis, respectively. Asparagine
407 plays a central role in nitrogen storage and transport in
408 plants, which is further facilitated by its biochemical
409 properties (Lea et al. 2007).
410Glutamine, aspartic acid, c-aminobutyric acid (Gaba)
411and alanine are accumulated at lower levels than aspara-
412gine (Table 1). Glutamines and glutamic acid are precur-
413sors of other amino acids (Radwanski and Last 1995). In
414addition, exogenous glutamine is commonly supplemented
415to the basal culture medium in several protocols of somatic
416embryogenesis, including A. sellowiana (Dal Vesco and
417Guerra 2001). In the present work alanine showed high418levels at 45, 75 e 90 DAP, and the IAA levels showed an
419inverse proportion comparatively to alanine. This suggests
420that synthesis and degradation of this amino acid could be
421associated with the IAA synthesis when this auxin is higly
422required. The L-alanine and e-lysine conjugates were also
423found to be useful for induction and development of
424Oenothera leaf callus and in tomato cell-suspension cul-
425ture, two systems which require highly active sources of
426auxin (Magnus et al. 1992).
427Unlike the rest, Gaba is a non-proteic amino acid which
428results from glutamic acid decarboxylation (Satya-Naraian
429and Nair 1990), normally being accumulated in response to4302,4-D, or under stress conditions (Snedden and Fromm
4311998). In the present work, Gaba was the fourth largest
432amino acid accumulated, mostly from the 75 to 90 DAP,
433decreasing thereafter (Table 1). The presence of these
434amino acids at such levels suggests its role in A. sellowiana
435embryogenesis. This amino acid was also accumulated in
436the early somatic embryogenesis of carrots as shown by
437Kamada and Harada (1984).
438Polyamines (PAs)
439The total PAs level was low at zero-time, increasing
440significantly up to 21 DAP. In subsequent stages (30, 45 and
44160 DAP), increased levels of conjugated PAs were
442observed, these progressively decreasing until embryos
443reached maturity (Fig. 5a). Conversely, total free PA levels
444increased according to progression to further developmental
445stages, peaking by 105 DAP (Fig. 5a). This variation was
446different from that observed in O. catharinensis where the
447levels of free PAs were higher than those of conjugated PAs,
448both types of PAs reaching high levels in mature zygotic
449embryos (Santa-Catarina et al. 2006). The metabolic role of
450conjugated PAs has not yet been fully elucidated. Bais and
451Havishankar (2002) suggested that free PA levels in plants
452can be regulated by the formation of reversible conjugated
453PAs. It has also been suggested that a combination of PAs
454with cinnamic acid and phenols may regulate the pool of
455free PAs in plant cells (Mader and Hanke 1997).
456The PA ratio [Put.(Spd ? Spm)-1] was low both at
457zero-time and 21 DAP (Fig. 5b), corresponding, respec-
458tively, to fertilization and formation of the zygotic embryo.
459Geoffriau et al. (2006) showed the involvement of PAs in
460the gynogenesis of Allium cepa, where the Put/Spd-Spm
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
13/18
UNCO
RREC
TED PR
OOF
Table1
Aminoacidscontent(lg
g-
1
FW)inzygoticembryosduringseeddevelopmentinA.sellowiana
Daysafterpo
llination(DAP)anddevelopmentstages
0
21
30
45
60
75
90
105
120
Globular
Heart
Torpedo
Earlycotyle
donary
Cotyledonary
Maturecotyledonary
Asparticacid
22.5
0.7
24.1
8.2
27.5
7.8
75.5
6.4
19.9
4.3
95.2
6
.0
88.6
11.4
30.8
3.3
54.5
5.3
Glutamicacid
50.2
2.7
17.3
6.0
39.3
6.0
121.0
2.8
54.9
13.2
41.1
1
3.5
26.9
4.2
9.4
0.1
9.3
0.8
Asparagine
123.2
3.8
141.8
43.5
110.4
30.8
417.8
33.0
293.9
87.4
982.2
1
37.2
629.5
96.4
126.7
4.7
157.2
3.5
Serine
32.3
0.5
23.7
7.5
27.7
5.6
83.3
5.3
45.3
11.2
131.6
9
.9
81.8
8.9
21.6
3.5
33.8
7.3
Glutamine
34.3
1.9
16.9
5.5
35.7
12.0
357.7
79.3
229.5
44.8
708.7
2
7.2
481.8
77.4
78.2
8.4
62.2
8.6
Histidine
3.5
0.1
3.6
0.4
5.5
1.3
24.9
3.6
25.3
7.0
91.5
1
1.0
83.1
13.0
27.7
3.7
53.2
4.2
Glycine
2.5
0.1
3.7
0.9
4.8
0.3
12.7
2.0
5.9
1.3
16.0
2
.2
18.9
5.3
2.4
0.2
10.2
1.9
Arginine
14.9
1.4
27.8
7.1
14.5
2.4
13.9
3.0
5.3
1.4
123.9
1
5.2
324.9
54.1
28.7
5.0
22.8
3.3
Threonine
10.5
0.1
5.9
2.2
11.1
2.2
33.1
1.3
15.1
4.6
48.9
3
.3
45.1
6.1
6.7
1.0
19.0
4.4
Alanine
21.0
0.1
27.8
8.0
30.4
10.6
94.8
11.9
40.5
6.6
167.0
2
1.1
128.4
-16.2
11.3
