Somatic embryogenesis and plant regeneration from cotyledonary explants of green gram [Vigna radiata (L.) Wilezek.]—A recalcitrant grain legume

Summary This study reports a protocol for plant regeneration from cultured explants of green gram [Vigna radiata (L.) Wilezek] via somatic embryogenesis. Somatic embryos were induced from nature cotyledons of var., TAP-7 and Pusa Baisaki when cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 2,4-dichlorophenoxyacetic acid, α-naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid singly or in combination with 2.22–8.88 μM N ⁶-benzylaminopurine (BAP) or 2.32–9.38 μM kinetin. The type and concentration of auxin and plant genotype influenced the frequency of somatic embryogenesis. NAA was the most effective auxin for somatic embryo induction. The well-developed, cotyledonary shaped embryos of var. TAP-7 germinated into plantlets at a frequency of 56.6% on MS medium supplemented with 1.88 μM abscisic acid and 6.66 μM BAP. Regenerated plants were transferred to soil and grown to maturity with 90% survival. The protocol described here offers a good potential for genetic improvement using gene transfer techniques and the production of synthetic seeds of V. radiata.     

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.     

Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.     

Somatic embryogenesis in the medicinal legume <Emphasis Type="Italic">Desmodium motorium</Emphasis> (Houtt.) Merr.

An efficient protocol was established for regeneration of Desmodium motorium via somatic embryogenesis. Embryogenic calli were induced from cotyledon segments (6 mm, 16 days old) lacking embryo axis, excised from seedlings grown in vitro on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (2.9 μM) in combination with 6-benzyladenine (BA) (4.44 and 8.88 μM). Differentiation of embryogenic calli into globular and heart-shaped somatic embryos was achieved on transfer to hormone-free MS medium. When incubated for 4 days on MS medium supplemented with BA (8.88 μM), 95% of the globular and heart-shaped somatic embryos matured into torpedo and cotyledonary stages with minimum (10%) abnormalities. Modified MS basal medium without hormones and containing half-strength macronutrients and 0.88 M sucrose was suitable for germination of mature somatic embryos. Regenerated plantlets were successfully transferred to earthen pots with survival rate of 50%. Secondary embryogenesis was observed when pre-existing somatic embryos at globular and heart-shaped stages were cultured on MS medium supplemented with various concentrations of BA, adenine sulphate (AdS) and abscisic acid (ABA) individually.     

Plant regeneration of common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis> L.) by somatic embryogenesis

This is the first report on somatic embryogenesis in common ash (Fraxinus excelsior L.). Experiments on somatic embryogenesis induction were carried out on zygotic embryos at different phases of development and maturation. The embryo axes were isolated and cultured on media containing different plant growth regulators (PGRs). Embryogenic tissues were obtained from embryos collected at an incomplete maturation phase and cultured on a modified Murashige and Skoog medium containing 8.8 μM 2,4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine (BA). Embryos isolated from seeds at an advanced stage of maturation showed only organogenetic phenomena. Embryogenic tissues were successfully subcultured and multiplied on medium containing a reduced concentration of PGRs. After their isolation, somatic embryos were induced to develop and mature by transfer to PGR-free medium and subsequent culture on medium containing 0.1 μM BA. Somatic embryos developed completely and also germinated spontaneously. Embryo germination and conversion were significantly improved when subjected to a period of storage at 4°C and transplant onto woody plant medium. Plantlets were successfully transferred to soil and acclimatized in a “misted” greenhouse.     

Primary and repetitive secondary somatic embryogenesis of <Emphasis Type="Italic">Lepidosperma drummondii</Emphasis> (Cyperaceae) and <Emphasis Type="Italic">Baloskion tetraphyllum</Emphasis> (Restionaceae) for land restoration and horticulture

Somatic embryogenesis was developed as a method of mass propagation for Lepidosperma drummondii (Cyperaceae), a difficult to propagate but important species for post-mining restoration in a region of high plant biodiversity, in the southwest of Western Australia. Cultures were initiated from excised zygotic embryos, shoot cultures to rhizomes. Only zygotic embryos of L. drummondii developed somatic embryos, with half strength Murashige and Skoog basal medium (BM) and 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) being the most effective combination. The first culture cycle yielded a mean of 30 somatic embryos per excised zygotic embryo forming an embryo cluster. After a further 6 wk in culture (on fresh BM with 1 μM 2,4-D), approximately 350 somatic embryos per starting embryo cluster were recorded. Following regular sub-culturing of primary somatic embryo clusters onto fresh media (every 4 wk), more than 74,000 secondary somatic embryos were estimated to have been produced after eight subculture periods. This translates to between 1,000 and 2,000 somatic embryos produced from an estimated 45 mg of starting tissue per culture plate or potentially 22,0000–44,000 somatic embryos per gram of tissue. This is a significant improvement over all previous methods used to propagate L. drummondii, in which typical in vitro shoot multiplication rates are as low as 1.43 per 8 wk. This also compared favourably with published data and concurrent experiments undertaken in this study (as an extra control measure) on somatic embryo production for a related species Baloskion tetraphyllum (using the same BM with 1 μM 2,4-D and coleoptile segments as explants). Various media combinations were investigated for efficacy in converting somatic embryos into plants with best results ranging from 86% to 100% conversion for B. tetraphyllum on BM without plant growth regulators. Development of L. drummondii somatic embryos into plants was not observed on BM without plant growth regulators. However, a best result of 39% conversion to plants was observed on BM with 1 μM thidiazuron. This is the first report of successful development of somatic embryogenesis and conversion of somatic embryos into plants using thidiazuron for the Australian cyperale L. drummondii.     

