Induction of somatic embryos from cultures of <Emphasis Type="Italic">Agave vera-cruz</Emphasis> Mill.

Somatic embryogenesis from cultures of shoot apices, cotyledon and young leaves of in vitro shoots of Agave vera-cruz Mill. was studied. Embryogenic callus was obtained when explants were cultured on Murashige and Skoog’s (MS) medium (1962) supplemented with L₂ vitamins, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-d) or 5.37 μM ∝-naphthalene acetic acid (NAA). Somatic embryos differentiated from this embryogenic callus upon subculture to maturation/conversion medium containing cytokinin either alone or with auxin and l-glutamine. The best combination of growth regulators for development of somatic embryos was found to be 5.37 μM naphthalene acetic acid plus 0.91 μM zeatin and 40 g/l sucrose. The conversion frequency of somatic embryos to plantlets varied from 46–50%. Rooted plantlets were transferred directly to pots containing a soil, sand, and manure mixture without any hardening phase with 96–98% survival of the plantlets. Based on the histological observations, the potential origin of the somatic embryo is discussed.     

Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures

The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the culture treatments and, in general, low light intensities (50 μmol m^–2 s^–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart- and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively. Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997     

Somatic embryogenesis from mature leaves of rose (Rosa sp.)

Several plant growth regulators (0.3–53.3 μm 6-benzyladenine, 2,4-dichlorophenoxyacetic acid, gibberellic acid, 3-indoleacetic acid, p-chlorophenoxyacetic acid, kinetin and α-naphthylacetic acid), alone or in combination, and culture conditions were tested for their capacity to induce somatic embryogenesis from mature leaf and stem explants of rose (Rosa sp.) of four commercial rose cultivars (Baccara, Mercedes, Ronto and Soraya). Somatic embryos were only induced from mature leaf explants derived from Soraya on Murashige and Skoog (MS) medium supplemented with 53.5 μm p-chlorophenoxyacetic acid and 4.6 μm kinetin, although satisfactory callus induction rates were obtained from all cultivars. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto a MS medium supplemented with 5.2 μm 6-benzyladenine and 5.7 μm 3-indoleacetic acid. Germination of mature embryos took place after subculturing them onto medium of the same composition. Plantlets regenerated from embryos and bearing three to four leaves were transferred to a greenhouse. Received: 4 February 1997 / Revision received: 28 August 1997 / Accepted: 1 October 1997     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Induction of somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of <Emphasis Type="Italic">eruca sativa</Emphasis> mill

Summary A protocol was developed for high frequency somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of Eruca sativa. Explants grown on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-D formed embryogenic callus after 4 wk of culture. Secondary somatic embryos were also produced from primary somatic embryos on MS medium containing 0.56 μM 2,4-D. Somatic embryos developed into mature embryos on MS medium in the presence of 45 gl⁻¹ polyethylene glycol. After desiccation, somatic embryos developed into plantlets by culturing the mature somatic embryos on 1/2 x MS medium containing 0.24 μM indole-3-butyric acid.     

In vitro morphogenesis of Ceratozamia hildae and C. mexicana from megagametophytes and zygotic embryos

Organogenesis and somatic embryogenesis were induced from megagametophyte and zygotic embryo explants of 2 cycad species, Ceratozamia hildae and C. mexicana, cultured on modified B5 medium containing kinetin (0–13.9 M) and 2,4-d (0–9.0 M). Organogenesis occurred from megagametophyte explants of both species on the range of media tested. Somatic embryogenesis was largely restricted to zygotic embryo explants. Somatic embryos germinated in vitro: however, rooting of adventitious shoots was unsuccessful.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid R-014515.     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.     

Induction of somatic embryogenesis in cultured cells of <Emphasis Type="Italic">Chenopodium quinoa</Emphasis>

A major limiting factor for quinoa cultivation as a grain crop on a large scale are virus diseases, in particularly seed borne diseases. Therefore, a somatic embryogenesis protocol is a necessary tool to produce virus free plants. Somatic embryogenesis offers the possibility of mass production of transgenic plants and therefore can be used easily to study the effect on plants resulting from breeding processes. An in vitro protocol has been developed for somatic embryogensis from calluses and cell cultures of Chenopodium quinoa. Callus was induced from hypocotyl explants within 2 weeks of culture on a modified Murashige and Skoog (MS) medium supplemented with 0.45 M 2,4-D. Calluses were cultured on solid or liquid MS medium and later the development of somatic embryos was observed on both employing the same MS medium without 2,4-D. To our knowledge this is the first report of somatic embryogenesis in Chenopodium quinoa.     

