Maturation of somatic embryos in two embryogenic cultures of <Emphasis Type="Italic">Picea morrisonicola</Emphasis> Hayata as affected by alternation of endogenous IAA content

Embryogenic culture lines T4 and T2 were initiated from two mature zygotic embryos of Picea morrisonicola Hay. Mature somatic embryos (SEs) were produced in culture line T2 but not in line T4 after 8-week abscisic acid (ABA) treatment. High performance liquid chromatography (HPLC) analysis of the endogenous indole-3-acetic acid (IAA) content has shown 7.5 times higher IAA production in T4 line than in T2 line during the proliferation phase. However, after ABA incubation the line T4 produced much less IAA than line T2. The application of an anti-auxin, 2,3,5-triiodobenzoic acid (TIBA) or 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB) induced culture line T4 to produce mature SEs. Both 1 μM TIBA and 5 μM PCIB increased the production of stage 2 SEs in T4 culture line when cultures were treated during the proliferation stage for 8 weeks. Occasionally cotyledonary (stage 3) SEs were even produced from treated T4 culture line. Both chemicals have also been demonstrated to significantly decrease the amount of IAA in the treated T4 and T2 embryogenic lines. However the decrease of the IAA level was not beneficial for SE production in the T2 embryogenic line. These results indicated the importance of endogenous IAA level in manipulating the process of SE maturation in spruce embryogenic cultures.     

The role of auxins in somatic embryogenesis of <Emphasis Type="Italic">Abies alba</Emphasis>

The somatic embryogenesis of conifers is a process susceptible to exogenous phytohormonal treatments. We report the effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the auxin inhibitor p-chlorophenoxyisobutyric acid (PCIB) on the endogenous level of the auxin indole-3-acetic acid (IAA) and on the anatomical composition of early somatic embryos of Abies alba (European silver fir). The embryogenic suspensor mass (ESM) of Abies alba proliferated on a medium supplemented by 2,4-D as well as on an auxin-free medium. The endogenous level of IAA was significantly higher in the ESM cultivated on a medium supplemented by 2,4-D. The decrease in the endogenous level of IAA in the first week of maturation is one of the most important stimuli responsible for the subsequent development of embryos. However, suppression of IAA synthesis by an auxin inhibitor did not stimulate the development of embryos. The maturation of somatic embryos from the globular to the cotyledonary stage occurs when the concentration of endogenous auxin in the ESM (including the embryos) increases. Early somatic embryos proliferating on a medium supplemented by auxin had an increased probability of maturing successfully. Exogenous auxin treatment during maturation did not compensate for the auxin deficiency during proliferation.     

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.     

Clonal plant production from self- and cross-pollinated seed families of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.     

Improvement of somatic embryogenesis in wild cherry (Prunus avium). Effect of maltose and ABA supplements

Three different types of morphogenesis were identified in embryogenic cultures of Prunus avium grown on a proliferation medium containing 0.54 μM NAA, 0.46 μM kinetin and 0.44 μM BA: a friable hyperhydric callus, repetitive embryogenesis and an embryogenic tissue. Translucent and white somatic embryos were produced from the three types of morphogenesis but mainly from the embryogenic tissue. These somatic embryos showed histological and cytological teratological features such as highly differentiated cells with shrunken cytoplasm and destructured nuclei. For the four lines studied, somatic embryo production was improved by transferring the embryogenic tissue to developmental media without auxin and cytokinin but supplemented with maltose alone or maltose and 10 μM ABA. Three weeks after transfer, the line showing the most embryogenesis produced 1404 somatic embryos per gram of embryogenic tissue. A concentration of 263 mM maltose significantly increased the number of white somatic embryos for L 10 line, while translucent somatic embryo production was improved by 88 mM maltose for L 16 line. The combination of maltose and ABA produced different effects with each line. When used with 88 mM maltose, 10 μM ABA significantly increased white somatic embryo production for two lines but decreased the production for one line. When combined with 263 mM maltose, ABA had no effect on white somatic embryo production but significantly decreased the number of translucent somatic embryos. Cells of white somatic embryos contained protein storage reserves and numerous lipid bodies, while those of translucent embryos did not contain storage reserves or lipid bodies. After a two-month cold treatment conversion rate of white and translucent somatic embryos reached 8.5% and 35.2% respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of various partial pressures of oxygen and carbon dioxide on different stages of somatic embryogenesis in Picea abies

