Identification of a high-regeneration Malus robusta Rehd. genotype and establishment of an efficient transformation...

Transgenic apple cultivars may pose environmental safety concerns, particularly regarding pollen dispersion. Grafting non-transgenic scions onto transgenic rootstocks offers a promising strategy to combine desirable trait regulation with minimized risk of transgene flow via pollen. In China, Malus robusta Rehd. is one of the most widely used apple rootstocks. However, their transformation efficiency and reproducibility remain limited. In this study, we identified a high-regeneration genotype BL-57 from seedlings of Malus robusta Rehd., optimized the leaf regeneration and Agrobacterium-mediated genetic transformation system, and finally established a stable transgenic system for both gene knockout and overexpression. Leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 0.3 mg/L 6-benzylaminopurine (6-BA), 0.2 mg/L indole-3-acetic acid (IAA), 0.1 mg/L gibberellic acid 3 (GA₃), 30 g/L sucrose, and 7.5 g/L agar. Following leaf transection, the explants were transferred to MS medium containing 2 mg/L thidiazuron (TDZ), 0.5 mg/L naphthaleneacetic acid (NAA), 30 g/L sucrose, and 7.5 g/L agar, where the highest regeneration efficiency was observed, with an average of 15 shoots per explant. Leaf explants infected with Agrobacterium were cultured on bud induction medium supplemented with 6 mg/L kanamycin and 250 mg/L cefotaxime. The regenerated plantlets were identified and verified, demonstrating that the transgenic systems for gene knockout and overexpression have been successfully established in BL-57. In summary, we identified a M. robusta germplasm with high regeneration ability, and established an efficient leaf regeneration and transformation system. This platform provides a valuable tool for advancing molecular breeding and functional genomics research in apple.