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l⁻¹) and BA (1.5 mg l⁻¹). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.     

Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination

Commercial deployment of clonal trees via somatic embryogenesis (SE) could increase forest productivity over conventional tree breeding techniques. However, some technical advances need to be made to use SE in clonal forestry with Pinus radiata. For example, the conversion of embryonal mass (EM) into plants is at present a major bottleneck. For this reason, maturation experiments were carried out to determine the effect of the initial amount of EM, activated charcoal (AC) and the best combination of abscisic acid (ABA), sucrose and amino acid concentration in the maturation medium. Germination was evaluated on different media formulations with and without AC. When 100 mg of EM were suspended in liquid medium without AC, cotyledonary somatic embryos were obtained in all the maturation media tested. Maturation medium supplemented with 60 μM ABA, 6% sucrose, and embryo development medium amino acid mixture produced the highest number of cotyledonary somatic embryos, between 10 and 1,550 embryos per gram of EM fresh weight. Approximately half of the tested 25 lines produced more than 600 embryos per gFW. Embryo development was the best when somatic embryos were germinated in half strength modified Quoirin and Lepoivre medium supplemented with 2 g L⁻¹ AC. This protocol simplified and improved SE maturation and germination due to the elimination of subcultures, the large number of somatic embryos obtained from a very low amount of EM, and the elimination of pre-germination treatments, resulting in a significant saving of cost and labor.     

Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

Effect of exogenous ABA on somatic embryo maturation and germination in Persian walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of embryo maturation, germination and conversion to plantlets is a major problem in many species including Persian walnut. We studied the effects of abscisic acid (ABA) and sucrose, on the maturation and germination of Persian walnut (Juglans regia) somatic embryos. Individual globular somatic embryos were grown on a maturation medium supplemented with different combinations of ABA and sucrose for ca. 1 month, until shoot meristems and radicles had developed. White and opaque embryos in late cotyledonary stage were subjected to desiccation after the culture period on maturation media. The number of germinated somatic embryos was influenced by the concentrations of ABA in the maturation medium. The best treatment for germination, in which both shoot and root were developed contained 2 mg l⁻¹ ABA and resulted in 41% conversion of embryos into plantlets. Regeneration was reduced at higher levels of ABA. While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos. On the other hand, sucrose had little influence on maturation. Normal and abnormal embryos were verified anatomically.     

Maturation,germination, and conversion of norway spruce (Picea abies L.) somatic embryos to plants

Summary Quantitative data are presented on the efficiency of three stages of plant regeneration from somatic embryos of Norway spruce (Picea abies L.): 1) Maturation, the development of immature embryos to the cotyledonary stage; 2) Germination, primary root growth; and 3) Conversion, plantlet survival and continued growth in nonaxenic conditions. Maturation frequency was calculated relative to the number of immature somatic embryos induced to develop on the basal medium of von Arnold and Eriksson (1981). The average number of immature somatic embryos was 700 per gram of embryogenic callus, on medium supplemented with ABA and IBA (1 μM each). Maturation was the least efficient stage of regeneration; an average of 3% of the embryos induced to develop reached the cotyledonary stage. Mean germination frequencies were improved on treatments which avoided immersion of the radicle in medium solidified with agar. Whereas, 27% of the somatic embryos germinated when radicles were immersed in agar medium, 45% germinated when placed on the surface of the medium, and 56% germinated when cotyledons were immersed in agar medium and the culture vessel inverted. Twenty-nine percent of the somatic embryos germinatedin vitro were converted to plants. Under greenhouse conditions these plants set dormant buds, subsequently survived overwintering (to −5°C), and renewed vegetative growth synchronously with seedlings grown under the same conditions. Our results verified long-term (2 year) growth and development potential of conifer somatic embryo plants.     

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1^−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1⁻¹) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N⁶-benzyladenine (BAP, 0.75 mg 1^−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1^−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.     

Abscisic acid and ancymidol promote conversion of somatic embryos to plantlets and secondary embryogenesis inAsparagus officinalis L.

