An efficient leaf regeneration and genetic transformation system for Pyrus betulifolia

Abstract Pears rank as the third most produced fruit in China, with widespread cultivation throughout the country. Asexual propagation, primarily through grafting onto Pyrus betulifolia rootstocks, is the predominant method of cultivation. However, the absence of an efficient genetic transformation system for P. betulifolia significantly hinders genetic enhancement efforts for pear rootstocks. In this study, TDL39, a P. betulifolia genotype, was identified for its remarkable regenerative capacity. Leaf strips derived from 60-day-old TDL39 seedlings were cultured abaxial side up on a regeneration medium consisting of NN 69 , 3.0 mg/L thidiazuron (TDZ), 0.1 mg/L indole-3-butyric acid (IBA), 30 g/L sucrose, and 6.6 g/L agar. The strips were kept in the dark for 28 days and subsequently exposed to light for 30 days, resulting in the formation of adventitious shoots with an average regeneration rate of 81.5%. Employing this regeneration protocol, transgenic P. betulifolia plants overexpressing the Green Fluorescent Protein ( GFP ) gene were successfully generated, with 15 mg/L kanamycin (Kan) used for selection. The highest transformation efficiency achieved was 4.2%. This study successfully establishes a regeneration system for P. betulifolia and facilitates the production of transgenic plants, thereby advancing molecular breeding and the functional characterization of P. betulifolia and other pear genotypes.