Establishment of an efficient regeneration system and in vitro polyploid induction based on the bulblet centre in Lilium rosthornii Diels

Lilium rosthornii Diels, commonly known as the Nanchuan lily in China, is a unique wild lily species with potential medicinal or ornamental value. The development and utilization are mainly restricted to its highly efficient regeneration and the scarcity of new germplasm. In this study, an efficient plant regeneration method for Nanchuan lily was established based on embryogenic callus induction, adventitious bud sprouting, and shoot proliferation and rooting. The results showed that Murashige and Skoog (MS) media supplemented with 0.5 mg L⁻¹ picloram (PIC) and 1.0 mg L⁻¹ 1-naphthaleneacetic acid (NAA) were suitable for embryogenic callus induction using outer bulblet scales (78.89% rate), and MS supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (6-BA), 1.0 mg L⁻¹ NAA, and 75.0 mg L⁻¹ ascorbic acid in darkness was optimal for embryogenic callus proliferation (2.62 coefficient). Moreover, callus inoculation in MS medium supplemented with 1.0 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA significantly promoted shoot induction (3.16 coefficient) under relatively low light conditions. The shoot proliferation and rooting of MS plants treated with 0.1 mg L⁻¹ NAA and 0.5 mg L⁻¹ TDZ were greatest under light conditions at 93.33% and 98.89%, respectively. In addition, bulblet growth was induced by treatment with different combinations of colchicine at different concentrations for different durations. Focusing on the bulblet centre, 100% morphological changes were achieved for in vitro induction using 0.15% colchicine for 72 h of treatment. Compared with the control, three stable mutagenic plants with greater guard cells and reduced stomatal density were generated and were identified as chimaeras by flow cytometry analysis. These results provide support for improving tissue reproduction and breeding autotetraploid germplasm in Nanchuan lily.