Establishing An Efficient Genetic Transformation System Of Apple And Transformation With MiR156and RSPL9Genes

With the completion of the genome sequencing of cultivated apple, the apple gene function analysis become an important part of the study of molecular biology. But this depends on the efficient genetic transformation system. Although Agrobacterium tumefaciens-mediated genetic transformation in some apple genotypes have been reported, but the transformation rate usually is low. In this study, an efficient Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants was developed, the clones with high regeneration ability and more sensitive to Agrobacterium were screened from the seedling population of M. micromalus and M. domestica cv. Gala, and miR156and rSPL9genes were transferred into apple. The main results are as follows:1. The effects of explant types, explant size, dark-culture time, the basic medium and the contact style of explants with medium were studied, using the cotyledons M. micromalus as explants. The bud regeneration frequency of proximal cotyledon explants near was2.3fold compared distal cotyledon explants, and the number of buds per explant of proximal cotyledon was3.2fold compared distal cotyledon explants. When the explants were cultured on SC medium, the cotyledon regeneration bud frequency was90%, and the number of buds per explant reached9.14. While on the MS medium, the cotyledon regeneration bud frequency and the number of buds per explant were72.5%and6.28, respectively. The number of buds per1/3proximal cotyledon explant was significantly higher than that of1/2proximal cotyledon explant.2. On the base of establishment of the efficient regeneration system of M. micromalus cotyledons, using nearly1/3proximal cotyledon as the transformed explants, effects of the concentration of bacteria, the inoculation time, the co-culture time and the delayed selection time on transformation efficiency were studied. An efficient Agrobacterium-mediated transformation system in M. micromalus using cotyledons as explants was developed. In this system, the concentration of bacteria was OD6oo=0.5, the explanted were immersed in bacteria broth for8min, the co-culture time was3days, and there was no delayed selection. The percentage of explants with resistant buds reached11.67%.3. Two seedling populations were developed from the seeds of M. micromalus and M. domestica cv. Gala. Two clones, BT-9and GL-3, with high regeneration ability were screened. On the medium supplemented with BA, the regeneration frequency of adventitious buds of BT-9leaves reached to100%, and the number of buds per explant was18.4. The adventitious bud regeneration ability of GL-3was higher than that of BT-9, Gala and Hanfu. On the medium supplemented with TDZ, the regeneration frequencies of4genotypes were100%, but the number of buds per explant of GL-3was22.36, which was significantly higher than that of BT-9(15.5), Gala (13.8) and Hanfu (10.16).4. The sensitivity to Agrobacterium tumefaciens of GL-3, BT-9, Gala and Hanfu were compared by analyzing the GUS gene transient expression. The GUS gene transient expression rate of GL-3explants was the highest, and it means that GL-3is more sensitive to Agrobacterium and suitable for genetic transformation.5. MiR156and SPL9genes were isolated from Arabidopsis and M. domestica, respectively. The rSPL9gene was obtained by mutating the miR156target site on SPL9gene. MiR156and rSPL9genes were transferred into GL-3and Hanfu by Agrobacterium-mediated transformation with leaves as explants. CL-3is very suitable for genetic transformation. The regeneration rate of resistant buds with miR156gene reached14.49%, while for Hanfu, it was up to7.06%. The apple plants transformed with miR156gene produced more shoots. The apple plants transformed with miR156gene with high regeneration ability.