Transferring of rofcbcd genes in some citrus rootstock through Agrobacterium rhizogenes

摘要

Agrobacterium-mediated transformation represents an effective and universal method for transferring target genes to plant species. The obtainment of dwarf rootstocks is one of the essential goals in breeding of fruit tree species and especially for citrus. In the present study, non-recombinant wild-type A rhizogenes strain ATCC15834 was used to obtain dwarf citrus rootstock in in vitro conditions. Tuzcu 3131 Sour Orange, Carrizo citrange, Gou Tou Sour Orange, Swingle citrumelo and Troyer citrange were used as plant materials. Internodal stem explants were obtained from seedlings. Two different methods were applied for transformation. In the first treatment, explants were excised and inoculated with A. rhizogenes suspension and shaken at 80 rpm for 30 min, and explants were transferred to MS medium for 2 days in darkness at 24°C. After co-cultivation, the explants were cultured on MS medium containing 500 mg L1 cefotaxime. In the second treatment bacterial cells were pelleted by centrifugation at 5000 rpm for 10 min, resuspended and diluted in bacterial suspension medium. The explants were immersed for 15 min in bacterial suspension and transferred to Petri dishes containing cocultivation medium (MS, 2 mg L1 IAA, 1 mg L12IP, 2 mg L1 2,4-D) for 3 days at darkness. Then the explants were transferred to shoot regeneration medium (MS supplemented with 500 mg L1 of cefotaxime and 1 mg L1 of BAP). The presence of rol genes in the putative transformed plants was confirmed by PCR analysis. Based on molecular analysis, 13 clones containing rol genes were recovered from Troyer citrange, they were rooted in in vitro conditions, and maintained their vitality and continued to develop. Four clones containing rol genes were recovered from Carrizo citrange, they were rooted in in vitro conditions, but these clones were not survived during acclimatization.