Efficient production of celery embryos and plantlets released in culture of immobilized gel beads

Celery embryos and plantlets were found to be selectively released in a culture of immobilized Ca-alginate gel beads in which celery callus was entrapped under regeneration conditions. We studied the feasibility of use of this process for celery embryogenesis in an artificial seed system. The cells released from the gel beads were larger than those obtained in suspension culture. The optimal concentration of alginate gel for embryo and plantlet production was 2% for the immobilized cell culture. Considering the maintenance of the gel bead structure and detrimental effect of CaCl₂ on plantlet development, 5 mM CaCl₂ supplementation gave the best result in terms of the number of heart and torpedo embryos and plantlets. The ratio of the number of heart embryos, torpedo embryos and plantlets to total number of cells in the immobilized cell culture was higher than that in the suspension culture. Repeated batch culture with 5 mM CaCl₂ provided long-term (more than 154 d) embryo and plantlet production without gel beads disruption. Productivity of plantlets in the immobilized cell culture with 5 mM CaCl₂ was 2.2-fold as high as that in the suspension culture.     

Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by -cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones b and d of esterase and zone b of phosphogluconate dehydrogenase coincided with vascular bundle formation.Abbreviations ADH alcohol dehydrogenase - CD -cyclodextrine - CS cell suspension culture - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraaeetie acid - LiBo lithium hydroxide/boric acid - PEG polyethylene glycol - PVP polyvinylpyrrolidone - SEg somatic embryo at the globular stage - SEh heart stage - SEte early torpedo stage - SEtl late torpedo stage - SEce early cotyledonary stage - SEcl late cotyledonary stage - SECD somatic embryo blocked at the torpedo stage with -cyclodextrine - EST esterase - GOT aspartate aminotransferase - IDH isocitrate dehydrogenase - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) - PMS phenazonium methosulfate - PGD phosphogluconate dehydrogenase - PGI glucosephosphate isomerase - PGM phosphoglucomutase - SO dry seed - S1–3 seed after 1–3 days of germination - SP1–2 young and old somatic plantlets - ZE zygotic embryo - ZP4–5 4–5 day-old seedlings     

An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.     

Somatic embryogenesis and plant regeneration from immature embryo explant of papaya (Carica papaya L. cv. washington and honey dew)

Immature zygotic embryo explants of Carica papaya were cultured on MS medium supplemented with 2,4-D (2.0 mg/l) and formed globular embryos on explants without callus formation in 4-6 weeks. Maturation and conversion of somatic embryos was also achieved on the same medium. Cotyledonary stage embryos germinated to 63.66 and 68.33% in cv. honey dew and washington respectively in MS basal medium supplemented ABA (0.5 microm/l). Robust development and proliferation of plantlet roots in vitro was obtained on MS basal medium. Hardened plantlets have 60% survival rate.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.     

Bioreactor studies of the effect of medium pH on carrot (Daucus carota L.) somatic embryogenesis

Daucus carota cell differentiation was examined under different medium pH conditions in a controlled bioreactor. Somatic embryogenesis was affected by pH changes. Embryo production was greatest when the pH of the hormone-free medium was maintained at 4.3. However, the same level was not favourable to development since most embryos did not progress to the torpedo and plantlet stages. In contrast, although there was about a threefold decrease in embryo yield in cultures on the same free 2,4-dichlorophenoxyacetic acid medium maintained at pH 5.8, cells differentiated into fully developed plantlets. Changes in embryo development appeared to be associated with alterations in ammonium loss from the medium and sugar uptake.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Alfalfa embryo production in airlift vessels via direct somatic embryogenesis

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.Abbreviations ABA abscisic acid - B5 Gamborgs B5 medium (Gamborg et al. 1968) - COT cotyledon embryo state - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - ID internal diameter - MS Murashige and Skoog medium (Murashige & Skoog 1962) - PEG polyethylene glycol - POLY polyembryos - VVM volume of gas/volume of bioreactor     

Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis

In vitro somatic embryogenesis of Glehnia littoralis Fr. schm. was observed when zygotic embryos were cultured on a medium containing 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (0.01-10 μM), with 1 μM being the optimum. Microscopic observations revealed globular, heart-shaped and torpedo-shaped embryo formations and plantlet regeneration. These somatic embryos seemed to be produced directly from cells of the zygotic embryos used as explants. Of seven types of media tested, Nitsch's medium showed the highest rate of somatic embryogenesis. Somatic embryos developed into normal plantlets and were able to be potted. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphohistological analysis and histochemistry of Feijoa sellowiana somatic embryogenesis

