Recurrent Somatic Embryogenesis Research Articles

Induction of somatic embryogenesis in Tecomella undulata (Sm.) Seem using a pistillate explant

Desert teak (Tecomella undulata (Sm.) Seem) a multipurpose ornamental tree native to the arid and semi-arid tropics has entered the endangered plant category mainly as a result of the species' ineffective seed reproduction system. The tree usually reproduces through a few root suckers in old stands. Conventional methods of plants multiplication could not offer a viable practice for its mass multiplication. Low adventitious rooting of the cuttings has been the principal cause of failure in its vegetative propagation. Hence, the present research was planned and conducted to evaluate the feasibility of somatic embryogenesis in this species, the pathway that bypasses the need for rooting stage by developing bipolar embryos. Ovary explant was cultured in modified Murashigue & Skoog (MS) medium supplemented with different auxins and cytokinins. The results showed α-naphthalene acetic acid (NAA) was superior in inducing embryogenic callus. NAA ranging 5.4-21.5 μM could induce the highest embryogenic calli which exhibited developing pro-embryogenic masses (PEM) and globular somatic embryos. The calli which were induced by the use of 2,4-dichlorophenoxyacetic acid (2,4-D) were poor in quality and showed no morphogenesis potency. Individual application of Thidiazuron (TDZ) and N6-benzyladenine (BA) induced good callogenesis at low concentrations but the calli were non-embryogenic with both. The proliferation of embryogenic calli was the best in a hormone-free medium. However, the media containing 40.5 and 54 μM NAA alone could induce somatic embryos along with callus proliferation. Low BA-contained medium (0.9-4.44 μM) led to recurrent somatic embryogenesis. Neither BA and GA3, nor the elevating sucrose concentration could cause further development and maturation of the somatic embryos induced during previous stages (callogenesis and callus proliferation). More research is required to optimize the maturation stage. The findings of the present study can be useful for future studies in the micropropagation of this recalcitrant specieslationships in Indian mustard under heat stress and the differential remobilization efficiencies in the advanced breeding lines.

Introduction of the Nicotiana protein kinase (NPK1) gene by combining Agrobacterium-mediated transformation and recurrent somatic embryogenesis to enhance salt tolerance in cauliflower

Cauliflower is exposed to various biotic and abiotic stresses, including increased salinity due to the intensive irrigation of crops. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play important roles in regulating innate immune responses in plants. Based on involvement of tobacco MAP kinase kinase kinase (NPK1) in stress response, the effect of the expression of NPK1 transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. The Agrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring the NPK1 and phosphinothricin N-acetyltransferase (bar) genes, the cyclic somatic embryogenesis regeneration pathway, the application of acetosyringone (AS) during co-cultivation and a delayed phosphinothricine (PPT) selection procedure provided sufficient transformation efficiency of 7.33% without escapes. PCR analysis indicated the integration of both NPK1 and bar transgenes in regenerated cauliflower lines. Transgenic cauliflower lines, exposed to NaCl stress in vitro, showed higher growth rates, greater ability to retain chlorophyll and carotenoids, and increased osmotic regulation capacity compared with non-transformed control plants. The tolerance level of transformed lines correlated with the level of NPK1 gene expression estimated by RT-qPCR, and the L2 line with the highest NPK1 expression displayed the greatest tolerance to NaCl stress. None of the obtained cauliflower transformed lines grown in greenhouses showed any morphological or yield differences compared with non-transformed plants. Furthermore, the expression of the bar gene facilitated the tolerance of transformed lines to the total herbicide PPT, applied at concentrations 2–3 times higher than those routinely used for weed control in the crop field. The results underlined that constitutively expressing NPK1 can significantly contribute to enhanced salt stress tolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerance cruciferous vegetable lines.

