Somatic embryo productions by liquid shake culture of embryogenic calluses in <Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper

The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed into plants.     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture

Plant regeneration in Arachis pintoi was obtained via two developmental pathways: organogenesis and somatic embryogenesis. Organogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with NAA or 2,4-D in combination with BA, KIN or 2iP. The most suitable combination for plant regeneration through organogenesis was an initial medium composed of 10 mg/l NAA+1 mg/l BA followed by transfer of the callus to a shoot induction medium (MS+1 mg/l BA). Rooting of regenerated shoots was readily achieved by culture on MS+0.01 mg/l NAA. Embryogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with PICL in combination with KIN, ZEA, BA or 2iP, and the most suitable combinations were 20 mg/l PICL+1 mg/l BA or 2iP. When pieces of embryogenic callus were subcultured on MS+1 mg/l BA, somatic embryos were differentiated and developed further into well-developed plants in MS+1 g/l AC followed by MS medium devoid of plant growth regulators. Received: 29 April 1999 / Revision received: 24 November 1999 / Accepted: 18 December 1999     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

Shoot regeneration and somatic embryogenesis from needles of redwood (Sequoia sempervirens (D.Don.) Endl.)

A rapid and effective system of somatic embryogenesis and organogenesis from the in vitro needles of redwood (Sequoia sempervirens (D.Don.) Endl.) had been established. The influences of plant growth regulators (PGRs) and days of seedlings in vitro on adventitious bud regeneration and somatic embryogenesis were studied. The process of somatic embryo formation was also observed. The results showed that embryogenic callus was induced and proliferated on Schenk and Hildebrandt (SH) medium with BA (0.5 mg/l), KT (0.5 mg/l) and IBA (1.0 mg/l). SH medium containing BA (0.5 mg/l), KT (0.2 mg/l) and IBA (0.2 mg/l) effectively promoted adventitious bud regeneration. The highest frequency (66.3%) of direct somatic embryogenesis was obtained in the combination of BA (0.5 mg/l) and IBA (0.5 mg/l). The optimal days of seedling in vitro for adventitious bud and somatic embryogenesis were 30 days and 30–40 days, respectively. The developments of somatic embryos were similar to that of zygotic embryogenesis. The result of histocytological studies indicated that proteins were gradually accumulated in the process of somatic embryo formation and there were two peaks of starch grains accumulation that one was in the embryogenic callus and the other was in the globular embryos. These results indicated that starch and protein were closely related with the energy supply and the molecular base of somatic embryogenesis, respectively.     

Isolation and functional characterization of two 5-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferases related to anthocyanin biosynthesis from <Emphasis Type="Italic">Freesia hybrida</Emphasis>

Kelussia odoratissima Mozaff. (or Kelus) is a medicinal plant native to the Zagros Mountains in Iran. This plant is widely used as a food flavoring and for its health-promoting properties. It has been considered an endangered species by the United Nations Development Programme. In this study, a somatic embryogenesis (SE) method was developed for mass propagation of Kelus. The green globular embryogenic callus was induced on cotyledonary leaves using the Murashige and Skoog (MS) medium supplemented with 1 mg/l 2,4-dichlorophenoxyaceticacid (2,4-D) and 0.25 mg/l Kinetin. Different treatments were assayed for proliferation of the embryogenic callus. The calli remained embryogenic in an MS medium containing 2,4-D (1 mg/l). The light treatments and carbon source showed significant effects (P?≤?0.05) on the proliferation and development of somatic embryos. These treatments improved the conversion rate of the cotyledonary-stage embryos by 100%. The average numbers of embryos in the globular, heart, torpedo, and cotyledonary stages decreased by the addition of 3 g/l case in hydrolisate. The genetic stability among tissue culture-derived plants and the mother plant were assessed using the amplification fragment length polymorphism. No polymorphic band was observed among all the plants, exhibiting the genetic stability during in vitro multiplication. This research provides a promising approach for true-to-type plant multiplication of K. odoratissima through SE.     