Summary. Morphohistological analysis and histochemical studies were carried out during the induction and development of Feijoa sellowiana somatic embryos. Zygotic embryos were cultured on LPm medium containing 2,4-dichlorophenoxyacetic acid (20µM) and glutamine (8mM). Somatic embryogenesis could be induced from embryogenic cells that originated in meristematic centers or from clusters of cells. The presence of few starch grains and abundant protein bodies was observed in the globular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associated with the accumulation of reserves to be used in the conversion of the embryos to plantlets. Proteins were predominantly observed in protoderm cells, as well as in the meristematic apical region of torpedo and cotyledonary-stage somatic embryos.Correspondence and reprints: Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, C.P. 476, 88034-001 Florianópolis, SC, Brazil.     

Turnover of cell-wall polysaccharides during somatic embryogenesis and development of celery (Apium graveolens L.)

Non-embryogenic cells (NEC) and embryogenc cells (EC) were separated from cell clusters derived from the hypocotyl segments of celery seedlings, which had been suspension-cultured in MS medium supplemented with 10⁵ M 2,4-D. The EC formed globular embryos in medium without 2,4-D. The globular embryo developed through heart-shaped, torpedo to cotyledonary embryos within 10 days. The EC and developing embryos were fractionated into symplastic [MeOH, hot water (HW), starch (S)] and apoplastic [pectin, hemicellulose, TFA (trifluoroacetic acid)-soluble and cellulose] fractions. The EC contained lower levels of sugar in the MeOH fraction and higher levels of starch than NEC. In the apoplastic fractions, there were no differences of total sugar amounts between NEC and EC. Cellulose contents were about 10% of the wall polysaccharides. During somatic embryogenesis, total sugar contents of the MeOH and HW fractions increased till the heart-shaped embryo stage, and then decreased during the torpedo and cotyledonary embryo stages. The sugar contents of the starch, pectin, TFA-soluble, and cellulose fractions did not change during the stages mentioned above. However, the hemicellulose substances remarkably increased during embryogenesis, and then decreased as the development proceeded. The neutral sugar components of the hemicellulosic fractions were analyzed. Arabinose increased markedly in EC to the globular embryo stage, but decreased as the development proceeded. Galactose increased only at the torpedo and cotyledonary embryo stages. Xylose was present at lower levels in all stages of embryogenesis than in the differentiated hypocotyl cell walls. These results suggest that there was a high turnover of arabinogalactan polysaccharides during embryogenesis, and that xylan accumulated in the cell walls of differentiated cells     

花生体细胞胚的诱导及其植株再生

采用不同成熟度的花生胚轴为外植体进行体细胞胚诱导及植株再生研究,结果表明,成熟胚轴在高浓度2,4－D的MS培养基中,经过30d左右的培养,可直接诱导产生出大量的体细胞胚,含40mgL~－12,4－D的培养基中体细胞胚的诱导率达100％,平均每个外植体产生11．58个体细胞胚．体细胞胚的继代培养需降低2,4－D的浓度（1－20mgL~－1）．未成熟胚轴的体细胞胚诱导及继代培养的2,4－D浓度宜为10mgL~－1．将诱导的体细胞胚转接到合5－10mgL~－1BA的MS培养基中,体细胞胚能够萌发再生成无根小植株,将其转接到生根培养基中可获得完整小植株．     

Photoautotrophic culture of Coffea arabusta somatic embryos: photosynthetic ability and growth of different stage embryos

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 micromol m(-2) s(-1)) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90-130 microg g(-1) fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300-500 microg g(-1) fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar-free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20-25% of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50%, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100-150 micromol m(-2) s(-1)) and increased CO2 concentration (1100 micromol mol(-1)) were found to be necessary for the development of plantlets from cotyledonary stage embryos.     

Promotion of plantlet formation from somatic embryos of carrot treated with a high molecular weight extract from a marine cyanobacterium

Summary Somatic embryos of Daucus carota L. developed into plantlets at high frequency after addition of an extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. High molecular weight, nondialyzing fraction, separated from the extract, possessed enhanced plantlet formation promoting activity. Plantlet formation frequency was 60 % after addition of nondialysate (100 mg/l) compared to 28 % without addition. Embryos treated with the nondialysate contained five times more chlorophyll than nontreated embryos after 6 days of culture. The chlorophyll a/b ratio of 4-day old treated somatic embryos was found to be similar to that of zygotic embryos. However, the chlorophyll a/b ratio of plantlets induced from nontreated somatic embryos was variable. Nondialysate was fractionated by ultracentrifugation and an active component obtained, which gave a maximum plantlet formation frequency of 71 %, and induced rapid greening of shoots.Abbreviations MS Murashige and Skoog medium - 2,4D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - E Einstein - Chl Chlorophyll - vvm volume of air, volume of medium per minute     