1.6
21.7
2.8
c-Aminobutiricacid
20.4
0.1
48.5
16.1
33.1
8.9
112.2
8.2
35.6
7.9
173.8
6
.9
206.5
17.6
44.0
3.9
43.5
-6.2
Tyrosine
2.5
.5
4.8
1.4
5.4
0.7
23.8
2.5
11.3
1.8
26.3
1
.0
16.0
1.2
4.1
0.7
12.3
2.9
Tryptophan
0.7
0.1
2.6
0.9
1.2
0.2
3.6
0.4
2.0
0.5
7.2
0
.4
5.4
0.6
1.5
0.2
3.8
0.4
Methionine
0.0
0.0
0.2
0.0
0.5
0.1
1.4
0.2
12.2
0
.7
9.2
0.8
2.4
0.1
6.7
1.1
Valine
3.0
0.0
3.1
0.3
3.0
0.3
14.7
2.4
7.2
2.1
32.9
3
.1
26.0
3.2
5.7
0.4
16.4
3.0
Phenylalanine
1.1
0.1
4.0
0.3
4.2
0.3
16.3
2.8
6.1
1.7
25.3
1
.4
20.1
2.4
5.9
0.7
11.7
2.2
Isoleucine
1.7
0.2
2.3
0.4
2.5
0.4
11.7
2.1
4.8
-1.5
23.5
2
.2
26.2
2.9
6.0
0.7
11.8
2.6
Leucine
3.0
0.2
5.1
0.5
5.9
0.5
26.6
4.5
10.8
3.3
53.7
5
.2
39.0
5.1
11.0
0.5
21.1
4.4
Ornithine
2.3
0.1
1.4
0.0
1.7
0.0
2.2
0.5
1.8
0.1
2.0
-
0.2
3.2
0.2
1.7
0.2
1.9
0.0
Lysine
3.6
0.3
3.6
0.9
5.9
0.9
18.0
2.6
6.7
0.8
28.9
6
.0
25.2
2.0
7.3
1.4
19.1
-5.2
Totalaminoacid
353.0
2.3E
368.1
71.7E
369.7
91.4E
1,464.2
174.4C
823.4
196.3D
2,791.8
1
34.4A
2,285.8
329.0B
433.0
40.5E
592.0
70.1DE
Dataarepresentedasmean
stan
darddeviation(n=
3).Valuesfollowedbydifferentletters,insideofparametersindicatingsignificantdifferencesaccordingto
theSNKtest(P\
0.05)
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
14/18
UNCO
RREC
TED PR
OOF
461 ratio was low during flowering. In the present work, the
462 proportion of PAs [Put.(Spd ? Spm)-1] was high during463 embryo development (30, 45 and 60 DAP), corresponding,
464 respectively, to the globular, heart and torpedo stages, the
465 highest being detected at the heart stage then decreasing
466 until the cotyledonary stage (Fig. 5b). These results are in
467 agreement with those reported in A. angustifolia (Astarita
468 et al. 2003b), P. taeda (Silveira et al. 2004b), and
469 O. catharinensis (Santa-Catarina et al. 2006), Thus, the PA
470 ratio could be considered as a reliable marker of embryonal
471 development in A. sellowiana.
472In the present work, at zero-time and at the time of
473fertilization and formation of the zygotic embryo (DAP47421), free Spd was predominant as compared to other PAs
475(Fig. 5c). It has been shown that high Spd levels are
476associated with induction and floral development (Aribaud
477and Martin-Tanguy 1994). Spd was the most abundant free
478PA in Brassica rapa, followed by Put and Spm (Puga-
479Hermida et al. 2003). These authors also pointed out
480that free PAs content did not change when the flower was
481still closed, changing the profile only after synthesis of
482carotenoids in petals.
a
a a
b b
b
b
bc
c
aa a
a
b
b
b
b
b
0
20
40
60
80
100
120
0 21 30 45 60 75 90 105 120
g/g
FW
free total PAs conjugated total PAs
a
b bb
bc
c
ddd
0,0
0,3
0,6
0,9
0 21 30 45 60 75 90 105 120
Put/(Spd+S
pm)
aa abab
bc bccd
dd
a
bbcbcd bcd
cdecde
de e
a
bbc bccd
dee e e
b
a
aa
b b b
bc
c
0
20
40
60
80
100
120
0 21 30 45 60 75 90 105 120
Days after pollination
Concentration(g
/gFW)
PUT SPD SPM Total
a
b
c
Fig. 5 The mean concentration
a total polyamines (lg/g) of
fresh weight (FW), b ratio of
PAs: Put (Spd ? Spm)-1 and
c free PAs (lg/g) of fresh
weight (FW) during the
development and maturation of
the zygotic embryo of
A. sellowiana (n = 3). Values
followed by different letters,
inside of parameters indicating
significant differences
according to the SNK test
(P\ 0.05)
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
15/18
UNCO
RREC
TED PR
OOF
483 In the present work, similar patterns of variation
484 between free PAs, mainly for Put at all stages, were noted
485 in subsequent developmental stages (Fig. 5c). This could
486 be attributed to Put conversion to Spd and Spm by means
487 of Spd and Spm synthases, as suggested by Puga-Hermida
488 et al. (2003). At 60 DAP the ratio was quite the same,
489 although levels were lower, ranging further (105 and 120
490 DAP) for Spd (Fig. 5c).491 It is important to note that arginine and ornithine are
492 direct precursors, whereas methionine is an indirect pre-
493 cursor, of PAs biosynthetic routes in plants (Antognoni
494 et al. 1998; Bouchereau et al. 1999). In the present study,
495 these amino acids were those that presented the highest
496 levels at 75 and 90 DAP, the later corresponding to the
497 cotyledonary stage. Accordingly, the accumulation of these
498 amino acids as detected in this developmental stage could
499 be due to the synthesis of PAs, as revealed by analysis.
