An efficient protocol for in vitro regeneration and conservation of Shirui lily (Lilium mackliniae Sealy): a lab-to-land approach to save the rare endangered Asiatic lily species

Abstract

An efficient protocol for direct and indirect shoot regeneration and proliferation from bulb scales of Shirui lily (Lilium mackliniae Sealy), an endangered Asiatic lily species endemic to the Shirui hill peak, Manipur, India, has been developed. Bulb scales were isolated from mature bulbs and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ). For direct shoot regeneration from bulb scale explants, 0.5 mg L⁻¹ BAP yielded the highest shoot induction (3.5 shoots per scale; a 96.7% response). For indirect de novo organogenesis, optimum callus induction was achieved with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and shoot organogenesis was higher (16.2) when subcultured onto 0.5 mg L⁻¹ BAP medium. Multiple shoot regeneration and pseudo-bulb formation protocols were assessed; the highest shoot proliferation (10.1) occurred with 0.5 mg L⁻¹ BAP and 1.0 mg L⁻¹ gibberellic acid (GA₃). Rooting response was 96% with 0.5 mg L⁻¹ 1-naphthalene acetic acid (NAA), with multiple roots per shootlet. Plantlet survival was increased to 92.5% during the hardening-off process by using hydroponics with Hoagland’s solution in a mist chamber. Clonal fidelity was assessed through random amplified polymorphic DNA (RAPD) analysis comparing the mother plant and regenerated plantlets. After confirming genetic uniformity, the pseudo-bulblets with four to six leaves and three to four roots were successfully established at the Shirui hills peak. This in vitro regeneration and ex vitro conservation approach could be helpful to save this rare endangered species in a sustainable way.