Haploid plant production and flowcytometric evaluation of Lilium ledebourii (Baker) Boiss

• Evaluation of growth regulator combinations on callogenesis, embryogenesis, and regeneration of L. ledebourii via anther culture. • Successful induction of callus formation and embryogenesis using specific hormone combinations. • Identification of optimal conditions for callus induction and embryo development. • Determination of ploidy levels in regenerated bulblets. • Potential for producing homozygous L. ledebourii plants through the proposed anther culture protocol. Lilium ledebourii (Baker) Boiss. is a distinct ornamental species, with its breeding program being of significant importance. Due to its wild nature, cultivation of this rare plant has been explored in limited studies. This investigation aimed to assess the impact of growth regulator combinations on callogenesis, embryogenesis, and regeneration of L. ledebourii through anther culture, alongside evaluating the ploidy level of the regenerated plants. The experiment followed a factorial design, employing a wholly randomized setup with five replications. Callogenesis was initiated using MS, B5 , and NLN culture media supplemented with 3 % sucrose and 2,4-D (0, 0.5, 1, 2, and 4 mg/L) and thidiazuron ( TDZ ) (0, 0.5, 1, 2, and 4 mg/L). Embryogenesis was induced using Picloram ( Pic ) (0, 0.5, 1, 2, and 4 mg/L) and Kinetin ( Kn ) (0, 0.5, 1, 2, and 4 mg/L) in NLN medium. Regeneration was facilitated on a solid, hormone-free NLN . Calli obtained from the treatments were transferred to an embryogenesis medium and subjected to darkness treatment to foster embryo development. Results revealed that NLN . yielded the highest rate of callus induction (28.57 %), followed by B5 (24.06 %), with MS medium yielding the lowest (13.41 %). T he NLN yielded the largest callus size, followed by B5, while the MS medium yielded the smallest. The optimal hormonal combination for callogenesis comprised 1 mg/L of 2,4-D and 1 mg/L of TDZ , achieving a callus induction percentage of 79.68 % and a callus size of 44.4 mm. The highest embryogenesis percentage (76.65 %) was attained with 2 mg/L of Pic and 1 mg/L of Kn . Ploidy level evaluation of the regenerated bulblets revealed three haploid bulblets, the remainder being diploid, consistent with the control, determined through chromosome count, and confirmed by flow cytometry. In summary, the anther culture protocol proposed in this study holds promise for producing homozygous L. ledebourii plants.