Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Callus induction and plant regeneration from maize mature embryos

Calli were induced from mature embryos of maize (Zea mays L.) inbred lines A632, B73 and Mol7 on MS medium supplemented with 1–2 mg/1 2,4-dichlorophenoxyacetic acid. Callus induction frequency ranged from 23–100%, with Mol7 having the highest frequency. Plants were regenerated from 4–5% of the B73 and Mol7 explants. Embryogenic and organogenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R₀ plant with abnormal pollen was detected, and no morphological variants were observed in the R₁ progeny.Abbreviations Dicamba 3,6-Dichloro-o-anisic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Ze zeatin     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

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

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

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm^?3 N⁶-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm^?3) and BA or kinetin (1–5 mg dm^?3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.     

Screening of diploid Medicago sativa germplasm for somatic embryogenesis

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP iso-pentyladenine - NAA -naphthaleneacetic acid Contribution No. 772 Ottawa Research Station     

High frequency somatic embryogenesis and synthetic seed production of the endangered species Swertia chirayita

An efficient protocol for plant regeneration through somatic embryogenesis was established from in vivo leaf explants of Swertia chirayita, a critically endangered medicinal herb. The highest frequency (76%) of embryogenic callus was induced on Murashige & Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (Kn) from in vivo leaf explants. Globular somatic embryos were induced and further matured from such embryogenic calli by subsequent culture on the same medium. The highest number of somatic embryos (48.83 ± 4.6) was recovered from embryogenic calli derived from leaf explants after 6 weeks of culture. Synthetic seeds were produced by encapsulating of torpedo stage embryos in sodium alginate (4% W/V) gel, dropped into 100 mM calcium chloride (CaCl₂ · 2H₂O) solution. The synthetic seeds were germinated on MS medium. The highest frequency of synthetic seed germination (84%) was observed on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA. Regenerants were successfully acclimatized under ex vitro condition. This is the first report on synthetic seed production of S. chirayita. Application of these protocols would be helpful in reducing stress in natural habitat, and in long-term storage of elite genotypes through synthetic seed production.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander (Coriandrum sativum L.)

Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS medium supplemented with 1 mgl^–1 2,4-D. Calli were transferred to MS liquid medium with 1 mgl^–1 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the medium as the subculture proceeded. Cultures were transferred to a nitrogen compound enriched liquid MS medium containing 2% sucrose and 0.1 mgl^–1 2,4-D, and cultured two weeks before plating on MS basal medium. Approximately 75% of cell aggregates (1 to two mm in diameter) underwent development into globular to cotyledonary somatic embryos after two weeks of plating. Most of the embryos were subsequently regenerated into plantlets. Regenerants were successfully transplanted to potting soil and grown to maturity in a phytotron.Abbreviations MS Murashige and Skoog - MS1D MS medium + 1 mgl^–1 2,4-D     

Thidiazuron required for efficient somatic embryogenesis from suspension-cultured cells ofPimpinella brachycarpa

To maintain embryogenic cell lines ofPimpinella brachycarpa, we suspension-cultured friable and rapidly growing yellowish calli in an MS liquid medium containing 0.2 ~ 2,4-D and 0.5pM BAP. Efficient somatic embryogenesis was achieved when selected cells were then transferred to an MS medium (0.2% gelrite) that contained 0.2gM 2,4-D, 0.5 uM BAP, and 10.0 laM TDZ (thidiazuron). These cells were cultured at 27°C under continuous illumination (21.5 I~E m^-2 s^-l). Embryogenic calli expanded about four-fold, and developed into pale yellow calli. Somatic embryogenesis was initiated only from glossy and nodular-type calli. After two more weeks of culture, globular embryos appeared on the surface of calli grown in the MS medium that contained 10.0 /aM TDZ only, or in combination with 0.5 gM NAA. Experimenting with 2,4-D, an auxin, to promote embryogenic calli resulted in excessive browning and death. We overcame this problem by growing glossy embryogenic and nodular calli on media that contained 10.0 gM TDZ. Calli that were not treated with TDZ turned dark brown and were not viable. Up to 74% of the calli showed somatic embryos when the medium was supplemented with 10.0 uM TDZ and 0.5 uM NAA. Embryos from these TDZ-induced, somatic embryogenic calli grew efficiently, forming multiple shoots and developing into normal plants. Therefore, efficient differentiation of suspension-cultured cell clusters into embryogenic calli, along with treatment of subsequent somatic embryos by TDZ, suggests that TDZ probably helps in establishing the optimum cytokinin-auxin ratio required for induction and expression of somatic embryogenesis.     

Effect of 2,4-dichlorophenoxyacetic acid concentration on somatic embryogenesis and heritable variation in soybean [Glycine max (L) merr.]

Summary The frequency and quality of embryogenic response from cotyledons of immature zygotic soybean embryos varied with 2,4-dichlorophenoxyacetic acid (2,4-D) concentration in the culture medium. The frequency of variants among progeny of regenerated plants decreased with an increase of 2,4-D concentration. Teratogenic effects on embryo morphology and development were greatest at 22.5μM 2,4-D and decreased with increasing 2,4-D. At the lowest 2,4-D concentration tested, 22.5μM, morphologically abnormal, cotyledonary-stage somatic embryos were produced. Ten percent or less of these embryos converted to plants. Over the nine genotypes tested, 40% of the families derived from plants regenerated under a low 2,4-D concentration manifested heritable variation. In contrast, embryogeny was suppressed at the globular stage by the highest 2,4-D concentration tested, 200μM. Eighty to one-hundred percent of the embryos organized under this latter 2,4-D level converted to plants. Only 3% of the families from the progeny of plants regenerated under a high 2,4-D concentration exhibited heritable variation. This is Journal Paper No. J-14217 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2974. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or Iowa State University and does not imply its approval to the exclusion of other products that may be suitable. This work was supported, in part, by American Soybean Association grant no. 400-46-73-15-2763.     

