Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis>

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm⁻³ kinetin and 0.2, 0.5 mg dm⁻³ indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.     

Somatic embryogenesis from leaf- and petiole-derived callus of Vitis rupestris

Somatic embryogenesis from leaf- and petiole-derived calli of Vitis rupestris was obtained with an efficiency of 3.2% and 4.2% of plated explants, respectively on two combinations of 6-benzyladenine and 2,4-dichlorophenoxyacetic acid (1/0.1 and 1/1 mgl^–1) added to MS medium. Embryogenic callus, embryo subcultures and somatic embryogenesis from somatic embryos were obtained either in the presence of 1 mgl^–1 indole-3-acetic acid or 0.1 mgl^–1 indole-3-butyric acid added to MS or NN media. Within a 4-month culture, embryo germination occurred at a frequency of 13% of explanted embryos when chilling at 4°C was provided for two weeks and a combination of 6-benzyladenine (1 mgl^–1) with indole-3-butyric acid (0.1 mgl^–1) was added to NN medium supplemented with casein hydrolysate (250 mgl^–1). A higher frequency (51%) was obtained in a longer culture time (9 months) when only indole-3-butyric acid was present in the medium and in absence of chilling.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NN Nitsch and Nitsch (1969) - NOA 2-naphthoxyacetic acid     

Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.)

Summary Somatic embryos of carob (Ceratonia siliqua L.) were induced from cotyledonary segments excised from immature seeds when cultured on Murashige and Skoog media supplemented with several combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The best frequencies of induction (33.8%) were obtained when 4.4 μM BA and 0.5 μM IBA were used. Shoots were also sporadically formed in the same media. When IBA was replaced by other auxins in the induction media, only α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce somatic embryogenesis, although at lower rates than IBA. 2,4-Dichlorophenoxyacetic acid and 4-amino-3,5,6-trichloropicolinic acid were completely ineffective. Besides culture media composition, the developmental stage of the explants at the time of culture showed a strong influence on somatic embryogenesis induction, with cotyledons from stage II pods providing the highest levels of induction. By contrast, the genotype of the explant did not determine a significant role in the induction process. Attempts to achieve somatic embryo germination were mostly unsuccessful, since only shoot development was observed; the highest frequencies of development occurred on media containing only gibberellic acid (3.0 μM). For plant regeneration, the developed shoots were further rooted on IBA-supplemented media, and the plantlets obtained were transferred to soil, where c. 88% of them survived. Histological observations showed the presence of morphologically normal and abnormal somatic embryos, the latter displaying an abnormal pattern of vascular bundles. Ultrastructural analysis showed that the cells of the globular embryos had a dense cytoplasm, whereas those not involved in somatic embryo formation showed signs of senescence. Histological studies were also used to distinguish between somatic embryos and shoots originated in the same media.     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.)

Somatic embryogenesis and plant regeneration were obtained from immature leaflet callus of chickpea. Numerous globular embryos developed on the surface of callus on Murashige and Skoog's (1962) medium containing 25 μM 2,4-dichlorophenoxyacetic acid. These globular embryos differentiated into mature somatic embryos upon removal of 2,4-dichlorophenoxyacetic acid. The maturation of embryos was significantly affected by pH, photoperiod, abscisic acid and genotype. Callus continued to produce somatic embryos for over 8 subcultures at 4 week intervals. Two per cent of the embryos formed plants on medium containing 15 μM gibberellic acid and 1 μM indole-3-butyric acid. Desiccation of embryos for a period of 3 d increased their rate of conversion into plants from 0.9 to 2.8%. All regenerated plants showed normal morphological characteristics.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Adventitious root induction inCorylus avellana L. cotyledon slices

Summary An experimental rooting system was developed to study in vitro adventitious root formation in hazelnut cotyledons. Experiments involveda) assay of several culture media,b) use of different developmental status of the seeds (germinated and ungerminated),c) cotyledons subjected to various light regimes, andd) different size of cotyledons slices. It was observed that higher rooting was induced in cotyledonary portions of 5- or 7-mm thickness (250 and 350 mm³, respectively) cultured on half-strength basal medium supplemented with 50 μM indole-3-butyric acid and 5 μM kinetin. Rooting was affected by light and the developmental state of seeds. Preinitiation, initiation, and root manifestation stages were defined according to specific culture periods and in relation with morphologic and histologic changes. The first histologic changes (cell division and root primordia induction) were observed after 12 days in culture. At 30 days of culture in rhizogenic medium root primodia were fully differentiated with well-developed vascular tissues.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