500 These PAs could be important during maturation of zygotic
501 embryos.
502 IAA and ABA
503 The role of IAA in zygotic embryogenesis is well-known
504 (Ribnicky et al. 2002; Bassuner et al. 2007), and is mainly
505 associated with cell division and elongation, and differen-
506 tiation of the vascular system (Gaspar et al. 1996). Many of
507 its effects are dependent on transport across tissues and
508 organs (Fischer-Iglesias et al. 2001; Friml et al. 2003).
509 In the present work, IAA and ABA levels between the
510 heart and cotyledonary stages were inversely proportional
511 (Fig. 6). Dynamic changes in these hormones could be
512 associated with changes in the patterns of histodifferenti-
513 ation and the establishment of embryonal radial and axial
514 symmetry, as suggested by (Michalczuck et al. 1992). The
515 dynamics of variation at IAA levels follows the same
516 pattern observed in the development of seeds, in which
517 high IAA levels occur during embryo development, to then
518decrease in mature seeds (Bewley and Black 1994). In the
519present work, the highest IAA levels were observed in the
520torpedo stage (60 DAP), followed by a continuous decrease
521(Fig. 6). The same pattern of IAA accumulation was
522observed in seeds of O. catharinensis (Santa-Catarina et al.
5232006). High IAA levels were found in Quercus roburat the
524heart stage, with a decrease in more advanced develop-
525mental stages (Prewein et al. 2006). In A. angustifolia526seeds, high IAA levels occurred in tissues of the embryonic
527axis and in the early stages of development (Astarita et al.
5282003c).
529In P. glauca and P. taeda the high levels of IAA
530observed during initial seed development were linked with
531growth of the zygotic embryo (Kong et al. 1997; Silveira
532et al. 2004b). In orthodox seeds, as in the case of A. sel-
533lowiana, reduction in hormone levels in mature seeds is a
534common feature caused by the high degree of seed dehy-
535dration. These hormones can be conjugated for further
536release during seed germination (Kong et al. 1997).
537Tryptophan is considered the main precursor of IAA538biosynthesis (Bandurski et al. 1995). The biosynthesis of
539tryptophan is important for establishing embryo polarity in
540early stages of development (Astarita et al. 2003c). In the
541present work, a decrease in tryptophan levels in the torpedo
542stage (60 DAP) was observed (Table 1), this occurring
543concomitantly with the IAA peak, thus suggesting that
544tryptophan was consumed in IAA biosynthesis.
545ABA also plays a major role in embryonal development.
546Among other effects ABA prevents early germination
547(Kermode 1995). In the present study, the highest levels of
548ABA (Fig. 6) were observed at the cotyledonary stage
549(90, 105 and 120 DAP). The patterns of ABA accumulation
550in the present work are common in most angiosperms,
551which show an increase in ABA content during seed
552development and a decline in the mature seed (Bewley and
553Black1994). In embryos of Quercus robur, ABA peaked at
554the cotyledonary stage and then decreased progressively.
555Concomitant with ABA, the water content declined as the
556embryo reached the maximum size (Prewein et al. 2006).
557Rock and Quatrano (1995) showed that at the end of barley
558zygotic embryogenesis, a decrease in IAA levels and an
559increase in ABA levels was observed. This ran parallel
560with the accumulation of storage compounds. In the pres-
561ent case, similar features were observed regarding the
562dynamics of IAA and ABA accumulation.
