Objective To screen the hormone proportion suitable for callus differentiation of Litsea cubeba and clarify its tolerance to antibiotics, and preliminarily establish the genetic transformation system of L. cubeba.

Method The effects of different hormone concentrations on adventitious bud induction and adventitious bud rooting of L. cubeba callus were studied. The critical screening concentrations of hygromycin and cefotaxime were discussed, and the foreign gene was introduced into L.cubeba callus by Agrobacterium mediated method.

Result The optimum medium for inducing adventitious bud differentiation of callus was MS + 2.0 mg·L⁻¹ 6-BA + 0.01 mg·L⁻¹ IBA + 0.05 mg·L⁻¹ TDZ, and the differentiation rate was 16.67%～36.67%; The optimum medium for adventitious bud rooting was 1/2MS + 0.5 mg·L⁻¹ IAA, and the rooting rate was 97.33%. The initial concentration of hygromycin for resistant callus screening was 5 mg·L⁻¹ (about 7~10 days), and then the critical screening culture was carried out by gradually increasing the hygromycin screening concentration to 30 mg·L⁻¹. the optimum concentration of cephalosporin was 300 mg·L⁻¹. Finally, foreign gene was transferred into the callus by Agrobacterium mediated method, and PCR primers were designed for identification. A total of 16 resistant seedlings contained the target band, indicating that the target gene had been inserted into the L. cubeba genome, with a transformation rate of 0.67%. In addition, our research group obtained the resistant calli of multiple genes through this method. Southern detection showed that the target fragment had been inserted into the calli of L. cubeba.

Conclusion The regeneration and genetic transformation system of L. cubeba has been preliminarily established, and the resistant calli of multiple genes have been obtained, which provides technical support for further gene function research and genetic improvement. The next step is to optimize the genetic transformation system and improve its transformation efficiency.