Recurrent somatic embryogenesis and development of somatic embryos in Akebia trifoliata (Thunb.) Koidz (Lardizabalaceae)

A simple and effective protocol was established for recurrent somatic embryogenesis and plant regeneration in Akebia trifoliata (Thunb.) Koidz. Somatic embryos directly formed from the root zone of the immature zygotic embryos cultured on MS medium devoid of plant growth regulator (PGR). The induction frequency of immature zygotic embryos was 52.5%, and the mean number of somatic embryos reached up to 19.5. Secondary somatic embryos arose on the primary embryos at a high frequency (95.8%), and this process maintained in a recurrent way on PGR-free MS medium. The highest mean number of somatic embryos also appeared in the primary embryos which can reach up to 44.7. While both of the embryogenic potential and mean number of embryos per explant displayed a gradual diminution with subculturing. The addition of 0.5 mg l−1 6-benzyladenine in the 1/2 MS regeneration medium can significantly improve the frequency of somatic embryos convert to plantlets. The microscopic analysis revealed that the development process of somatic embryos via the globular, heart, torpedo and cotyledonal stages in A. trifoliata, the histological analysis showed that somatic embryos directly initiated from root epidermal cells and there are no vascular connections between the somatic embryos and maternal tissue. Regenerated plantlets acclimated successfully to greenhouse conditions. Approximately 65% plantlets survived and displayed no morphological characteristics differences with seed-derived plants. The simple and effective protocols established in this study will promote large-scale clonal propagation and genetic improvement of A. trifoliata. Somatic embryogenesis has never been reported in Akebia trifoliata (Thunb.) Koidz. The study showed the origin and development process of somatic embryos in Akebia trifoliata (Thunb.) Koidz.

Somatic embryogenesis from stamen filaments of Aesculus flava Sol. and peroxidase activity during the transition from friable to embryogenic callus

Species of the genus Aesculus are among the most attractive ornamental woody plants. Conventional propagation methods of these species are either inefficient (stem cuttings) or unsuitable for clonal propagation (seeds). The aim of the present study was to develop an efficient protocol for clonal propagation of elite specimens of yellow buckeye (Aesculus flava) by somatic embryogenesis. For this purpose, stamen filaments of yellow buckeye were cultivated on media supplemented with 1, 5 or 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 0, 1, 5 or 10 μM 6-furfurylaminopurine (Kin), either under light or dark conditions, for 8 weeks, and then subcultivated on plant growth regulator (PGR)-free medium with 400 mg/l of glutamine. The highest somatic embryo (SE) initiation rates were achieved for the explants cultivated in darkness on medium containing 1 μM 2,4-D + 10 μM Kin during callus induction (CI) phase. Embryogenic calli (EC) were initiated from friable calli, starting from the 7th week of culture initiation, while SEs appeared two weeks later, following a week of subcultivation of the explants on PGR-free medium. EC and SEs were observed only in the explants grown in darkness during CI phase. Minimal duration of CI phase and darkness necessary for SE induction was four weeks, while the highest embryogenic response was achieved when each lasted for 8–10 weeks. Obtained SEs were efficiently multiplied on medium supplemented with 0.05 μM 2,4-D + 5 μM Kin by recurrent somatic embryogenesis. SEs at globular stage of development exhibited the highest capacity for secondary SE regeneration. High germination and conversion rates were attained in cotyledonary-stage SEs cultivated on medium with 0.05 μM 2,4-D + 5 μM Kin, but this phase needs to be further optimised, since the obtained plants failed to acclimatize to greenhouse conditions. During the transition of calli from friable to embryogenic state, total peroxidase (POX) activity significantly decreased, indicating their involvement in the acquisition of embryogenic capacity. The presented protocol is suitable for clonal propagation and genetic transformation of this ornamental species, and POX activity may be used as a marker for SE initiation.

Recurrent Somatic Embryogenesis and Plantlet Regeneration in Psidium guajava L.