563Conclusions
564In this study relevant information about the biochemical and
565physiological changes occurring during the formation and
566development of the zygotic embryo of A. sellowiana was
567acquired. Starch is the predominant storage compound
A
A
A
ccc
B B
Bc
b
a
0
4
8
12
16
20
24
45 60 75 90 105 120
Days after pollination
Concentration(g/gFW)
AB A IA A
Fig. 6 Mean concentration of IAA and ABA (lg g-1) in fresh weight
(FW) for the development of the zygotic embryo ofA. sellowiana
(n = 3). Values followed by different letters, inside of parameters
indicating significant differences according to the SNK test (P\
0.05)
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
8/6/2019 Bio EZ Cangahuala Et Al 09
16/18
UNCO
RREC
TED PR
OOF
568 during embryonal development. All amino acids are syn-
569 thesized during embryogenesis with an increased accumu-
570 lation in the heart and cotyledonary stages. Asparagine is
571 the major amino acid observed. Free PAs synthesis during
572 early developmental stages, as well as the accumulation of
573 PAs conjugates in the cotyledonary stage, may be consid-
574 ered as reliable biochemical markers of embryonal devel-
575 opment in A. sellowiana. IAA and ABA levels were
576 inversely proportional between the heart and cotyledonary
577 stages, suggesting their involvement with histodifferentia-
578 tion patterns, mainly the establishment of embryonal sym-579 metries. The information here obtained will help to explain
580 the biochemical and physiological changes that occur dur-
581 ing zygotic embryogenesis and may assist in the develop-
582 ment of improved somatic embryogenesis protocols in this
583 and other woody plant species (Fig. 7).584
585 References
586 Antognoni F, Fornale S, Grimmer C, Komor E, Bagni N (1998) Long-587 distance translocation of polyamines in phloem and xylem of588
Ricinus communis L. plants. Planta 204:520527. doi:589 10.1007/s004250050287590 Aribaud M, Martin-Tanguy J (1994) Polyamine metabolism, floral591 initiation and floral development in chrysanthemum (Chrysan-592 themum morifolium Ramat.). Plant Growth Regul 15:2331. doi:593 10.1007/BF00024673594 Arnold CD, Mitrenga D, Mayresbach H (1975) Gefriertro und595 einbsttung in glycolmethacrylat (GMA)Ergehnisse histochem-596 ischer reaktionen. Acta histochem 14:271277597 Astarita LV, Floh EIS, Handro W (2003a) Free amino acid, protein598 and water content changes associated with seed development599 in Araucaria angustifolia. Biol Plant 47:5359. doi:10.1023/600 A:1027376730521
601Astarita LV, Handro W, Floh EIS (2003b) Changes in polyamines602content associated with zygotic embryogenesis in the Brazilian603pine, Araucaria angustifolia (Bert.) O. Ktze. Rev Bras Bot60426:163168. doi:10.1590/S0100-84042003000200003605Astarita LV, Floh EIS, Handro W (2003c) Changes in IAA,606tryptophan and activity of soluble peroxidases associated with607zygotic embryogenesis in Araucaria angustifolia (Brazilian608pine). Plant Growth Regul 39:113118. doi:10.1023/A:1022542609618945610Bais HP, Havishankar GA (2002) Role of polyamines in the ontogeny611of plants and their biotechnological applications. Plant Cell612Tissue Organ Cult 69:134. doi:10.1023/A:1015064227278613
Bandurski RS, Cohen JD, Slovin JP, Reinecke DM (1995) Hormone 614biosynthesis and metabolism. In: Davies PJ (ed) Plant hormone.615Physiology, biochemistry and molecular biology. Kluwer, Dordr-616echt, pp 3965617Baron K, Stasolla C (2008) The role of polyamines duringin vivo and618in vitro development. In Vitro Cell Dev Biol Plant 44:384395.619doi:10.1007/s11627-008-9176-4620Bassuner BM, Lam R, Lukowitz W, Yeung EC (2007) Auxin and root621initiation in somatic embryos of Arabidopsis. Plant Cell Rep62226:111. doi:10.1007/s00299-006-0207-5623Bewley JD, Black M (1994) Seeds: physiology of development and624germination, 2nd edn. Plenum Press, New York625Black M (1991) Abscisic acid in germination and dormancy. In:626Davies WJ, Jones HJ (eds) Abscisic acid physiology and627biochemistry. Bios Scientific Publishers, Oxford, pp 8198628
Bouchereau A, Aziz A, Larher F, Martin-Tanguy J (1999) Polyamines 629and environmental challenges: recent development. Plant Sci630140:103125. doi:10.1016/S0168-9452(98)00218-0631Bradford MM (1976) A rapid and sensitive method for the632quantification of microgram quantities of protein utilizing the633principle of protein-dye binding. Anal Biochem 72:248254.634doi:10.1016/0003-2697(76)90527-3635Cailloux F, Julien-Guerrier J, Linissier L, Coudret A (1996) Long-636term somatic embryogenesis and maturation of somatic embryos637in Hevea brasiliensis. Plant Sci 120:185196. doi:10.1016/638S0168-9452(96)04491-3639Cangahuala-Inocente GC, Dal Vesco LL, Steinmacher DA, Torres A,640Guerra MP (2007) Improvements in somatic embryogenesis
Fig. 7 Summary of
biochemical changes of
A. sellowiana zygotic
embryogenesis
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
