High efficiency Agrobacterium-mediated transformation of sour orange (Citrus aurantium L.) using gene encoding Citrus Tristeza Virus coat protein

Citrus trees are widely grown in tropical and subtropical climates due to their luscious taste, nutritional and medical benefits. Citrus fruits are native to southeastern Asia and are among the oldest fruit crops domesticated by humans. Breeding programs including the incorporation of genetic resistance to insect pests and diseases are necessary in this crop. Citrus tristeza virus (CTV) is of particular importance due to its rapid epidemic resulting in severe plant damage. The present research aimed to transform Citrus aurantinum with a gene encoding coat protein of CTV through Agrobacterium-mediated transformation. Thep25 coat protein gene was isolated from two native CTV isolates. Two conserved regions from the two isolates, were identified and subcloned as a single chimer into a pFGC5941 silencing vector. Epicotyls-originated explants of C. aurantium were transformed by EHA105 strain of Agrobacterium tumefaciens. Some of the effective factors in gene transformation were examined by inoculation methods with Agrobacterium such as Acetosyringon effect (0, 50, and 100 μM), inoculation time (5, 10, 15, 20, and 25 min), and co-cultivation period (1, 2, 3 and 4 days). Based on our results, maximum number of transformed plants ( 13.7%) were obtained under combined treatment of 50 μM acetosyringone after 15 min inoculation time and 2 days of co-cultivation with Agrobacterium. One of the advantages of the current protocol is regeneration of explants through direct organogenesis which avoid callus phase and consequently somaclonal variation.