A simple and efficient protocol for recurrent somatic embryogenesis and plant regeneration is one of the prerequisites for genetic improvement of guava. An efficient reproducible regeneration somatic embryogenesis protocol was developed in four genotypes of Psidium guajava L. using immature zygotic embryo as starter explant. Somatic embryogenesis induction was obtained on MS basal medium supplemented with 2.0 mgL-1 2, 4-D, 400 mgL-1 L-glutamine, 6% sucrose and 500 mgL-1 Malt extract. Following SE induction different developmental stages of somatic embryos (Globular, heart-shaped, torpedo, cotyledonary) was directly obtained and further recurrent embryogenesis also obtained upon prolonged incubation in induction media. Addition of polyethylene glycol (50 mgL-1) significantly improved the embryos maturation in MS supplemented with and 3% sucrose. The regeneration in MS medium supplemented with BAP (0.5 mgL-1), NAA (0.2 mgL-1), casein hydrolysate (100 mgL-1) and 3% sucrose. High plant regeneration frequency and intensity of somatic embryos (58.5%) obtained. Plant maturation on MS medium supplemented with BAP 2.0 mgL-1 with 2% sucrose. The rooted plants was successfully acclimatize in the greenhouse with a survival rate of 85%. This somatic embryogenesis protocol developed would be helpful in establishment of genetically modified guava aimed at seedlessness, increased shelf life and wilt disease.

Recurrent somatic embryogenesis and plant regeneration from seedlings of Hepatica nobilis Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.

Stress induced acquisition of somatic embryogenesis in common bean Phaseolus vulgaris L.

Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. We used an alternative strategy to induce SE in common bean based upon the use of a cytokinin (BAP) coupled with osmotic stress adaptation instead of SE response that is induced by auxins. Explants derived from zygotic embryos of common bean were subjected to osmotic stress (sucrose 12 % w/v, 0.5 M) in the presence of BAP 10 mg/L and adenine free base 40 mg/L to induce somatic embryos from specific competent cells of the apical meristem and cotyledonary node. Somatic embryos were obtained from the competent cells in a direct response (direct SE). In a secondary response (secondary SE), those somatic embryos formed proembryogenic masses (PEM) that originated/developed into secondary somatic embryos and showed the SE ontogeny. Maturation of somatic embryos was achieved by using different osmolality media and converted to plants. Full-visible light spectrum was necessary to achieve efficient plant regeneration. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and bioballistics as well as for basic biochemical and molecular biology experiments.

INDUCTION OF DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE EMBRYOS OF BRASSICA OLERACEA VAR. SABAUDA L.

An efficient plant propagation system through somatic embryogenesis by using immature zygotic embryos was established in Savoy cabbage (Brassica oleracea var. sabauda L.). The effects of zygotic embryo age, presence of 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) (B5-D) in the culture medium, and the pH (5.0 and 5.8) of the B5 plant growth regulator (PGR)-free (B5-0) induction media were investigated. It appeared that the developmental stage of the immature zygotic embryos used as explants was the most important factor for somatic embryogenesis. Highest frequency of somatic embryogenesis (86.67%) was achieved directly on zygotic embryos at cotyledonary stage (1.8 mm long) of development that cultured on B5-0 medium. Optimal pH of the medium was 5.0. Mean number of primary somatic embryos per explant on B5-0 pH 5.0 medium was 7.2. The inclusion of 2,4-D into the induction medium significantly reduced both frequency of somatic embryogenesis (to 53.34 %) and mean number of embryos per explant (to 2.62) in cotyledonary zygotic embryos. After transferring to PGR-free Murashige and Skoog (MS-0) medium 69.69% of the primary somatic embryos of the Savoy cabbage produced secondary somatic embryos (SSEs) Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of Savoy cabbage.

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l−1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.