Summary The effects of abscisic acid (ABA) (0, 0.09 μM, 0.19 μM, 0.28 μM, and 0.38 μM) or ancymidol (0, 0.98 μM, 1.95 μM, 2.93 μM, 3.90 μM) in embryo germination medium on the conversion of primary embryos to plantlets and secondary embryogenesis were evaluated for asparagus. ABA and ancymidol each significantly enhanced both responses. ABA was more effective than ancymidol in promoting the conversion of primary embryos to plantlets, while the converse was true for the production of secondary embryos. The most effective treatments for embryo conversion were 0.19 and 0.28 μM ABA; 75–77% bipolar and 55–57% globular embryos converted to plantlets. For secondary embryogenesis, the most effective treatments were 1.95 and 2.93 μM ancymidol; 99–101 and 84–86 somatic embryos were produced from 10 globular and 10 bipolar embryos, respectively. Bipolar embryos generally converted to plantlets better than globular embryos, but more secondary embryos were produced from globular embryos than from bipolar embryos in all treatments. ABA and ancymidol also affected the morphology of the plantlets produced. The plantlets from the embryos incubated on the medium with ancymidol had strong and thick shoots and roots, while those on the medium with ABA had long, thin shoots and short thin roots.     

Unfertilized ovary: a novel explant for coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) somatic embryogenesis

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 μM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 μM abscisic acid, followed by plant regeneration medium (with 5 μM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.     

Development and germination of American chestnut somatic embryos

American chestnut (Castanea dentata (Marsh.) Borkh.) plants were regenerated from developing ovules through somatic embryogenesis. On an initiation medium containing 18.18 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine (BA), 25 out of 1,576 ovules were induced to form proembryogenic masses (PEMs). These PEMs were cultivated on a development medium for 4 weeks. Individual somatic embryos were then grown on a maturation medium for at least one month, until shoot meristems and radicles were developed. Both development and maturation media consisted of Gamborg's B-5 basal medium, 0.5 μM BA, and 0.5 μM α-naphthaleneacetic acid, but the former contained 20 g l⁻¹ sucrose and the later contained 60 g l⁻¹ sucrose. A range of 86 to 586 embryos per gram PEMs was observed beyond the cotyledonary stage. These embryos then germinated, resulting in plantlets with a 3.3% conversion rate. An additional 6.3% of the mature embryos produced shoots, which could also result in plantlets by rooting of microcuttings. Proembryogenic masses that were established in continuous culture and maintained on initiation medium for 17 months retained regenerability, though the embryo yield decreased over time. Twenty plantlets were acclimatized and grown in potting mix in a greenhouse. The largest 6 were transplanted, along with seedling controls, into a nursery bed in 1997. As of July, 1999, 4 out of the 6 were surviving. This revised version was published online in June 2006 with corrections to the Cover Date.     

Repetitive somatic embryogenesis in carnation on picloram supplemented media

An efficient method of repetitive somatic embryogenesis and plant regeneration of two carnation cultivars (Sagres and Impulse) was established using a two-step protocol. In the first step, embryogenic tissue were induced from petal explants on MS culture medium containing 9% sucrose (w/v), 9 μM 2,4-D and 0.8 μM BA. In the second step, embryogenic tissue transferred onto the MS medium containing 3% sucrose supplemented with different concentrations of picloram (0.8, 2, 4, 8 and 16 μM) to produce primary somatic embryos. Precotyledonary clumps and cotyledonary somatic embryos were then isolated and subcultured onto the same media as the second step where they formed secondary somatic embryos in repetitive cycles. Cotyledony somatic embryos were converted into plantlets when they transferred onto the growth regulator-free half-strength MS medium followed by being acclimated to the greenhouse conditions.     

Somatic embryogenesis and plant regeneration from callus culture of Acacia catechu-a multipurpose leguminous tree

Plant regeneration via somatic embryogenesis was achieved from callus derived from immature cotyledons of Acacia catechu Willd. on Woody Plant Medium (WPM) supplemented with 13.9 M kinetin and 2.7 M 1-naphthaleneacetic acid. The addition of 0.9–3.5 mM L-proline to the medium influenced development of somatic embryos and also promoted secondary somatic embryogenesis. The light-green somatic embryos germinated on half-strength MS medium supplemented with 2% (w/v) sucrose. Somatic embryos germinated into plantlets that were acclimatized in the greenhouse and subsequently transferred to the field.     

Somatic embryogenesis and plantlet regeneration from leaf segments of Piper colubrinum

Direct somatic embryogenesis from in vitro-cultured leaf segments of multiple disease-resistant pepper, Piper colubrinum Link is reported. Somatic embryos were initiated on Murashige and Skoog's (1962) basal medium containing 2.2 μM benzyladenine+0.46 μM kinetin and multiplied profusely through secondary embryogenesis on the same medium. Some 91% somatic embryos converted into plantlets on MS medium supplemented with 4.4 μM benzyladenine+0.23 μM kinetin and plantlets developed on half-strength MS+2.4 μM indole-3-butyric acid. Plantlets were hardened, transferred to soil, and 100% of plants survived. Various developmental stages of somatic embryogenesis were studied using histological methods. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.