In vitro somatic embryogenis and plant regeneration from cell suspension culture of endangered plant ‘Virali’ Dodonaea viscosa

Abstract

An effective protocol was developed for in vitro propagation of ‘Virali’ via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium by a relatively moderate concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (4 mg/L), high concentrations of proline and glutamine (both 300 mg/L) and coconut water to develop induction of calli from explant. 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 resulted in synchronously proliferating cells with the capacity to be induced into somatic embryos on an 8g/L agarose, 20 g/L maltose, 10 g/L dextrose, 40 g/L sucrose, 1 mg/L naphthaleneacetic acid (NAA), and 0.2 mg/L. On an 8 g/L agarose-solidified MS 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 young somatic embryos differentiated into regenerable mature somatic embryos. In 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, the mature somatic embryos regenerated into plantlets (TDZ). For the formation of plantlets with a height of 10 to 12 cm that could endure the hardening process, it took a total of 6 months from the explant stage. The plantlets’ regular morphology suggests that there was no somaclonal variation in their development. The current regeneration approach for “Virali” has a significant deal of potential for micro propagating endangered germplasm and improving it through transgenic technology.