http://dx.doi.org/10.1007/s004250050287http://dx.doi.org/10.1007/BF00024673http://dx.doi.org/10.1023/A:1027376730521http://dx.doi.org/10.1023/A:1027376730521http://dx.doi.org/10.1590/S0100-84042003000200003http://dx.doi.org/10.1023/A:1022542618945http://dx.doi.org/10.1023/A:1022542618945http://dx.doi.org/10.1023/A:1015064227278http://dx.doi.org/10.1007/s11627-008-9176-4http://dx.doi.org/10.1007/s00299-006-0207-5http://dx.doi.org/10.1016/S0168-9452(98)00218-0http://dx.doi.org/10.1016/0003-2697(76)90527-3http://dx.doi.org/10.1016/S0168-9452(96)04491-3http://dx.doi.org/10.1016/S0168-9452(96)04491-3http://dx.doi.org/10.1016/S0168-9452(96)04491-3http://dx.doi.org/10.1016/S0168-9452(96)04491-3http://dx.doi.org/10.1016/0003-2697(76)90527-3http://dx.doi.org/10.1016/S0168-9452(98)00218-0http://dx.doi.org/10.1007/s00299-006-0207-5http://dx.doi.org/10.1007/s11627-008-9176-4http://dx.doi.org/10.1023/A:1015064227278http://dx.doi.org/10.1023/A:1022542618945http://dx.doi.org/10.1023/A:1022542618945http://dx.doi.org/10.1590/S0100-84042003000200003http://dx.doi.org/10.1023/A:1027376730521http://dx.doi.org/10.1023/A:1027376730521http://dx.doi.org/10.1007/BF00024673http://dx.doi.org/10.1007/s0042500502878/6/2019 Bio EZ Cangahuala Et Al 09
17/18
UNCO
RREC
TED PR
OOF
641 protocol in Feijoa ( Acca sellowiana (Berg) Burret): induction,642 conversion and synthetic Seeds. Sci Hortic (Amsterdam)643 11:228234. doi:10.1016/j.scienta.2006.10.030644 Cangahuala-Inocente GC, Steiner N, Maldonado SB, Guerra MP645 (2009) Patterns of protein and carbohydrate accumulation during646 somatic embryogenesis ofAcca sellowiana. Pesq. agropec. bras.,647 Braslia (in press)648 Canhoto JM, Cruz GS (1996) Feijoa sellowiana Berg (pineapple649 guava). In: Bajaj YPS (ed) Tree IV. Biotechnology in agriculture
650 on forestry. Springer, Berlin, pp 156172651 Corte VB, Lima De, e Borges EE, Pontes CA, De Almeida Leite IT,652 Ventrella MC, Mathias AA (2006) Mobilizacao de reservas653 durante a germinacao das sementes e crescimento das plantulas654 de Caesalpinia peltophoroides Benth.(Leguminosae-caesalpinoi-655 deae). Rev Arvore 30(6):941949656 Coruzzi G, Last R (2000) Amino acids. In: Buchanan BB, Gruissem657 W, Jones RL (eds) Biochemistry and molecular biology of658 plants. American Society of Plant Physiologists, Rockville,659 pp 358410660 Dal Vesco LL, Guerra MP (2001) The effectiveness of nitrogen661 sources in Feijoa (Feijoa sellowiana Berg) somatic embryogen-662 esis. Plant Cell Tissue Organ Cult 64:1925. doi:10.1023/663 A:1010635926146664 Dam S, Laursen BS, Ornfelt JH, Jochimsen B, Strfeldt HH, Friis
665 C, Nielsen K, Goffard N, Besenbacher S, Krusell L, Sato S,666 Tabata S, Thogersen IB, Enghild JJ, Stougaard J (2009) The667 proteome of seed development in the model legume Lotus668 japonicus. Plant Physiol 149:13251340. doi:10.1104/pp.108.669 133405670 Dodeman VL, Ducreux G, Kreis M (1997) Zygotic embryogenesis671 versus somatic embryogenesis. J Exp Bot 48:14931509672 Etienne H, Sotta B, Montoro P, Miginiac E, Carron MP (1993)673 Relation between exogenous growth regulators and endogenous674 indole-3-acetic acid and abscisic acid in expression of somatic675 embryogenesis in Hevea brasiliensis (Mull. Arg.). Plant Sci676 88:9196. doi:10.1016/0168-9452(93)90113-E677 Feirer RP (1995) The biochemistry of conifer embryo development:678 amino acids, polyamines, and storage proteins. In: Jain SM,679 Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody680 plants. v.1. Kluwer, Dordrecht, pp 317336681 Fischer-Iglesias C, Nauhaus G (2001) Zygotic embryogenesis682 hormonal control of embryo development. In: Bhojwani SS,683 Soh WY (eds) Current trends in the embryology of angiosperms.684 Kluwer, Dordrecht, pp 223247685 Fischer-Iglesias C, Sundberg B, Neuhaus G, Jones AM (2001) Auxin686 distribution and transport during embryonic pattern formation in687 wheat. Plant J 26:115129. doi:10.1046/j.1365-313x.2001.688 01013.x689 Friml J, Vieten A, Sauer M, Weijers D, Scwarz H, Hamann T,690 Offringa R, Jurgens G (2003) Efflux-dependent auxin gradients691 establish the apical-basal axis ofArabidopsis. Nature 426:147692 153. doi:10.1038/nature02085693 Gaspar T, Kevers C, Penel C, Greppin H, Reid DM, Thorpe TA694
(1996) Plant hormones and growth regulators in plant tissue695 culture. In Vitro Cell Dev Biol Plant 32:272289. doi:696 10.1007/BF02822700697 Geoffriau E, Kahane R, Martin-Tanguy J (2006) Polyamines are698 involved in the gynogenesis process in onion. Physiol Plant699 127:119129. doi:10.1111/j.1399-3054.2006.00597.x700 Gibson SI (2005) Control of plant development and gene expression701 by sugar signaling. Curr Opin Plant Biol 8:93102. doi:702 10.1016/j.pbi.2004.11.003703 Goldberg RB, Paiva G, Yadegari R (1994) Plant embryogenesis:704 zygote to seed. Science 266:605614. doi:10.1126/science.266.705 5185.605