Effects of various partial pressures of oxygen (5, 20 and 45 kPa) and carbon dioxide (0.03 and 6 kPa) on initiation, proliferation and maturation of somatic embryos in Picea abies were studied. The pO₂ had a significant effect on the initiation of embryogenic tissue from mature zygotic embryos. However, the effect of pO₂ was dependent on the strength of the basal medium. Low pO₂ stimulated the formation of embryogenic tissue when the zygotic embryos were incubated on full strength medium, but was inhibitory when half-strength medium was used. Proliferation of embryogenic tissue was stimulated by higher partial pressures of both CO₂ and O₂. The effect of the gas phase on maturation of somatic embryos varied between different cell lines. However, there was a general tendency for 5 kPa O₂ and 6 kPa CO₂ to stimulate maturation.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ET embryogenic tissue     

Abscisic acid induces somatic embryogenesis and enables the capture of high-value genotypes in Douglas fir (Pseudotsuga menziesii [MIRB.] Franco)

Douglas fir (Pseudotsuga menziesii) is one of Europe’s most important non-native tree species due to its drought tolerance as well as timber quality and yield. To obtain superior seed from selected parental trees, breeding programs had been established in seed orchards. Douglas fir seed is used as source material for somatic embryogenesis with the aim to select elite genotypes invaluable for clonal mass propagation. To improve given protocols for somatic embryo initiation, we used immature Douglas fir zygotic embryos as explants and abscisic acid (ABA) as plant growth regulator in contrast to the application of auxins and cytokinins. With ABA supplementation, induction frequencies were slightly but in mean higher than with auxin/cytokinin, showing also a strong genotype effect. This offered the possibility to capture SE cultures from otherwise recalcitrant crosses. Furthermore, we observed remarkable differences between the two sets of plant growth regulators concerning the morphological development of the explants, including the absence of non-embryogenic callus by using ABA as inducer. This simplifies the detection of events and the handling of the obtained cultures. Nevertheless, a histological approach suggested, that the same competent cells are addressed by the different hormonal stimulation. Besides, we studied the influence of different points in time of cone harvest, two different basal media and different genetic backgrounds of the explants as well as the maturation ability of the induced embryogenic cultures. In sum, we were able to improve the first steps of somatic embryogenesis and to maintain a significantly higher number of high-value genotypes.

     

Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

Genotype effects on ABA consumption and somatic embryo maturation in interior spruce (Picea glaucaxengelmanni)

Abscisic acid (ABA) plays an important role during somatic embryo development and maturation in coniferous species. The purpose of this research was to study ABA utilization by genotypes with different embryo maturation capabilities in interior spruce. Cell lines ISP11 and ISP48 were of high embryo maturation capability. By contrast, the tissue of line ISP16 contained numerous immature embryos, but only a few mature embryos developed. Exogenous ABA, i.e. S-ABA [(+)-cis, trans-ABA], racemic ABA, or ABA isomers were added into suspension cultures at a final concentration of 30 microM. In comparison to racemic ABA and ABA isomers, S-ABA reduced tissue proliferation the most. In all cell lines, about half of the racemic ABA was used within 2 weeks; the remaining ABA was (-)-cis, trans-ABA. The concentration of ABA showed little change thereafter. In the cultures supplied with ABA isomers, about half of (+/-)-cis, trans-ABA was utilized during 22 d. By contrast, (+/-)-trans, trans-ABA was hardly used, especially in line ISP16. S-ABA was almost completely metabolized by line ISP11. However, approximately 28% and 22% of the S-ABA remained in the culture of cell lines ISP16 and ISP48, respectively. Cell line ISP16 grew the fastest in culture. By 3 weeks, S-ABA consumption by ISP11 and ISP48 on the basis of tissue growth was, respectively, 2.2-fold and 3.4-fold greater than that of ISP16. A higher ratio of dihydrophaseic acid to phaseic acid existed with cell lines of higher embryo maturation capability, especially when the exogenously supplied ABA was chemically synthesized.     

