Agroinfiltration is a key factor to improve the efficiency of apple and pear transformation

摘要

Genetic transformation of apple and pear is mainly performed via Agrobacterium tumefaciens. The average efficiency of transformation remains low in most laboratories and the success of the transformation experiments is very variable. Improving the efficiency and the reproducibility of apple and pear transformation is thus highly desirable. Adventitious regeneration ability of the explants is not a limiting factor for apple or pear transformation. The present study focuses on improving the frequency of stably transformed cells in the explants following A. tumefaciens inoculation. We report here the results of 36 independent transformation experiments on 'Gala' apple, performed with 10 different binary plasmids, comparing three methods of A. turnefaciens inoculation. Agroinfiltration (Agrobacterium-mediated vacuum infiltration) of the explants in a bacterial suspension containing a surfactant (Silwet L-77) at a low concentration (0.002% v/v) significantly increased the average transformation efficiency (mean rate of 5.8% and maximum rate of 30%) and reduced the number of failed experiments (16%) compared to the method of crushing the explants with non-traumatic forceps prior to immersion into the inoculum. The success of the agroinfiltration method was associated with a very high level of GUS expression one month after inoculation.In addition, agroinfiltration dramatically increased the transformation efficiency of pear, reaching rates of transformation between 50 and 80%, compared to inoculation with a scalpel dipped into the inoculum. Altogether, our results demonstrate that the production of large number of transgenic apple or pear lines in a short period of time is feasible using the agroinfiltration method.