706Kamada H, Harada H (1984) Changes in endogenous amino acids707compositions during somatic embryogenesis in Daucus carota.708Plant Cell Physiol 25:2738709Kermode AR (1995) Regulatory mechanisms in the transition from710seed development to germination: interaction between the711embryo and the seed environment. In: Kigel J, Galili G (eds)712Seed development and germination. Marcel Dekker, New York,713pp 273332714Kong L, Attree SM, Fowke LC (1997) Changes of endogenous
715hormone levels in developing seeds, zygotic embryos and716megagametophytes in Picea glauca. Physiol Plant 101:2330.717doi:10.1111/j.1399-3054.1997.tb01815.x718Kusano T, Yamaguchi K, Berberich T, Takahashi Y (2007) Advances719in polyamine research in 2007. J Plant Res 120:345350. doi:72010.1007/s10265-007-0074-3721Lea PJ, Sodek L, Parry MAJ, Shewry PR, Halford NG (2007)722Asparagine in plants. Ann Appl Biol 150:126. doi:10.1111/723j.1744-7348.2006.00104.x724Liu CM, Xu ZM, Chua NH (1993) Auxin polar transport is essential725for the establishment of bilateral symmetry during early plant726embryogenesis. Plant Cell 5:621630727Mader JC, Hanke DE (1997) Polyamine sparing may be involved in728the prolongations of cell division due to inhibition of phenyl-729propanoid synthesis in cytokinin starved soybean cells. J Plant
730Growth Regul 16:8993. doi:10.1007/PL00006983731Magnus V, Nigovic B, Hangarter RP, Good NE (1992) N - (indol-3-732ylacetyl) amino acids as sources of auxin in plant tissue culture. J733Plant Growth Regul 11:1928. doi:10.1007/BF00193839734Mansfield SG, Briarty LG (1991) Early embryogenesis inArabidopsis735thaliana. II. The developing embryo. Can J Bot 69:461476. doi:73610.1139/b91-063737Matsumoto TK, Kuehnle AR, Webb DT (1998) Zygotic embryogen-738esis in anthurium (araceae). Am J Bot 85:15601568. doi:73910.2307/2446482740McCready RM, Guggolz J, Silveira VE, Owens HS (1950) Determi-741nation of starch an amylose in vegetables. Anal Chem 22:11567421158. doi:10.1021/ac60045a016743Merkle SA, Parrott WA, Flinn BS (1995) Morphogenic aspects of744somatic embryogenesis in plants. Dordrecht. Kluwer Acad745Publishers Cap 5:155203746Michalczuck L, Cooke TD, Cohen JD (1992) Auxin levels at different747stages of carrot somatic embryogenesis. Phytochemistry 4:10977481103. doi:10.1016/0031-9422(92)80241-6749Misra S, Attree SM, Leal I, Fowke LC (1993) Effect of abscisic acid,750osmoticum, and desiccation on synthesis of storage proteins751during the development of white spruce somatic embryos. Ann752Bot (Lond) 71:1122. doi:10.1006/anbo.1993.1002753Mordhorst AP, Toonen MAJ, De Vries SC (1997) Plant embryogen-754esis. Crit Rev Plant Sci 16:535576. doi:10.1080/713608156755Nuutila AM, Kurten U, Kauppinen V (1991) Optimization of sucrose756and inorganic nitrogen concentrations for somatic embryogen-757esis of birch (Betula pendula Roth.) callus cultures: a statistical758approach. Plant Cell Tissue Organ Cult 24:7377. doi:759
10.1007/BF00039733 760OBrien TP, Feder N, McCully ME (1965) Polychromatic staining of761plant cell walls by toluidine blue O. Protoplasma 59:368373.762doi:10.1007/BF01248568763Ortiz-Lopez A, Chang HC, Bush DR (2000) Amino acid transporters764in plants. Biochim Biophys Acta 1465:275280. doi:10.1016/765S0005-2736(00)00144-9766Pescador R, Kerbauy GB, Kraus JE, Ferreira WM, Guerra MP,767Figueiredo-Ribeiro C (2008) Changes in soluble carbohydrates768and starch amounts during somatic and zygotic embryogenesis of769Acca sellowiana (Myrtaceae). In Vitro Cell Dev Biol Plant77044:289299. doi:10.1007/s11627-008-9118-1
Plant Growth Regul
123Journal : Large 10725 Dispatch : 17-6-2009 Pages : 13
Article No. : 9393h LE h TYPESET
MS Code : GROW1340 h CP h DISK4 4
http://dx.doi.org/10.1046/j.1365-313x.2001.01013.xhttp://dx.doi.org/10.2307/2446482http://dx.doi.org/10.1139/b91-063http://dx.doi.org/10.1016/j.scienta.2006.10.030http://dx.doi.org/10.1023/A:1010635926146http://dx.doi.org/10.1023/A:1010635926146http://dx.doi.org/10.1104/pp.108.133405http://dx.doi.org/10.1104/pp.108.133405http://dx.doi.org/10.1016/0168-9452(93)90113-Ehttp://dx.doi.org/10.1046/j.1365-313x.2001.01013.xhttp://dx.doi.org/10.1046/j.1365-313x.2001.01013.xhttp://dx.doi.org/10.1038/nature02085http://dx.doi.org/10.1007/BF02822700http://dx.doi.org/10.1111/j.1399-3054.2006.00597.xhttp://dx.doi.org/10.1016/j.pbi.2004.11.003http://dx.doi.org/10.1126/science.266.5185.605http://dx.doi.org/10.1126/science.266.5185.605http://dx.doi.org/10.1111/j.1399-3054.1997.tb01815.xhttp://dx.doi.org/10.1007/s10265-007-0074-3http://dx.doi.org/10.1111/j.1744-7348.2006.00104.xhttp://dx.doi.org/10.1111/j.1744-7348.2006.00104.xhttp://dx.doi.org/10.1007/PL00006983http://dx.doi.org/10.1007/BF00193839http://dx.doi.org/10.1139/b91-063http://dx.doi.org/10.2307/2446482http://dx.doi.org/10.1021/ac60045a016http://dx.doi.org/10.1016/0031-9422(92)80241-6http://dx.doi.org/10.1006/anbo.1993.1002http://dx.doi.org/10.1080/713608156http://dx.doi.org/10.1007/BF00039733http://dx.doi.org/10.1007/BF01248568http://dx.doi.org/10.1016/S0005-2736(00)00144-9http://dx.doi.org/10.1016/S0005