The Influence of Auxin and Cytokinin on Proliferation and Morphology of Somatic Embryos of Picea abies (L.) Karst

Embryogenic cell-lines of Picea abies were initiated from maturezygotic embryos and cultured on medium containing 2,4-D andBA The cell-lines were categorized into two main groups (solar/polarand undeveloped embryos), based on the morphology of the somaticembryos and their ability to go through a maturation processwhen treated with ABA The cell-lines were transferred to mediacontaining (1) 2,4-D and BA, (2) only BA, (3) only 2,4-D or(4) no growth regulators When cultured on a medium containingboth 2,4-D and BA new somatic embryos were continually formedIn contrast, when they were cultured without one or both ofthe growth regulators no new somatic embryos were formed Solar/polarand undeveloped embryos responded in the same way On a mediumcontaining only BA the somatic embryos already present increasedin size and developed an extremely large embryonic region Ona medium containing only 2,4-D the embryos already present becamedisorganized into loose aggregates When transferred from a mediumcontaining both 2,4-D and BA to one containing ABA, mature somaticembryos developed from the solar/polar type but not from theundeveloped type The ability of the solar/polar somatic embryosto go through a maturation process decreased when they wereprecultured on a medium lacking auxin and was lost when theywere precultured on medium lacking cytokinin The cell-linescontaining undeveloped somatic embryos produced mature somaticembryos in one cell-line out of three that had been culturedon the medium containing only BA Auxin, cytokinin, embryogenic cultures, Norway spruce, somatic embryos, Picea abies (L.) Karst     

Screening of soybean, Glycine max L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons

Summary Seventeen breeding lines of soybean, Glycine max (L.) Merrill, and cv. Jack, from relative maturity groups 0.3–7.5 were assessed for their ability to undergo somatic embryogenesis. The goal of this study was to determine which lines had high embryogenic capacity. We also sought to understand the relationship between relative maturity and embryogenesis. Embryos from immature cotyledons were initiated on solid MS medium with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D). Qualitative and quantitative measures of initiation, proliferation, differentiation, and maturation were recorded. The breeding lines differed significantly with respect to percent induction, number of embryos induced, and quality of induced embryos. After 1 mo, of proliferation, two early maturing lines, the control, Jack, and NK-5, had the best overall performance. High percent response of proliferating embryos was positively associated with lower maturity groups. Relatively high concentrations of 2,4-D (compared with that used in prolifcrating medium, e.g., 226 μM; 50 mg l⁻¹) in the initiating medium reduced numbers of embryo clusters per cotyledon initiated and percent initiation, and the concentration of 2,4-D affected the proliferation of somatic embryos in a breeding line-dependent manner. The breeding lines differed significantly in the time to produce mature somatic embryos. There was a positive correlation between immature embryo quality and number of differentiated somatic embryos produced. Retired.     

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst)

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.     

Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (White spruce)

Hakman, I. and von Arnold, S. 1988. Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (White spruce). - Physiol. Plant. 72: 579–587.
Plantlets were regenerated from long-term embryogenic cultures of Picea glauca (Moench) Voss. (White spruce). Embryogenic calli, initiated from immature zygotic embryos and maintained by monthly subculture for 16 months, were used to establish suspension cultures. Small somatic embryos were continuously produced in liquid culture medium containing auxin and cytokinin and the cultures showed a sustained regeneration capacity for >6 months. Somatic embryos propagated in the suspension cultures developed further into embryos bearing cotyledons, about 1 month after transfer to solidified medium containing abscisic acid. Electron microscopic examination revealed that storage nutrients, lipids, proteins and carbohydrates, accumulated in the somatic embryos during this treatment with abscisic acid (ABA). Upon subculture to medium lacking plant growth regulators such embryos could develop into small green plantlets.     

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba×Abies cephalonica,and Abies alba×Abies numidica)

Summary Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

     