Somatic embryogenesis from zygotic embryos and shoot-tips of the Chilean tree <Emphasis Type="Italic">Gomortega keule</Emphasis>

The endangered Chilean tree Gomortega keule (Mol.) Baillon produces edible fruit, making it a potential crop. However, its cultivation from seed or cuttings is extremely difficult. This paper reports the induction of somatic embryogenesis and the initiation of liquid cultures in this species. Callus was induced from zygotic embryos and field-collected shoots. Somatic embryogenesis on zygotic embryos occurred at a low frequency. Induction of somatic embryogenesis was accomplished on micropropagated shoots after 6.5 months on semi-solid Murashige and Skoog (MS) medium with 30 g/l sucrose, 1.0 mg/l 2,4-dichlorophenoxyacetic acid and 1.0 mg/l 6-(γ,γ-dimethylallylamino) purine (2iP). Liquid cultures of compact callus and small aggregates were obtained and showed optimum proliferation in MS medium with 20 g/l sucrose, 0.01 mg/l α-naphthaleneacetic acid and 0.1 mg/l 2iP. The proliferation of friable embryogenic callus was observed in liquid medium and will allow the propagation of selected genotypes of this tree on a large scale. Genetic variation in two embryogenic genotypes cultured in vitro was not detected in an assessment using microsatellites; this approach is suitable for tracing genotypes.     

High-frequency somatic embryo production and maturation into normal plants in cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>) through metabolic stress

A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton ( Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20-24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.     

<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.     

High-efficiency somatic embryogenesis and plant regeneration in California poppy, Eschscholzia californica Cham.

 The development of a rapid protocol for high-efficiency somatic embryogenesis and plant regeneration from seed-derived embryogenic callus cultures of California poppy (Eschscholzia californica Cham.) is reported. The optimized procedure required less than 13 weeks from the initiation of seed cultures to the recovery of plantlets and involved the sequential transfer of cultures onto solid Murashige and Skoog basal medium containing three different combinations of growth regulators. All steps were performed at 25  °C. Friable primary callus was induced from seeds of E. californica cultured on medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The primary callus was transferred to medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid and 0.5 mg l⁻¹ 6-benzylaminopurine to establish embryogenic callus and promote somatic embryogenesis. Regenerated plantlets were recovered after the conversion of somatic embryos on medium containing 0.05 mg l⁻¹ 6-benzylaminopurine and showed normal development. Embryogenic callus was induced at a frequency of 85%, an average of 45 somatic embryos were produced per callus, 90% of the somatic embryos converted, and about 70% of the plantlets were recovered in soil. The growth rate of somatic embryo-derived shoots could be increased by gibberellic acid treatment, but the resulting plantlets were hyperhydritic. Received: 14 February 1999 / Revision received: 27 April 1999 / Accepted: 14 May 1999     

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

Somatic embryogenesis and organogenesis from mature caryopses of North African barley accession “Kerkena” (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The regeneration ability of a Tunisian barley accession originated from Kerkena islands was monitored through somatic embryogenesis and organogenesis. To prevent or to reduce normal germination, longitudinally bisected as well as base-wounded mature caryopses were cultured on a modified Chée and Pool-based medium (CP) enriched with different phytohormonal combinations. The greatest embryogenesis response was obtained when base-wounded caryopses were cultured on CP enriched with 2 mg/l chlorophenoxyacetic acid + 2.5 mg/l kinetin (76.85%). The same combination coupled to longitudinally bisected caryopses led to the embryogenic induction at the hypocotyl base of the germinated caryopses (61.9%). Embryogenic calluses differentiated into globular, heart-shaped, torpedo, and fully differentiated stages of somatic embryos on hormone-free Murashige and Skoog-based medium. Rooted plantlets were successfully transferred to soil and grown to maturity in the greenhouse and produced fertile seeds within 3 mo. On the other hand, organogenesis was achieved on CP enriched with 2 mg/l 2,4-dichlorophenoxyacetic acid + 2.5 mg/l kinetin. Histological aspects and scanning electron microscopy of both regeneration methods confirmed further the embryogenic and organogenic nature of the established processes. This efficient plant regeneration system provides a foundation for generating transgenic plants and germplasm preservation of “Kerkena” barley accession.     

