Optimization of the regeneration and Agrobacterium-mediated transformation in pear

Abstract

Conventional breeding in pears is inefficient due to a long juvenile phase and self-incompatibility. Genetic transformation offers a promise to expedite the breeding process. However, the frequencies of regeneration and genetic transformation in most Pyrus spp. are relatively low. This study investigated various factors influencing regeneration and genetic transformation using leaves from Pyrus ussuriensis Maxim “Shanli” and P. communis L. “Conference” as explants. The optimum regeneration medium for “Shanli” and “Conference” was NN69 containing 3.0 mg L−1 thidiazuron (TDZ) and 0.3 mg L−1 indolybutyric acid (IBA) for the former or 1.0 mg L−1 TDZ and 0.5 mg L−1 naphthalene acetic acid (NAA) for the latter. Sectioning the leaves from 30-day-old plantlets transversely and placing them with their abaxial side facing downward could significantly improve the regeneration ratio in both accessions. Moreover, a two- or four-week culture under the dark was beneficial for the regeneration of “Shanli” and “Conference” respectively. The optimal infection time was 12 and 8 min, while the time of the delayed screening test was two and one day for “Shanli” and “Conference” respectively. Moreover, a coculture of two days was recommended for both accessions. Post-transformation, the optimal concentrations of antibiotics were 16 mg L−1 kanamycin (Kan), 150 mg L−1 timentin (Tim), and 300 mg L−1 cefotaxime (Cef). The optimized regeneration and transformation system can be an effective alternative for either gene function analysis or genetic improvement in pear.