adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

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

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

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.     

Overcoming phenolic accumulation during callus induction and in vitro organogenesis in water chestnut (Trapa japonica Flerov)

Summary Procedures for callus induction and subsequent organogenesis in the aquatic plant, water chestnut (Trapa japonica Flerov), were established. Phenolics exuded from explants at the callus-induction stage adversely affect callus growth. For cotyledonary node-derived callus cultured in Murashige and Skoog (MS) medium (full, half or quarter strength) containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with benzyladenine (BA), the accumulation of phenolics was reduced and callus induction increased by the addition of 10.8 μM phloroglucinol (PG) to the medium. Ascorbic acid was also effective in reducing phenolic accumulation, but less effective for callus induction than PG. Half-strength MS medium supplemented with 2.7 μM 2,4-D, 108.0 μM casein hydrolyzate, and 10.8 μM PG supported maximum callus induction. Plant organogenesis was increased by addition of vitamins (0.27 μM biotin and 2.7 μM folic acid) to half-strength MS medium supplemented with 0.27 μM BA. Many shoots developed from the regenerated nodal shoot explants in liquid half-strength MS salts medium supplemented with 1.08 μM BA and 0.27 μM naphthaleneacetic acid. Individual shoots were excised and cultured in liquid half-strength MS medium supplemented with 5.4 μM IBA and rooted plantlets (108) were transferred and acclimatized in plastic pots. After 3 wk, the plantlets were transplanted in a water chestnut field and the survival rate was 100%.     

Somatic embryogenesis and plant regeneration in Medicago arborea L.

Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N⁶-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.     

Regeneration of pineapple plants via somatic embryogenesis and organogenesis

Summary We have developed efficient methods for plant regeneration, via both embryogenesis and organogenesis, of Smooth Cayenne pineapple, Ananas comosus (L.) Merr. Leaf bases and core (stem) sections of in vitro shoots, produced from culture of crown tip meristem, were used as explants for plant regeneration as follows: (1) Leaf base and core section explants cultured on Murashige and Skoog (MS) medium containing 41 μM 4-amino-3,5,6-trichloropicolinic acid (picloram, P) or thidiazuron (T)/P combinations produced embryogenic tissues. Different types of embryogenic tissues (friable emryogenic tissue, embryogenic cell cluster, and chunky embryogenic tissue) have been developed with varying properties in terms of growth rate and state of development. The embryogenic tissues regenerated shoots upon culture on MS medium containing 13 μM 6-benzylaminopurine (BA) and 1μM α-naphthaleneacetic acid (NAA) followed by culture on MS medium containing 4 μM BA. (2) Crown tip meristems cultured on MS medium containing 13 μM BA followed by leaf explants cultured on MS medium with 27 μM NAA and 1 μM BA produced shoots via direct organogenesis. (3) Explants cultured on MS medium containing 5 μM T and 0.5 μM indole-3-butyric acid (IBA) produced nodular globular structures, which produced shoots upon culture on MS medium containing 1 μM BA and 1 μM gibberellic acid. Shoots obtained from all of the above methods were rooted in half-strength MS medium containing 3 μM NAA and 2.5 μM IBA. Plants were transferred to the greenhouse or shipped to Costa Rica for field trials. Somatic embryo-derived plants exhibited 21 % spininess, and organogenic-derived plants exhibited 5% spininess in the field trials.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> roxb

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency for shoot regeneration (85%) and maximum number of shoots per explant (9.5) were obtained on the medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid (IBA) after 25 d of culture. Fifty percent of shoots were also directly rooted as microtuttings on a peat moss, soil, and compost mixture (1∶1∶1). About 52% of plantlets were successfully acclimatized and established in pots.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

Multiplication and germination of somatic embryos of American ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets

Summary Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 μM α-naphthaleneacetic acid (NAA) and 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants callused at a frequency of 18.2–100%, while somatic embryos were produced from these calluses at a frequency of 25–87.5% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 μM, respectively, and maintained by subcultures every 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1% activated charcoal, and roots >5 mm in length developed within 3 wk. A 7-d exposure to 3 μM gibberellic acid and 5 μM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4 yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.     

Efficient plant regeneration in <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> Wall., from shoot segment-derived callus

Summary A procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica, through shoot segment-derived callus. Explants used for callus induction were shoot segments derived from 14-d-old axenic plants on Murashige and Skoog (MS) medium supplemented with 15 μM N⁶-benzyladenine (BA). A white friable type of callus was obtained in 4.52 μM 2,4-dichlorophenoxyacetic acid and 2.32 μM kinetin which did not have the potentiality to regenerate. High-frequency shoot differentiation was achieved on transferring the friable callus to MS medium supplemented with 17.8 μM BA and 8.0 μM naphthaleneacetic acid. The highest percentage of calluses forming shoots (65.06±2.26) was achieved in this medium. The organogenetic potential of the regenerating callus was influenced by the age of the culture. Rooting was achieved on the shoots using MS medium with 25 μM indolebutyric acid. The plantlets were acclimatized and established in soil. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