Recurrent somatic embryogenesis in long-term cultures of Gentiana lutea L. as a source for synthetic seed production for medium-term preservation

Our aim was to establish an efficient and reproducible system for producing synthetic seeds from recurrent somatic embryogenesis in long-term cultures of Gentiana lutea L. This species is a vulnerable medicinal plant, protected both at the national and international levels, and is included in different Red Lists and Books. In vitro culture, as an alternative to classical methods of preservation, allows for the cyclic multiplication of plant material and short-, medium- and long-term preservation of tissue collections. Biotechnological approaches allow for maintenance of the plant material in a confined space and protection against biotic and abiotic factors. Somatic embryogenesis (SE) is the most efficient way to regenerate plants, ensuring material for preservation and fundamental research. In our experiment, recurrent somatic embryogenesis was developed in long-term cultures in the presence of sugar alcohols (mannitol, sorbitol) and in the absence of growth factors. This process proceeded at a high rate, with adventive somatic embryos being generated in a continuous process, followed by maturation, germination and development into plants. To follow the somatic embryogenesis process, histological samples were made. We used these embryogenic cultures for synthetic seed production and medium-term conservation. The viability of somatic embryos after moderate osmotic stress treatment was tested using TTC. Our methodology relied on the induction of somatic embryogenesis in the presence of auxins in the first cycle of in vitro cultures, long-term high embryogenic culture maintenance in the presence of sugar alcohols and synthetic seed production.

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

SummarySecondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.

Regeneration and molecular analysis of date palm ( Phoenix dactylifera L.) plantlets using RAPD markers

Clonal plants of date palm (Poenix dactylifera L.) were regenerated from juvenile leaves on regimes consisting of the use of 2,4-D. Success depended on the concentrations of 2,4-D tested. The cultures produced adventitious shoot buds directly at the basal part of leaves as well as excessive calli. Somaclonal variation in plantlets which can be induced by 2,4-D during recurrent somatic embryogenesis were tested by random amplified polymorphic DNA (RAPD) profiles. Nine arbitrary 10- mer primers were used to amplify DNA from 180 plantlets. RAPD patterns of the plantlets were identical with the original plant mother, indicating that 2,4-D did not induce somaclonal variation that can be detected by the RAPD technique.

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l −1 KNO 3, 0.29 g l −1 NH 4NO 3, 3 mg l −1 thiamine HCl, 0.5 mg l −1 pyridoxine HCl, and 5 mg l −1 nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.

2,4,5-Trichlorophenoxyacetic acid promotes somatic embryogenesis in the rose cultivar ‘Livin’ Easy’ (Rosa sp.)

Somatic embryogenesis (SE) offers vast potential for the clonal propagation of high-value roses. However, some recalcitrant cultivars unresponsive to commonly employed SE-inducing agents and low induction rates currently hinder the commercialization of SE technology in rose. Rose SE technology requires improvement before it can be implemented as a production system on a commercial scale. In the present work, we assessed 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a synthetic auxin not previously tested in rose, for its effectiveness to induce SE in the rose cultivar "Livin' Easy" (Rosa sp.). We ran a parallel comparison to the commonly used 2,4-dichlorophenoxyacetic acid (2,4-D). We tested each auxin with two different basal media: Murashige and Skoog (MS) basal medium and woody plant medium (WPM). MS medium resulted in somatic embryo production, whereas WPM did not. 2,4,5-T induced SE over a greater concentration range than 2,4-D's and resulted in significantly greater embryo yields. 2,4,5-T at a concentration of 10 or 25 microM was better for embrygenic tissue initiation than 2,4,5-T at 5 microM. Further embryo development occurred when the tissue was transferred to plant growth regulator (PGR) free medium or media with 40% the original auxin concentration. However, the PGR-free medium resulted in a high percentage of abnormal embryos (32.31%) compared to the media containing auxins. Upon transfer to germination medium, somatic embryos successfully converted into plantlets at rates ranging from 33.3 to 95.2%, depending on treatment. Survival rates 3 months ex vitro averaged 14.0 and 55.6% for 2,4-D- and 2,4,5-T-derived plantlets, respectively. Recurrent SE was observed in 60.2% of the plantlets growing on germination medium. This study is the first report of SE in the commercially valuable rose cultivar 'Livin' Easy' (Rosa sp.) and a suitable methodology was developed for SE of this rose cultivar.

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and theβ-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl(-1)) was an important part of our transformation protocol. Transformed lines still have strongβ-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).