Maturation and conversion ofLiriodendron tulipifera somatic embryos

Summary Embryogenic suspension cultures of the hardwood forest tree yellow-poplar (Liriodendron tulipifera) have the potential to produce millions of plantlets. However, low conversion frequencies limit the realization of this potential. Using 4 embryogenic yellow-poplar lines, we first tested the ability of somatic embryos, selected for their similarity to mature zygotic embryos, to convert to plantlets, then tested physical and chemical treatments for their effects on promoting maturation of somatic embryos and subsequent plantlet production. Embryos selected based on resemblance to mature zygotic embryos and transferred to a hormone-free basal medium without casein hydrolysate (CH) produced plantlets at a frequency of 63%. Populations of synchronized somatic embryos were obtained by repeated fractionation of liquid medium-cultured proembryogenic masses (PEMs) on stainless steel sieves. These fractionated embryos failed to mature properly when cultured in liquid basal medium, however. Development of embryos cultured in basal medium supplemented with 5×10⁻⁷ M abscisic acid (ABA) was slowed and embryos appeared to mature properly, with separated cotyledons and little precocious germination. However, ABA-treated embryos only rarely converted to plantlets, possibly due to residual effects of the ABA. PEMs fractionated on sieves, transferred to filter paper and placed on solidified basal medium gave a 60–70% synchronous population of mature embryos 10–12 days following plating. Mature embryos transferred to basal medium without CH converted at a frequency of 72%. The percentage of all embryos differentiating from PEMs on filter paper that formed plantlets was 32%. This material is based upon work supported by the U. S. Department of Agriculture Cooperative State Research Service under Agreement No. 85-FSTY-9-0117.     

Abscisic acid and methyl jasmonate as regulators of ethylene biosynthesis during somatic embryogenesis of Medicago sativa L.

Effects of abscisic acid (ABA) and methyl jasmonate (MeJA) on ethylene production, ACC oxidase (ACO) activity, and content of 1-aminocyclopropane-1-carboxylic acid (ACC) during indirect somatic embryogenesis (SE) of Medicago sativa L. were studied. ABA and MeJA, at 50 μM, were applied during the induction, proliferation, or differentiation phase. ABA decreased ethylene production at the beginning of callus and SE induction and during the differentiation of somatic embryos. The hormone inhibited ACO activity in explants with overgrowing callus during the first two weeks of induction, in embryogenic suspension and also in differentiating embryos. The ACC content was reduced by ABA in callus at the end of SE induction, in embryogenic suspension and in globular embryos, but elevated in cotyledonary embryos. MeJA had no significant effect on ethylene production during M. sativa SE, despite the fact, that it inhibited ACO activity during the first two weeks of induction and in torpedo and cotyledonary embryos. The ACC content was increased by MeJA in 14-day-old callus and embryogenic suspension but was inhibited in globular embryos. Both ABA and MeJA seem to be involved in the regulation of ethylene biosynthesis during distinct phases of SE in M. sativa. It might be considered that exogenous ABA, more probably than MeJA, exerts its inhibitory effect on M. sativa somatic embryo formation by modifying ethylene production.     

Somatic embryogenesis of <Emphasis Type="Italic">Myrciaria aureana</Emphasis> (Brazilian grape tree)

The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process.     

Embryology in Norway spruce (Picea abies). An analysis of the composition of seed storage proteins and deposition of storage reserves during seed development and somatic embryogenesis

Seed cones were collected from open-pollinated trees of Norway spruce ( Picea abies ) in a seed orchard from pollination until maturation of the seeds. Immature embryos were isolated for embryogenic tissue cultures that were maintained either on solidified medium or as liquid cultures. By transferring young somatic embryos to medium containing 90 m M sucrose and 7. 6 μ M ABA growth continued to mature embryos that accumulated storage reserves in both the hypocotyl-shoot axis and the cotyledons. Both zygotic and somatic embryos at different developmental stages were processed for microscopy as were the megagametophytes. Total protein was extracted from the seed material at intervals during development and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These analyses revealed that storage protein started to accumulate in the megagametophytes at the time when embryos were growing into the gametophytic tissue, while it occurred a few weeks later in the embryos at rapid embryo growth and organ differentiation. Lipid bodies also became abundant in the mature plant material. Although plastids with prominent starch grains were very frequent in both megagametophytes and embryos during development they were not observed in the desiccated tissues. Zygotic and somatic embryos displayed a similar developmental pattern.
By sequential salt-extraction and dilution two fractions highly enriched in storage protein were obtained. One fraction (G-1), requiring higher salt concentration for protein solubilization, was dominated by a protein migrating to around M, 55000–60000 when separated under non-reduced condition. After exposure to reducing agent this protein was replaced by two new ones with M, 33000 and 22000 giving evidence of disulfide bonded polypeptides. The other fraction (G-2), was dominated by polypeptides around M, 42000 and low molecular mass polypeptides (<14000).