Efficient regeneration of the endangered banana cultivar ‘Virupakshi’ (AAB) via embryogenic cell suspension from immature male flowers

Abstract

Plantlets of the banana cultivar ‘Virupakshi’ (AAB) were regenerated from somatic embryos derived from embryogenic cells of calli from immature male flower explants. Induction of calli from explants was favored by a relatively moderate concentration of2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/L), high concentrations of proline and glutamine (both 300 mg/L) and coconut water. A suspension culture of the callus-derived embryogenic cells in Murashige and Skoog (MS) basal medium (pH 5.3) with 2 mg/L 2,4-D, 1 mg/L indole-3-acetic acid (IAA), 1 mg/L NAA, 45 g/L sucrose and 20 g/L maltose produced synchronously proliferating cells with the potency to be induced into somatic embryos on an 8 g/L agarose-solified SH basal medium (pH 5.7) with recommended vitamins, 1 mg/L IAA, 1 mg/L naphthaleneacetic acid (NAA), 0.2 mg/L2-ip, 40 g/L sucrose, 20 g/L maltose, 10 g/L dextrose. The young somatic embryos differentiated into regenerable mature somatic embryos on an 8 g/L agarose-solidifiedMS basal medium (pH 5.7) with 1 mg/L NAA, 100 ml/L coconut water, 30 g/L sucrose,30 g/L maltose and 100 mg/L glutamine. The mature somatic embryos regenerated into plantlets on a 2.5 g/L gelrite-solidified MS-based medium with 2.5 mg/L 6-benzylaminopurine (BAP), 1 mg/L gibberellic acid (GA3), 100 mg/L L-glutamine, 30 g/L sucrose and 2.5 mg thidiazuron (TDZ). The total duration from explant stage, for the development of plantlets of 10 to 15 cm height, which could withstand hardening process, was 16 months. The plantlets were morphologically normal, suggesting normal development without somaclonal variation. The present regeneration protocol for ‘Virupakshi’ has great potential for preserving the endangered germplasm by micropropagation and its improvement by transgenic technology against the deadly Banana bunchy top virus and other economically important diseases.