-2736(00)00144-9http://dx.doi.org/10.1007/s11627-008-9118-1http://dx.doi.org/10.1007/s11627-008-9118-1http://dx.doi.org/10.1016/S0005-2736(00)00144-9http://dx.doi.org/10.1016/S0005-2736(00)00144-9http://dx.doi.org/10.1007/BF01248568http://dx.doi.org/10.1007/BF00039733http://dx.doi.org/10.1080/713608156http://dx.doi.org/10.1006/anbo.1993.1002http://dx.doi.org/10.1016/0031-9422(92)80241-6http://dx.doi.org/10.1021/ac60045a016http://dx.doi.org/10.2307/2446482http://dx.doi.org/10.1139/b91-063http://dx.doi.org/10.1007/BF00193839http://dx.doi.org/10.1007/PL00006983http://dx.doi.org/10.1111/j.1744-7348.2006.00104.xhttp://dx.doi.org/10.1111/j.1744-7348.2006.00104.xhttp://dx.doi.org/10.1007/s10265-007-0074-3http://dx.doi.org/10.1111/j.1399-3054.1997.tb01815.xhttp://dx.doi.org/10.1126/science.266.5185.605http://dx.doi.org/10.1126/science.266.5185.605http://dx.doi.org/10.1016/j.pbi.2004.11.003http://dx.doi.org/10.1111/j.1399-3054.2006.00597.xhttp://dx.doi.org/10.1007/BF02822700http://dx.doi.org/10.1038/nature02085http://dx.doi.org/10.1046/j.1365-313x.2001.01013.xhttp://dx.doi.org/10.1046/j.1365-313x.2001.01013.xhttp://dx.doi.org/10.1016/0168-9452(93)90113-Ehttp://dx.doi.org/10.1104/pp.108.133405http://dx.doi.org/10.1104/pp.108.133405http://dx.doi.org/10.1023/A:1010635926146http://dx.doi.org/10.1023/A:1010635926146http://dx.doi.org/10.1016/j.scienta.2006.10.0308/6/2019 Bio EZ Cangahuala Et Al 09
18/18
UNCO
RREC
TED PR
OOF
771 Prewein C, Endemann M, Reinohl V, Salaj J, Sunderlikova V,772 Wihelm E (2006) Physiological and morphological characteris-773 tics during development of pedunculate oak (Quercus robur L.)774 zygotic embryos. Trees (Berl) 20:5360. doi:10.1007/s00468-775 005-0012-8776 Puga-Hermida MI, Gallardo M, Matilla AJ (2003) The zygotic777 embryogenesis and ripening ofBrassica rapa seeds provokes778 important alterations in the levels of free and conjugated abscisic779 acid and polyamines. Physiol Plant 117:279288. doi:10.1034/
780 j.1399-3054.2003.00033.x781 Radwanski ER, Last RL (1995) Tryptophan biosynthesis and782 metabolism: biochemical and molecular genetics. Plant Cell783 7:921934784 Ribnicky DM, Cohen JD, Hu W, Coke TJ (2002) An auxin surge785 following fertilization in carrots: a mechanism for regulating786 plant totipotency. Planta 214:505509. doi:10.1007/s004787 250100639788 Rock CD, Quatrano RS (1995) The role of hormones during seed789 development. In: Davies PJ (ed) Plant hormones. Kluwer,790 Dordrecht, pp 671697791 Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and792 signaling in plants: conserved and novel mechanisms. Annu Rev793 Plant Biol 57:675709. doi:10.1146/annurev.arplant.57.032905.794 105441
795 Sallandrouze A, Faurobert M, Maataoui ME (2002) Characterization796 of the development stages of cypress zygotic embryos by two-797 dimensional electrophoresis and by cytochemistry. Physiol Plant798 114:608618. doi:10.1034/j.1399-3054.2002.1140415.x799 Santa-Catarina C, Silveira V, Balbuena TS, Viana AM, Estelita800 MEM, Handro W, Floh EIS (2006) IAA, ABA, polyamines and801 free amino acids associated with zygotic embryo development of802 Ocotea catharinensis. Plant Growth Regul 49:237247. doi:803 10.1007/s10725-006-9129-z804 Satya-Naraian V, Nair PM (1990) Metabolism, enzymology and805 possible roles of 4-aminobutyrate in higher plants. Phytochem-806 istry 29:367375. doi:10.1016/0031-9422(90)85081-P807 Shannon JC (1968) A procedure for the extraction and fractionation808 of carbohydrates from immature Zea mays kernels. Res Bull809 (Sun Chiwawitthaya thang Thale Phuket) 842:18
810Silveira V, Floh EIS, Handro W, Guerra MP (2004a) Effect of plant811growth regulators on the cellular growth and levels of intracel-812lular protein, starch and polyamines in embryogenic suspension813cultures ofPinus taeda. Plant Cell Tissue Organ Cult 76:5360.814doi:10.1023/A:1025847515435815Silveira V, Balbuena TS, Santa-Catarina C, Floh EIS, Guerra MP,816Handro W (2004b) Biochemical changes during zygotic embryo-817genesis in Pinus taeda L. Plant Growth Regul 44:147156818Silveira V, Santa-Catarina C, Balbuena TS, Moraes FMS, Ricart
819CAO, Sousa MV, Guerra MP, Handro W, Floh EIS (2008)820Endogenous abscisic acid and protein contents during seed821development of Araucaria angustifolia. Biol Plant 52(1):101822104. doi:10.1007/s10535-008-0018-3823Smeekens S (2000) Sugar-induced signal transduction in plants. Annu824Rev Plant Physiol Plant Mol Biol 51:4981. doi:10.1146/825annurev.arplant.51.1.49826Snedden WA, Fromm H (1998) Calmodulin, calmodulin-related827proteins and plant responses to the environment. Trends Plant828Sci 3:299304. doi:10.1016/S1360-1385(98)01284-9829Stefanello S, Dal Vesco LL, Ducroquet JPHJ, Nodari RO, Guerra MP830(2005) Somatic embryogenesis from floral tissues of feijoa831(Feijoa sellowiana Berg). Sci Hortic (Amsterdam) 105:117126.832doi:10.1016/j.scienta.2004.11.006833Thorp G, Bieleski R (2002) Feijoas: origins, cultivation and uses.
