Establishment of Highly Efficient Plant Regeneration, Callus Transformation and Analysis of Botrytis cinerea-Responsive PR Promoters in Lilium brownii var. viridulum.

Lilium brownii var. viridulum, commonly called Longya lily, is a well-known flower and vegetable plant in China that has poor tolerance to Botrytis fungal disease. The molecularimprovement has mainly been restricted to an efficient regeneration and transformation system. In this study, the highly efficient regeneration of Longya lily was established through the optimization of embryogenic callus, adventitious shoot and rooting induction. The major factors influencing transformation (antibiotics, Agrobacterium concentration, infection time, suspension solution and coculture medium) were examined. The expression responses of PR promoters (ZmPR4 and BjCHI1) to B. cinerea were assessed in transgenic calli. The results showed that Murashige and Skoog (MS) medium with 1.0 mg·L-1 picloram (PIC) and 0.2 mg·L-1 1-naphthaleneacetic acid (NAA) under light conditions and MS with 0.5 mg·L-1 6-benzylaminopurine (6-BA) and 1.0 mg·L-1 NAA under darkness were optimal for embryogenic callus induction (64.67% rate) and proliferation (3.96 coefficient). Callus inoculation into MS containing 2.0 mg·L-1 thidiazuron (TDZ), 0.4 mg·L-1 NAA, 1.0 mg·L-1 TDZ and 0.5 mg·L-1 NAA led to shooting induction (92.22 of rate) and proliferation (3.28 of coefficient) promotion, respectively. The rooting rate reached 99.00% on MS with 0.3 mg·L-1 NAA. Moreover, a transformation rate of 65.56% was achieved by soaking the callus in Agrobacterium at an OD600 of 0.4 for 10 min in modified MS without NH4NO3 as the suspension solution and coculture medium before selecting 75 mg·L-1 hygromycin and 300 mg·L-1 cefotaxime. Only the BjCHI1 promoter was obviously expressed in transgenic calli. These results could facilitate the generation of Longya lily transgenic plants with improved B. cinerea resistance.