A 5-methyltryptophan resistant rice mutant,MTR1, selected in tissue culture

Summary Cell lines resistant to tryptophan analogue 5-methyltryptophan (5MT) were selected in seed-derived calli of Oryza sativa L. var. Norin 8. Plants were regenerated (R₁ from one selected callus line (MTR1). In three out of the six R₁ plants, 5MT resistance was inherited in the R₂ and R₃ generations as a dominant nuclear mutation. Segregation ratios in the progeny of heterozygous plants were 11. Morphological and fertility variation seen in some of the R₂ plants were not correlated with 5-methyltryptophan resistance. Resistance in the MTR1 callus was due to the accumulation of high levels of free tryptophan (87-fold) that was associated with an increase in free phenylalanine content (9-fold). The leaves of resistant plants also contained elevated levels of free tryptophan and phenylalanine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - 5MT D,L-5-methyltryptophan - phe phenylalanine - trp tryptophan - tyr tyrosine     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Occurrence and frequency of precocious germination of somatic embryos is a genotpye — dependent phenomenon in wheat

Immature embryos of thirty-three genotypes of wheat were cultured on 2,4-D containing medium. Occurrence of precocious germination of the zygotic and somatic embryos simultaneously on the same medium was a striking feature observed during the course of work. The percentage of precocious germination was seen to vary extensively from 0–88% and 0–84% for zygotic and somatic embryos respectively. In the genotypes NI-5439 and NI-5643 which are characterized by a high tillering capacity, the phenomenon of precocious germination seems to take a different path from that observed in the other genotypes. This is evident since these two genotypes require total absence of hormone for shoot elongation although multiple shoot primordia are formed on auxin containing medium.Precocious germination also seems to be relevant to somatic embryogenesis and plantlet regeneration. This conclusion stems from the observation that a majority of the genotypes that show precocious germination of zygotic embryos have greater embryogenic potential. Consecutively, most of the genotypes that show precocious germination of somatic embryos exhibit a higher frequency and faster rate of plantlet regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Ki Kinetin - thi - HCl Thiamine hydrochloride - E calli Embryogenic calli NCL Communication No. 4456     

<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.     

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm⁻³) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 μM gibberellic acid (GA₃), plumular poles of few embryos elongated resulting in the development of shoots.     

Somatic embryogenesis and plant regeneration in callus culture of tef, Eragrostis tef (Zucc.) Trotter

The study was carried out to establish in vitro culture conditions for plant regeneration of tef, Eragrostis tef (Zucc.) Trotter. Mature seeds of two Ethiopian varieties, DZ-01-354 and DZ-01-196, were used to initiate callus cultures on Murashige and Skoog (MS) medium with different auxins. Four- and 8-week-old calli induced on a medium with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were subcultured onto various media to induce somatic embryogenesis. Compact, nodulated, embryogenic callus was observed after transfer onto MS-callus proliferating (CP) medium. Embryogenic tissue appeared on soft and amorphous callus and developed into somatic embryos during a subsequent subculture to MS embryo-promoting (EP) media. Various growth regulator combinations were tested in CP and EP media to obtain a high efficiency of somatic embryo formation. The highest frequency of calli forming somatic embryos (56.1–68.3%) was observed when CP media with 2.0 or 4.0 mg/l 2,3,5-triiodobenzoic acid were employed and then cultures were transferred to EP media with 0.5 mg/l 2,4-D and 0.5 mg/l kinetin followed by 0.5 mg/l indole-3-acetic acid and 0.5 mg/l N⁶-benzyladenine. Plant development from somatic embryos was obtained on MS medium supplemented with 1.0 mg/l gibberellic acid. On average, 71.2% of calli displaying somatic embryos converted into plants. Regenerated plants were successfully transferred to soil. Neither chlorophyll-deficient plants nor morphological variants were found among regenerants. All regenerated plants were fertile. Received: 9 May 1997 / Revision received: 25 September 1997 / Accepted: 3 January 1998     

Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids

Summary A method for the production of somatic embryos and subsequent plant regeneration for Anthurium andraeanum Linden ex André (Monocotyledonae) hybrids is described. Whole leaf blade explants, derived from plantlets grown in vitro, formed translucent embryogénic calli at their basal ends within one month of culture in the dark. Secondary somatic embryos formed frequently and without an intervening callus on surfaces of primary embryos. Embryogenesis was induced with three genotypes using a modified half-strength Murashige and Skoog (MS) medium supplemented with 1.0 to 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.33 to 1.0 mg l^–1 kinetin. A combination of 2% sucrose with 1% glucose in the medium favored embryogenesis over 3% sucrose alone. Whole leaf blades on medium solidified with 0.18% Gelrite produced more somatic embryos than leaves on medium with 0.7% Bacto-agar. Within two to three months after culture initiation, embryos were transferred to modified MS medium containing 0.2 mg l^–1 6-benzyladenine (BA) and 2% sucrose and placed in the light for conversion into plantlets. Rooted plantlets were recovered and transferred into pots with tree fern fiber medium and grown in the greenhouse.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

Somatic embryogenesis and plant regeneration in Cedrela fissilis

Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were transferred to fresh medium, embryogenic tissue appeared on MS + 45 μM 2,4-D, or 22.5 μM 2,4-D + 0.4 μM 6-benzyladenine (BA), or 20.7 μM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 μM 2,4-D resulted in the differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed typical characteristics of a somatic dicotyledonous embryo.