Organogenesis,embryogenesis, and synthetic seed production in <Emphasis Type="Italic">Arnebia euchroma</Emphasis>—A critically endangered medicinal plant of the Himalaya

Summary This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya. Root-derived callus showed only rhizogenesis, whereas leaf-derived callus showed simutaneous organogenesis and somatic embryogenesis. Organogenesis was optimal (12.2 shoots per culture) in 1 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine and induction of somatic embryogenesis (16.3 embryos per culture) occurred in 2.5 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine. Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 2.0 μM indole-3-butyric acid. Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 60.6% germination in MS medium. Rooted shoots transferred to a mixture of sterile soil, sand, and peat (1∶1∶1 by volume) showed 72% survival ex vitro. Application of these protocols would be helpful in reducing pressure in natural populations, in genetic transformation studies, and in long-term storage of elite genotypes through synthetic seed production.     

In vitro propagation and conservation of Indian sarsaparilla, Hemidesmus indicus L. R. Br. through somatic embryogenesis and synthetic seed production

A protocol has been developed for achieving somatic embryogenesis from callus derived from nodal cuttings and production of synthetic seeds in Hemidesmus indicus L. R. Br. a highly traded ethnomedicinal plant. Proembryogenic, friable, light yellowish callus was induced from the basal cut end of the nodal cuttings on Murashige and Skoog (MS) medium supplemented with 3 μM indole-3-butyric acid (IBA). The highest rate of somatic embryogenesis (92 %) was observed when the callus was subcultured on half strength MS medium supplemented with 2 μM IBA. On induction medium somatic embryos were developed up to the torpedo stage. Further elongation and germination of somatic embryos were obtained in MS medium supplemented with 4 μM 6-benzylaminopurine (BA) in combination with 1.5 μM gibberellic acid (GA₃). Somatic embryos were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V) dropped into 75 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds and later transferred to MS medium for germination. The synthetic seeds were successfully germinated on medium even after 120 days of storage at 4 °C. The plantlets were eventually transferred to soil with 92 % success.     

In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

Induction of somatic embryogenesis and embryo development in Rumex acetosella L.

Axillary buds of the dioecious plant Rumex acetosella L. were isolated and cultured in vitro. The callus tissue which developed at the basal parts of the explants displayed a high capacity for shoot formation. This morphogenetic pattern was predominant on Murashige and Skoog (MS) medium supplemented with 2% sucrose, 2.2 mgl^-1 benzylaminopurine and 0.17 mgl^-1 indole-3-acetic acid. Somatic embryogenesis was induced when the osmolality of the medium was increased by adding 6% sucrose instead of 2%, or hexitols in addition to 2% sucrose. Most of the embryogenic calli were formed on the basal parts of leaf laminae and bracts. Development and maturation was strongly promoted by transferring the tissue to a solid or liquid medium lacking benzylaminopurine and indole-3-acetic acid and supplemented with 10 mgl^-1 gibberellic acid. The embryos germinated and developed into normal rosette plants when transferred to vermiculite moistened with hormone-free, half-strength MS salt solution. The histology of successive embryogenic stages is presented.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

Factors affecting somatic embryogenesis in <Emphasis Type="Italic">Prunus incisa</Emphasis> cv. February Pink

Factors affecting somatic embryogenesis from root explants of Prunus incisa Thunb. cv. February Pink were investigated. Using a medium containing Murashige and Skoog salts and vitamins supplemented with 10 M 2,4-dichlorophenoxyacetic (2,4-D), we evaluated the effects of light, growth regulators, amino acids, carbohydrate source, and root induction medium. Explants cultured under light or dark conditions both resulted in the formation of embryos. Embryogenesis was inhibited by the addition of 6-benzyladenine, thidiazuron, or gibberellic acid to the medium. Amino acids were not effective in promoting embryogenesis, with high levels of amino acids actually inhibiting it. Sucrose and glucose effectively induced embryogenesis, while sorbitol and mannitol completely inhibited it. Sucrose and glucose also promoted secondary embryogenesis. Embryos that formed in medium containing 4% or 5% sucrose were abnormally shaped and did not fully develop, while those that formed in medium with sucrose concentrations of 2% or 3% were much more vigorous. Root explants that were induced on medium containing 1.0 M indole-3-butyric acid (IBA) produced more somatic embryos than explants induced on medium without IBA. Approximately 50% of the roots induced on medium containing 1.0 M IBA produced somatic embryos on medium containing 10 M 2,4-D and 3% sucrose.Abbreviations BA 6-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.