834David Baterman, New Zealand, p 87835Tobe PH, Raven H (1983) The embryology ofAxinandra zeylanica836(Crypteroniaceae) and the relationships of the genus. Bot Gaz837144(3):426432. doi:10.1086/337393838Umbreit WW, Burris RH (1960) Method for glucose and other sugars,839In: Umbreit WW, Burris RH, Stauffer JP (eds) Manometric840techniques, 4th edn. Burguers Publishing Co841Weber H, Borisjuk L, Wobus W (1997) Sugar import and metabolism842during seed development. Trends Plant Sci 2:169174. doi:84310.1016/S1360-1385(97)85222-3844Wise JM, Tunnacliffe A (2004) POPP the question: what do LEA845proteins do? Trends Plant Sci 9:1317. doi:10.1016/j.tplants.8462003.10.012
847
Plant Growth Regul
13
http://dx.doi.org/10.1016/S1360-1385(97)85222-3http://dx.doi.org/10.1016/j.scienta.2004.11.006http://dx.doi.org/10.1007/s00468-005-0012-8http://dx.doi.org/10.1007/s00468-005-0012-8http://dx.doi.org/10.1034/j.1399-3054.2003.00033.xhttp://dx.doi.org/10.1034/j.1399-3054.2003.00033.xhttp://dx.doi.org/10.1007/s004250100639http://dx.doi.org/10.1007/s004250100639http://dx.doi.org/10.1146/annurev.arplant.57.032905.105441http://dx.doi.org/10.1146/annurev.arplant.57.032905.105441http://dx.doi.org/10.1034/j.1399-3054.2002.1140415.xhttp://dx.doi.org/10.1007/s10725-006-9129-zhttp://dx.doi.org/10.1016/0031-9422(90)85081-Phttp://dx.doi.org/10.1023/A:1025847515435http://dx.doi.org/10.1007/s10535-008-0018-3http://dx.doi.org/10.1146/annurev.arplant.51.1.49http://dx.doi.org/10.1146/annurev.arplant.51.1.49http://dx.doi.org/10.1016/S1360-1385(98)01284-9http://dx.doi.org/10.1016/j.scienta.2004.11.006http://dx.doi.org/10.1086/337393http://dx.doi.org/10.1016/S1360-1385(97)85222-3http://dx.doi.org/10.1016/j.tplants.2003.10.012http://dx.doi.org/10.1016/j.tplants.2003.10.012http://dx.doi.org/10.1016/j.tplants.2003.10.012http://dx.doi.org/10.1016/j.tplants.2003.10.012http://dx.doi.org/10.1016/S1360-1385(97)85222-3http://dx.doi.org/10.1086/337393http://dx.doi.org/10.1016/j.scienta.2004.11.006http://dx.doi.org/10.1016/S1360-1385(98)01284-9http://dx.doi.org/10.1146/annurev.arplant.51.1.49http://dx.doi.org/10.1146/annurev.arplant.51.1.49http://dx.doi.org/10.1007/s10535-008-0018-3http://dx.doi.org/10.1023/A:1025847515435http://dx.doi.org/10.1016/0031-9422(90)85081-Phttp://dx.doi.org/10.1007/s10725-006-9129-zhttp://dx.doi.org/10.1034/j.1399-3054.2002.1140415.xhttp://dx.doi.org/10.1146/annurev.arplant.57.032905.105441http://dx.doi.org/10.1146/annurev.arplant.57.032905.105441http://dx.doi.org/10.1007/s004250100639http://dx.doi.org/10.1007/s004250100639http://dx.doi.org/10.1034/j.1399-3054.2003.00033.xhttp://dx.doi.org/10.1034/j.1399-3054.2003.00033.xhttp://dx.doi.org/10.1007/s00468-005-0012-8http://dx.doi.org/10.1007/s00468-005-0012-8