Ethylene inhibitor silver nitrate enhances regeneration and genetic transformation of Prunus avium (L.) cv Stella

The effects of the ethylene inhibitors, silver nitrate (AgNO3) and aminoethoxyvinylglycine (AVG), together with Agrobacterium co-cultivation were investigated for improving both regeneration and genetic transformation of leaf explants of the recalcitrant sweet cherry cultivar Stella. Co-cultivated young leaf explants with A. tumefaciens strain EHA105 harbouring a binary vector pROK containing both the phytochrome B of Arabidopsis thaliana and the nptII genes, were cultured on M2 MS-modified medium supplemented with either AVG (2.54 A mu M) or AgNO3 (296.08 A mu M) for 21 days in the dark, and then transferred onto ethylene-free inhibitors medium in the light. Efficiency, in terms of frequency on shoot organogenesis, was observed in explants subjected to co-cultivation with Agrobacterium and not co-cultivated in presence of each ethylene inhibitors. Ethylene inhibitors helped in the morphogenesis process and mass production of shoots. An increase in regeneration efficiency was observed in ethylene-inhibitor-free medium, which was almost four times greater than the control leaf explants. When ethylene inhibitors were added to the medium, a further regeneration efficiency enhancement for AVG (45 %) and AgNO3 (63.3 %) was observed. Whereas, in explants not co-cultivated with Agrobacterium, the resulting regeneration efficiency was 25 % for AVG and 23.3 %, for AgNO3. The regeneration frequency from leaf explants was also enhanced in treatments with AVG when combined with Agrobacterium co-cultivation, with 139 shoots regenerated from a total of 27 regenerating explants. The AgNO3 also contributed to control Agrobacterium growth in the post-co-cultivation phase. Only the lines originating from AgNO3-treated explants showed kanamycin resistance, 9 out of 141 regenerated lines, and polymerase chain reaction analysis confirmed both the insertion of the genes into the plant genome and the absence of bacterial contamination of the transgenic shoots.