Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Enhanced somatic single embryo formation by plasmolyzing pretreatment from cultured ginseng cotyledons

Cotyledon explants of Panax ginseng zygotic embryos directly produced somatic embryos on Murashige and Skoog medium without growth regulators. Somatic embryos were formed only near the proximal excised region of cotyledons. Multiple and/or single embryos were formed and the frequency of these formations differed according to the degree of maturity of the zygotic embryos used as the explant source. When cotyledon explants pre-plasmolysed (1.0 M sucrose for 24 h), the frequency of single embryo formation was enhanced regardless of cotyledon maturity. In addition, the distribution pattern of somatic embryos changed markedly because the embryos were formed over the whole surface of the cotyledons. Histological observation revealed that plasmolyzing pretreatment broke the plasmodesmatal connection between cells and when the embryogenic cell divisions commenced, plasmodesmatal strands were hardly observed except for newly formed cell walls. This indicates that the enhanced single embryo formation over the entire surfaces of cotyledon explants might be the result of an interruption of cell–cell interaction by plasmolyzing pretreatment.     

Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

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.     

栓皮栎体细胞胚胎发生的细胞组织学观察

以栓皮栎未成熟合子胚为外植体,在添加0.25mg/L 2,4-D和0.5mg/L 6-BA的MS培养基上6周可诱导产生2种类型的胚性愈伤组织,一种表面具光泽、白色;另一种表面光滑湿润具光泽,色泽淡黄或无色透明。组织切片表明,胚性愈伤组织的细胞体积小,细胞核大,细胞质浓,细胞排列紧密;非胚性愈伤组织细胞的体积大,细胞核小,细胞质稀薄。胚性细胞团培养在不含激素的培养基上可诱导产生体细胞胚。体细胞胚直接起源于胚性细胞团表皮或近表皮的单细胞,经历与合子胚相似的球形胚、心形胚、鱼雷胚和子叶胚发育阶段。所有发育时期的体细胞胚的胚轴、子叶均产生次生体胚,它们起源于细胞质较浓的表皮单细胞。     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of direct somatic embryogenesis from mature zygotic embryos ofPanax ginseng C. A. Meyer

Culture conditions were optimized for somatic embryogenesis ofPanax ginseng. The highest frequency of embryo formation was obtained when tissues were excised from the middle region of the cotyledon segments of zygotic embryos. Only treatment with light could stimulate the formation of single-type somatic embryos, whereas multiple-type somatic embryos and calli were observed under dark conditions. The highest production of somatic embryos was found with an NH₄ ⁺:NO₃ ratio of 21:39. Among the tested media (MS, B5, and SH), maximum formation of somatic embryos was obtained when cotyledon expiants were cultured on an 1% agar MS medium supplemented with 5% sucrose. Regenerated ginseng plantlets were transferred to an autoclaved soil mixture in the greenhouse. These transformants showed no detectable variations in their morphology or growth characteristics compared with the donor plant.     

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.     

Morphology and Anatomy of Pisum Sativum Somatic Embryos

The morphological and anatomical aspects of direct and indirect somatic embryogenesis in pea were described. Direct embryos were induced from shoot apical meristems of 3 to 5-d-old pea seedlings, embryogenic callus originated from immature pea zygotic embryos or shoot apices. Auxin (picloram, 2,4-dichlorophenoxyacetic acid) was necessary to induce somatic embryos. The developmental stages typical for pea zygotic embryos were detected. Globular and heartshaped somatic embryos were morphologically similar to their zygotic counterparts; in contrast, torpedo and cotyledonary somatic embryos displayed great morphological variation, which affected mainly cotyledons (size, shape, number). Based on anatomical sections, possible ways of somatic embryo formation and localization of initiation sites within primary explant tissue have been proposed. The multicellular origin of somatic embryos is supposed in both systems of pea somatic embryogenesis under investigation.     

Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.)

Summary Four scanning electron microscope techniques for preparing somatic and zygotic embryos of white spruce (Picea glauca [Moench] Voss.) were compared. Direct sputter coating without critical point drying worked well for desiccated embryos while conventional methods using chemical fixation were appropriate for hydrated somatic embryos. Low temperature scanning electron microscopy and plastic replicas provided excellent specimens of all embryos studied. Plastic replicas were used to document cotyledon formation and growth during maturation of somatic embryos. Apart from some differences in embryo size, orientation of cotyledons and surface wrinkling, the general morphology of mature somatic embryos of white spruce was very similar to zygotic embyros at a similar stage of development.     

Imbibition of white spruce seeds and somatic embryos: A study of morphological changes in an environmental scanning electron microscope and potassium leakage

Summary Potassium leakage and morphological changes during imbibition of white spruce [Picea glauca (Moench) Voss] seeds and somatic embryos were investigated. A single desiccated somatic embryo, a single somatic embryo exposed to a high relative humidity environment for 2 d, and a single dry zygotic embryo leaked similar amounts of potassium over a 120-min period of imbibition in liquid germination medium. A seed without a seed coat leaked two and eight times more potassium than a single whole seed and a single zygotic embryo, respectively. Nearly 50% of the potassium leaked for all tissues was leaked within the first 20 min of imbibition. Exposure of somatic embryos to an environment with high relative humidity resulted in a reduction in the percentage of potassium leaked after 80 and min to levels equivalent to those for zygotic embryos. Using an environmental scanning electron microscope, we found that desiccated somatic embryos and dry zygotic embryos had wrinkled surface cells, with cells in the surface of zygotic embryos being more shrunken in appearance. Imbibition of both types of embryos in water resulted in turgid surface cells after 2 h. Imbibition in liquid germination medium did not cause much hydration of surface cells, which still had wrinkled appearances after 2 h. Finally, imbibition on filter paper on semisolidified germination medium resulted in slower hydration of somatic and zygotic embryos. Cells near the medium appeared hydrated while cotyledon surface cells furthest from the medium resembled cells in desiccated embryos.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Somatic embryogenesis from immature zygotic embryos of annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl⁻¹ activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.     

Differences in protodermal cell wall structure in zygotic and somatic embryos of <Emphasis Type="Italic">Daucus carota</Emphasis> (L.) cultured on solid and in liquid media

The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.     

Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs

Embryogenic tissues have been initiated on cotyledon explants dissected from seedlings or emblings of hybrid firs. Cotyledons of seedling origin (Abies alba x A. cephalonica) gave a relatively low initiation frequency (1.94 percnt;). In embling-derived cotyledons (Abies alba x A. cephalonica, Abies alba x A. numidica), the initiation was cell-line dependent and reached values between 1.25 and 24.28 percnt;. The established embryogenic cell lines are being maintained in long-term cultures.The origin and development of the somatic embryos have been traced histologically. The early stages of somatic embryo development have been characterised by cell division activity (predominantly periclinal) in the epidermal and subepidermal layers of cotyledons and subsequently by development of nodular structures. Further differentiation led to the formation and emergence of somatic embryos on the surface of cotyledons.Somatic embryo development and plantlet regeneration occurred from proliferating tissues initiated from cotyledons of embling as well as seedling origin.