Induction,maturation and germination of holm oak (Quercus ilex L.) somatic embryos

Cotyledon explants of Panax ginseng produced somatic embryos directly on solid hormone-free MS medium containing 3% (w/v) sucrose while high concentration of NH₄NO₃ (60 mM) induced embryogenic callus. Ten subcultures of the embryogenic callus on hormone-free MS medium with 40 mM NH₄NO₃ gave hormone-independent proliferation of callus, which exhibited proliferation potential even on MS medium with a standard level of NH₄NO₃ (20 mM). Pulse treatment of callus with exogenous auxin or cytokinin (1.0 mg 1^–1 2,4-D, 1.0 mg 1^–1 kinetin) resulted in the loss of the hormone-independent characteristic and caused the callus to brown. For the suspension culture, embryogenic callus was transferred to MS liquid medium containing 3% (w/v) sucrose in an 500 ml Erlenmyer flask. Embryogenic cell clumps in full-strength MS liquid medium discharged toxic substances, resulting in strong suppression of cell growth. In 1/3-strength MS medium, exudation of toxic material did not occur. Embryogenic cell clumps were mass-grown on a large-scale in a bioreactor (20-1), showing a 7.1 increase of fresh weight in 1/3-strength MS medium with 3% (w/ v) sucrose after 5 weeks of culture. Total ginsenoside content of cultured embryogenic cell clumps was low and 6 times below naturally-cultivated ginseng roots.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Somatic embryogenesis in the medicinal legume <Emphasis Type="Italic">Desmodium motorium</Emphasis> (Houtt.) Merr.

An efficient protocol was established for regeneration of Desmodium motorium via somatic embryogenesis. Embryogenic calli were induced from cotyledon segments (6 mm, 16 days old) lacking embryo axis, excised from seedlings grown in vitro on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (2.9 μM) in combination with 6-benzyladenine (BA) (4.44 and 8.88 μM). Differentiation of embryogenic calli into globular and heart-shaped somatic embryos was achieved on transfer to hormone-free MS medium. When incubated for 4 days on MS medium supplemented with BA (8.88 μM), 95% of the globular and heart-shaped somatic embryos matured into torpedo and cotyledonary stages with minimum (10%) abnormalities. Modified MS basal medium without hormones and containing half-strength macronutrients and 0.88 M sucrose was suitable for germination of mature somatic embryos. Regenerated plantlets were successfully transferred to earthen pots with survival rate of 50%. Secondary embryogenesis was observed when pre-existing somatic embryos at globular and heart-shaped stages were cultured on MS medium supplemented with various concentrations of BA, adenine sulphate (AdS) and abscisic acid (ABA) individually.     

Somatic embryogenesis and regeneration of <Emphasis Type="Italic">Vigna radiata</Emphasis>

An efficient regeneration protocol via somatic embryogenesis was optimized for mung bean [Vigna radiata (L.) Wilczek; cv. Vamban 1]. Primary leaf explants were used for embryogenic callus induction in MMS medium (Murashige and Skoog salts with B5 vitamins) containing 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D), 150 mg dm⁻³ glutamine and 3 % sucrose. Fast growing, highly embryogenic cell suspensions were established from 21-d-old calli in MMS medium supplemented with 0.5 mg dm⁻³ 2,4-D and 50 mg dm⁻³ proline (Pro), and maximum recovery of globular (39.0 %), heart-shaped (26.3 %) and torpedo-stage (21.0 %) somatic embryos were observed in this medium. Mature cotyledonary-stage somatic embryos were cultured for 5 d in half strength B5 liquid medium containing 0.05 mg dm⁻³ 2,4-D, 20 mg dm⁻³ Pro, 5 μM abscisic acid, 1000 mg dm⁻³ KNO₃, 50 mg dm⁻³ polyethylene glycol (PEG 6000) and 30 g dm⁻³ D-mannitol. Mature somatic embryos were germinated after dessication for 3 d and complete development of plantlets accomplished in MMS medium containing 30 g dm⁻³ maltose, 0.5 mg dm⁻³ benzyladenine and 500 mg dm⁻³ KNO₃. Profuse lateral roots, and regeneration frequency (up to 60 %) were observed in half-strength MMS medium containing 0.5 mg dm⁻³ indolebutyric acid (IBA). The regenerated plants were grown to fruiting and were morphologically normal and fertile.