Establishment of an in vitro regeneration and Agrobacterium-mediated genetic transformation system for Lilium longiflorum ‘Sally’ stem segments

• Established an efficient stem-based regeneration system for lily. • Optimized Agrobacterium-mediated transformation for 'Sally' lily. • LiAGPase overexpression significantly increased bulb starch content. • CRISPR/Cas9-mediated LiAGPase editing reduced starch accumulation. • Validated LiAGPase as a key regulator in lily bulb development. Lilium longiflorum is a commercially important ornamental crop, yet its genetic improvement is constrained by the slow pace of conventional breeding. This study aimed to establish an efficient in vitro regeneration and Agrobacterium -mediated transformation system for L. longiflorum ‘Sally’ using stem segments. Results showed that Murashige and Skoog (MS) medium supplemented with 1.0 mg/L picloram (PIC) and 0.2 mg/L naphthaleneacetic acid (NAA) achieved the highest callus induction rate of 77.78%. For adventitious bud differentiation, 1.5 mg/L 6-benzylaminopurine (6-BA) combined with 0.5 mg/L NAA was optimal, reaching a 68.00% success rate. The most efficient transformation protocol utilized an Agrobacterium density (OD 600 ) of 0.8, a 10 min infection duration, and 300 mg/L cefotaxime for bacterial control. Using this system, the function of the ADP-glucose pyrophosphorylase ( LiAGPase ) gene in starch metabolism was validated. Overexpression lines exhibited significantly increased starch content, while CRISPR/Cas9-mediated gene editing produced a mutant with a missense mutation (Lys to Gln), reducing starch accumulation to 43.3% of the wild-type level. This study establishes a viable technical platform for lily genetic improvement and further elucidates the regulatory role of LiAGPase gene in starch biosynthesis, thereby providing essential support for molecular breeding programs aimed at enhancing bulb quality in Lilium .