Relationship between auxin-induced cell proliferation and somatic embryogenesis in culture of carrot cotyledons

We elucidated the relationship between cell proliferation and somatic embryogenesis in the culture of carrot cotyledons. Fresh weights of the cotyledon expiants were determined every five days while being cultured on a medium containing 2,4-D. Callus production increased exponentially from Day 20 to Day 25, showing a two-fold rate of proliferation. To examine the embryogenic potential of the callus, we pre-cultured cotyledon explants on an MS medium with 2,4-D, then transferred them to an MS basal medium at five-day intervals. Somatic embryos formed most frequently when the cotyledons were pre-cultured for 20 days on an MS medium that contained 5 μ2,4-D. The frequency of somatic embryo formation was 81%, while that of normal embryos with two cotyledons was 51% among those formed on a hormone-free medium. We used FACScan analysis to relate the embryogenic potential of the callus to the S phase in the cell cycle of cultured cells. The S phase was high after 25 days of culture on the medium with 5 μM 2,4-D. In contrast, the frequency of normal embryogenesis was higher at Day 20 of the pre-culture period. Culturing embryogenic calli on a medium with 5 μM 2,4-D was most favorable for producing somatic embryos with two cotyledons. We verified that active somatic embryogenesis was apparently related to cell division activity; somatic embryos induced from actively dividing cells were apt to accompany cotyledonary abnormality.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

Effect of thidiazuron on somatic embryogenesis of Cayratia japonica

Unpollinated ovary explants of Cayratia japonica (Thump.) Gagnep, were cultured on the revised Murashige & Skoog's medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) alone, or in combination with 0.009 M thidiazuron (TDZ) or 0.23 M kinetin for induction of embryogenic callus. The best results were obtained on medium containing 2.3 – 4.6 M 2,4-d and TDZ. When the calluses were subcultured on the basal medium (BM), somatic embryogenesis took place spontaneously at surfaces of the calluses, but only about 5% of the somatic embryos could develop to cotyledonary stage and most of the rest remained at the globular stage of development. If the calluses were transferred onto medium containing TDZ or TDZ combined with 0.27 M -napthaleneacetic acid, the number of cotyledonary somatic embryos increased up to 25%. When the somatic embryos of different stages were transferred onto fresh BM, only the cotyledonary embryos could convert into the plantlets. The results revealed that for the induction of embryogenic callus and somatic embryogenesis of Cayratia japonica, both cytokinin and auxin are required in the medium and the cytokinin activity of TDZ is much stronger than that of kinetin even when the concentration of TDZ used was only 4% of kinetin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea)     

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.     

A novel system for rapid high frequency somatic embryogenesis in Medicago sativa

A novel, genotype dependent system for rapid high frequency somatic embryogenesis in Medicago sativa L. was developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g l−¹ casein hydrolysate, 9 μ M 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.2 μ M kinetin. An efficient two-step, two-medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 μ M 2,4-D and 4.7 μ M kinetin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4-D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogenic and very little callus proliferation occurred when this method was used. Selected clones from three ssp. falcata -based M. sativa cultivars showed a response very similar to the highly regenerable falcata clone F1.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa , with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomile (Chamomilla recutita L.)

A protocol has been developed for the induction of somatic embryogenesis from flower explants of chamomile (Chamomilla recutita L.). The effects of several plant growth regulators [α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA) and kinetin (Kin), alone or in combination] and the flower type (disk or ray flower) were investigated. Both types of flowers responded to the callus and shoot induction treatments, but formation of globular somatic embryos took place only on disk-flower-derived explants after 2–4 weeks of culture on a Murashige and Skoog (MS) medium supplemented either with 8.87 μm BA and 1.07 μm NAA or with 26.8 μm NAA and 11.5 μm Kin. However, fully developed, cotyledonary-stage somatic embryos could be induced only on the NAA/Kin medium, 10 weeks after culture initiation. Germination of the embryos and plant regeneration took place after subculture for 4–5 weeks onto medium of the same composition. Plantlets regenerated from embryos flowered in vitro on a MS medium supplemented with 8.87 μm BA and 1.07 μm NAA. The significance of the results with respect to chamomile micropropagation and the utilization of wild populations in breeding programs is discussed. Received: 6 April 1998 / Revision received: 12 October 1998 / Accepted: 28 October 1998     

The embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.