Abstract: INTRODUCTION: In this experiment, the roots, tubers, leaves, buds and seeds of Bletilla striata ( Thunb. ) Reichb.f.were used as explants to explore the best induction conditions for embryonic callus and establish a genetic transformation system under the condition of multiple subculture selection.  RATIONALE: According to the experiment, the callus induction efficiency of different organs ofBletilla striata was studied to determine the optimal rapid propagation explants and the optimal concentration of plant growth regulators to promote the formation and regeneration of callus. This understanding is crucial for the development of genetic transformation related work in Bletilla striata.  RESULTS: The results showed that the best callus induction effect was obtained under the condition of MS + 0.5 mg/L 6-BA + 2 mg/L 2,4-D, and the induction rate was 86.1%. However, the callus induction period of tubers was longer. Compared with tubers, it was more convenient to obtain callus directly from the seeds of Bletilla striata, and the induction effect was not significantly different from that of tubers. The callus induction speed was fast, the structure was dense, the texture was soft, and it was white and green.Using seeds as explants, the best medium for callus proliferation was MS + 1.5 mg/L 2,4-D. The callus was dense, dry, clumpy and bright yellow-green. The average diameter after proliferation was 12.267 mm, and the browning rate was relatively low, about 19.4 %. The best medium for adventitious bud differentiation was MS + 0.1 mg/L 6-BA + 0.1 mg/L NAA, the differentiation rate was as high as 91.7 %, and the number of adventitious buds was large, and the browning rate was 8.3 %. The optimum medium for rooting was MS + 11 g/L agar powder + 0.3 mg / L NAA, which could effectively increase the rooting rate to 66.7 %. Based on the above regeneration system of Bletilla striata, this study used Agrobacterium infection method to overexpress the key enzyme genes BsPMM ( phosphomannose mutase gene ) and BsPGM ( glucose phosphomutase gene ) for polysaccharide synthesis of Bletilla striata, and successfully obtained positive B.striata seedlings. The success rate of BsPMM transformation was about 14.6 %, and the success rate of BsPGM transformation was about 8.3%.  CONCLUSION: In this study, the genetic transformation system of Bletilla striata was successfully established, including explant selection, callus induction, Agrobacterium-mediated genetic transformation, and the acquisition and identification of regenerated plants. Through the optimization of each link, the induction efficiency and differentiation quality of callus were improved. The genetic transformation system provides technical support for the research on the biosynthesis of active substances such as Bletilla striata polysaccharide and the genetic improvement of Bletilla striata. Follow-up research can further improve the technical system and expand its potential in the application of Bletilla striata genetic engineering.

Key words: Bletilla striata, Bletilla striata polysaccharides（BSP）, callus, transformation