Histodifferentiation of somatic embryos in cotyledons of pineapple guava (Feijoa sellowiana Berg)

Summary Somatic embryos of pineapple guava (Feijoa sellowiana Berg, Myrtaceae) were induced particularly well from the adaxial face of the cotyledons of zygotic embryos cultured on MS medium containing 1.0 mg/l 2,4-D and 0.3 M sucrose. Somatic embryos were never obtained from globular and heart-shaped zygotic embryos and embryos at the torpedo stage produced somatic embryos at lower frequencies than mature zygotic embryos. At the time of explantation, cotyledonary cells were rich in storage proteins and lipids but no starch was found. After the first 5 days of culture most of the reserves had been mobilized in cotyledons of germinating embryos, but were still present in large amounts in cotyledons undergoing embryogenie induction. In contrast to cotyledons following the normal pattern of development, cells of embryogenically-induced cotyledons accumulated starch, especially those cells not involved in the embryogenie process. Two patterns of somatic embryo differentiation were observed: (1) from single epidermal cells or (2) from groups of meristematic cells near the adaxial surface. Comparative observations on cotyledons from germinating embryos and those undergoing embryogenesis suggest that the meristematic layer arises as the result of successive divisions of cells that, under normal conditions, would form the palisade parenchyma. These were the only mesophyll cells that showed mitotic divisions during the normal development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FAA formalin/acetic acid/ethyl alcohol - PAS periodic acid-Schiff     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.     

Charged acrylamide copolymer gels as media for weak alignment

The use of mechanically strained acrylamide/acrylate copolymers is reported as a new alignment medium for biomacromolecules. Compared to uncharged, strained polyacrylamide gels, the negative charges of the acrylamide/acrylate copolymer strongly alter the alignment tensor and lead to pronounced electroosmotic swelling. The swelling itself can be used to achieve anisotropic, mechanical strain. The method is demonstrated for the alignment of TipAS, a 17 kDa antibiotic resistance protein, as well as for human ubiquitin, where alignment tensors with an A_ZZ,NH of up to 60 Hz are achieved at a gel concentration of 2% (w/v). The alignment can be modulated by the variation of pH, ionic strength, and gel concentration. The high mechanical stability of the swollen gels makes it possible to obtain alignment at polymer concentrations of less than 1% (w/v).     

三七体细胞胚胎发生及人工种子研制

三七为五加科的重要药用植物。以三七的种胚为材料接种于MS 补加2,4-D、IAA、NAA 各1 mg/L的培养基上。接种后约二个月,在MS 补加IAA 或NAA 的培养基上可以产生体细胞胚;在MS 加2,4-D 的培养基上只产生愈伤组织而无器官分化。体细胞胚在MS+IAA 0.5 mg/L+GA_3 1 mg/L 培养基上可发育成小植株。体细胞胚用4%海藻酸钠和2%氯化钙进行人工种皮包埋后,在无菌条件下,人工种子可转换成苗,转换率达89.7%。三七的体细胞胚起源于愈伤组织内或近表层的单个胚性细胞。体细胞胚经球形、心型、鱼雷形及子叶期等诸阶段发育成植株。通过PAS 法染色发现,胚性细胞、球形胚、早心形胚阶段有明显地淀粉积累,淀粉颗粒大而密集;到晚心形胚及子叶期体胚,淀粉颗粒小而少或消失。淀粉消长变化的规律与体细胞胚的发育有着密切的关系。     

Somatic embryogenesis and plant regeneration from immature seeds of Magnolia obovata Thunberg

We have tested plantlet formation by somatic embryogenesis using immature seeds of Magnolia obovata. Seed collection date appeared to be critical for embryogenic cell induction. The optimal collection date was 3–4 weeks postanthesis. The embryogenic cells proliferated, formed somatic embryos, and were subsequently converted into normal plantlets under optimized culture conditions. Somatic embryo formation from the embryogenic calli was better on sucrose medium than on glucose medium. The optimum level of sucrose appeared to be 3% with an average of 28 somatic embryos per plate. About 25% of somatic embryos were converted into normal plantlets in 1/2 MS medium containing gibberellic acid (GA₃). During somatic embryo germination, secondary embryogenesis was frequently observed in the lower part of the hypocotyl or radicle ends of germinating somatic embryos. Finally, about 85% of converted plantlets survived in an artificial soil mixture, were transferred to a nursery, and have grown normally.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.