Effect of carbon and nitrogen sources on in vitro flower and fruit formation and withanolides production in Withania somnifera (L.) Dunal.

We studied the influence of sucrose and nitrogen concentration on in vitro flowering and fruit setting in elongated shoots of Withania somnifera. BA (1.5 mg/l) and IAA (0.3 mg/l) on MS medium supplemented with 4% sucrose showed 67% of in vitro flower induction frequency, 9 flowers/shoot, 4 fruits/shoot and 11 seeds/fruit in elongated-shoots. Different concentrations of nitrogen sources (L-glutamine, adenine sulphate, ammonium nitrate, potassium nitrate and sodium nitrate 5-25 mg/l) were tested in combination with 4% sucrose and BA at 1.5 mg/l and IAA at 0.3 mg/l. Highest number of flowers (20 flowers/shoot; 2.2-fold) and fruits (16 fruits/shoot; 3.39-fold), fruit setting (12 seeds/fruit; 1.08-fold) at a higher frequency (88 %) were achieved on MS medium augmented with 15 mg/l adenine sulphate with same PGRs and sucrose concentration. The maximum production of withanolide A (0.68 mg/g DW) and withanolide B (0.77 mg/g DW) was recorded in in vitro fruits. Highest accumulation of withaferin A (2 mg/g DW) was quantified from in vitro flowers, whereas, it was low in in vitro fruits (0.49 mg/g DW withaferin A). However, withanone (0.23 mg/g DW) was found accumulated uniformly in both in vitro flowers and fruits compared to control.

Indirect Regeneration of Withania somnifera from Nodal Explants.

Withania somnifera is an important medicinal plant and used to cure many diseases. Indirect regeneration protocol for multiple shoots development was established using nodal explants of W. somnifera from 50-60 days old seedlings. The callus induction was observed from nodal explants, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn). Maximum level of callusing response (80.0%) was recorded on MS medium supplemented with a combinations of 2.0 mg/l 2,4-D and 0.2 mg/l Kn. The callus (greenish compact) was transferred into MS medium containing various concentrations (0.5–2.0mg/l) of 6-benzyl amino purine (BAP) alone and in combination (0.1–0.4mg/l) with indole-3-acetic acid (IAA) for shoot initiation and proliferation. The maximum number of shoots was initiated from callus on 1.0mg/l BAP along with 0.2 mg/l IAA and proliferation of shoots achieved by subsequent subcultures at 4 weeks interval in the same medium. The maximum of 31.4 shoots/explant were achieved in the second subculture. MS medium along with 1.0 mg/l gibberellic acid (GA3) induced maximum elongation (96.7%) of regenerated shoots and MS medium supplemented with 0.8 mg/l indole-3-butyric acid (IBA) induced maximum rooting (96.7%) from elongated shoots. After a hardening period, the plantlets were transferred to the field with 98% of survival.

Assessment of somatic embryogenesis potency in Indian soybean [Glycine max (L.) Merr.] cultivars.

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somatic embryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 µM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 µM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 µM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response.