Anther and microspore culture ofLupinus albus in liquid culture medium

Embryo-like structures (ELS) with clearly defined cotyledons and radicles have been regenerated fromLupinus albus L. microspores. ELS induction from microspores in liquid medium was successful, with a maximum of over 3,500 ELS per anther being produced from microspores predominantly at the early binucleate stage of development. Cytological analysis revealed that first pollen grain mitosis occurred in closed buds with maximum ELS production being obtained from buds at the point of first petal emergence. Generally there was a lack of synchronisation of microspores within anthers from the less mature bud germination from ELS has not been observed; recurrent somatic embryogenesis occurred following internal cleavage within the ELS or on the surface of the ELS. Methods of increasing the level of mature ELS capable of germination are under investigation.

Characterization of recurrent somatic embryogenesis of alfalfa on auxin-free medium

Callus cultures from 300 genotypes of alfalfa (Medicago sativa L.) were initiated from leaf, petiole, and internode explants placed on Blaydes medium containing 10.74 μM α-naphthaleneacetic acid, 11.42 μM indole-3-acetic acid, and 9.29 μM kinetin. Five genotypes produced somatic embryos. Upon transfer of these embryos to growth regulator-free Murashige and Skoog medium with B5 vitamins, new somatic embryos repeatedly formed directly on older somatic embryos without an intervening callus phase in a cycle lasting about 30 days. These cultures have been maintained for two years, during which time their embryogenic capacity has remained stable. New embryogenic cultures could be started repeatedly from these genotypes. The elimination of sugars from the medium could stop recurrent embryogenesis. Glucose, maltose, and fructose stimulated recurrent embryogenesis more effectively than sucrose. Sucrose was superior to lactose, while sorbitol and mannitol did not stimulate recurrent somatic embryogenesis. The absence of nicotinic acid in the medium, as long as sucrose was present, was lethal to embryos of three of the five tested genotypes. The ability of this system to propagate embryos exponentially offers potential for development of new gene transfer systems and application to artificial seed technology.

In VitroSomatic Embryogenesis and Plant Regeneration inClitoria ternatea

A sustainable plant regeneration system in vitro through somatic embryos from mature sexual embryos has been reported in Clitoria tematea. Somatic embryos developed through callus from seedling roots on hormone-free MS medium (MS,). Addition of growth hormones, KN 0-5 mg dm-3 (MS2) or K.N + 1AA 0-5 mg dm^3 of each (MS3) induced direct somatic embryos, in high frequency, on split root and hypocotyl systems. The embryogenie potential varied with the organ, roots or hypocotyls, and also with the medium. The morphogenetic capacity of the somatic embryos is retained for more than 2 years by subculturing at intervals of 4 weeks on MS3 in complete darkness. Somatic embryos, under the appropriate subculture conditions (16 h light/8 h dark photoperiod at 24+ 1 °C on media MS3, MS4 and MS5), resulted in recurrent-somatic embryogenesis and was profuse at the shoot and root apices of the somatic embryos. Mature somatic embryos were transplanted to MS, to stimulate germination and plantlet regeneration. Plantlets, developed from primary and secondary embryos on MS,, were successfully hardened and grown in natural outdoor conditions. The morphology and histology of the somatic embryo and plantlet and the culture conditions for continuous production of plantlets through direct somatic embryogeny are discussed.

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robot have been established and propagated on medium containing yeast extract. These cultures consisted of unorganized callus tissue bearing embryogenic centres which increased in size during subculture, yielding new regenerated somatic embryos at the end of each 20-d subculture. A development in the propagation of the embryogenic cultures was the establishment of single embryo culture in hormone-free medium where, in selected cases, the process of recurrent somatic embryogenesis (RSE) took place on the hypocotyl of explanted embryos. The process was independent of supporting callus tissue and occurred on simple defined medium. Single embryos underwent either plantlet development or continued RSE on the hypocotyl. One third of the regenerated plantlets showed RSE after the two to three trifoliate leaf stage. In these cases shoot development stopped and only somatic embryo production took place. In vitro cloning of regenerated plantlets allowed us to reproduce each particular genotype before transplantation into soil.