In vitro propagation of Typhonium flagelliforme (Lodd) blume

Summary An in vitro culture system was developed for Typhonium flagelliforme using buds from the rhizomes. The mineral salts of four media were tested. These were Murashige and Skoog (MS), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) of which MS medium was found to be the best medium for in vitro culture of T. flagelliforme. The addition of as low as 0.1 mg l⁻¹ (0.54 μM) α-naphthalene acetic acid (NAA) with the presence or absence of N⁶-benzyladenine (BA) in the MS medium caused abnormal shoot formation. The best medium for maximizing shoot number combined with normal complete plantlets from each bud was MS medium supplemented with 0.3 mg l⁻¹ (1.33 μM) BA and 0.5 mg l⁻¹ (2.46 μM) indole-3-butyric acid (IBA). The best acclimatization process was to transfer the normal plantlets, with all the leaves removed, into sand plus coconut husks substrate (1∶1) and placed in intermittent water mists house or shaded plant house with 50% light exclusion. Ninety two percent of the plantlets survived using this acclimatization method.     

Direct plantlet regeneration from male inflorescences of medicinal yam (Dioscorea floribunda Mart. & Gal.)

Summary Segments of male inflorescences of medicinal yam (Dioscorea floribunda) cultured on Murashige and Skoog (MS) medium supplemented with 13.94 μM kinetin (Kn) resulted in the conversion of floral buds into vegetative buds and these later developed into plantlets. Growth and multiplication of shoots could be obtained by culturing individual shoots in MS modified basal medium, replacing the MS standard three vitamins with 10.0 mgl⁻¹ thiamine in addition to 13.94 μM Kn. Root induction was also obtained simultaneously from the base of the shoots in the same medium. Such plantlets have been successfully transferred to potted soil, where they grew normally. Plantlets were also made to develop tubers on MS medium with 18.91 μM abscisic acid (ABA) and also with 2.68 μM α-naphthaleneacetic acid (NAA) and 40–50 gl⁻¹ sucrose.     

Micropropagation of <Emphasis Type="Italic">Clitoria ternatea</Emphasis> Linn. (<Emphasis Type="Italic">Fabaceae</Emphasis>)— An important medicinal plant

Summary An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9μM BA and 1.34μM NAA. The elongated shoots rooted within 7–8d in half-strength MS basal salts supplemented with 1.34μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

In vitro clonal propagation through mature nodes of Tinospora cordifolia (Willd.) Hook. F. & Thoms.: An important ayurvedic medicinal plant

Summary A protocol was developed for rapid clonal propagation of the important medicinal climber, Tinospora cordifolia, through in vitro culture of mature nodal explants. Shoots were initiated on both Murashige and Skoog (MS) medium and woody plant medium (WPM) supplemented with 2.32 μM kinetin (KIN). Of the two basal media tested, WPM was found to be superior to MS medium for the induction of multiple shoots. Among the cytokinins tested, N⁶-benzyladenine (BA) was more effective than KIN for axillary shoot proliferation. KIN was superior to BA in terms of shoot elongation. An average multiplication rate of 6.3 shoots per explant was obtained with WPM supplemented with 8.87 μM BA. Shoot clumps harvested from this medium were transferred to WPM supplemented with 2.22 μM BA and 4.65 μM KIN for shoot elongation. Elongated shoots were rooted in half-strength MS medium supplemented with 2.85 μM indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to sand and established with 80% survival.     

Effect of growth regulator and culture conditions on shoot multiplication and rhizome formation in ginger (Zingiber officinale Rosc.) in vitro

Summary Shoot multiplication of Zingiber officinale cv. V₃S₁₈ was achieved by meristem culture on a Murashige and Skoog (MS) basal medium supplemented with 26.6 μM 6-benzylaminopurine (BA), 8.57 μM indole-3-acetic acid (IAA), and 1111.1 μM adenine sulfate and 3% (w/v) sucrose. In vitro rhizome formation from in vitro-raised shoots was achieved on MS medium supplemented with 4.44 μM BA, 5.71 μM IAA, and 3–8% (w/v) sucrose after 8 wk of culture. Cultural variations such as photoperiod, carbohydrate, nutrient composition, and growth regulators were tested for the maximum yield of rhizomes. Among the different photoperiods used, a 24-h photoperiod helped in the formation of more rhizomes as compared with other photoperiods. Of the different carbohydrates used, sucrose helped to achieve rhizome formation as compared to other carbohydrates. The microrhizomes sprouted in a soil mixture within 2 wk of planting. The sprouted plantlets survived under field conditions with normal growth.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Plant regeneration from stem nodal segments of <Emphasis Type="Italic">Orthosiphon stamineus</Emphasis> benth., a medicinal plant with diuretic activity

Summary A micropropagation method for Orthosiphon stamineus, using stem nodal segments, has been developed. The highest number of regenerated shoots was obtained on Murashige and Skoog (MS) medium supplemented with 6.7 μM benzyladenine with the formation of an average of 6.1 shoots per explant over a period of 4 wk. The number of shoots increased with longer culture duration on proliferation medium. Multiple shoots which were maintained on the proliferation medium for 6 wk had the highest proliferation rate. Separation of multiple shoots and culturing in larger flasks significantly promoted the growth and formation of plantlets. All the in vitro plantlets survived when transferred to the field and showed no significant morphological differences from the mother plants.     

Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.