Improvement of genetic transformation with mature explants of sweet orange (Citrus sinensis)

High level of contamination, low morphogenetic potential and recalcitrance to Agrobacterium tumefaciens infection are the main problems in the transformation of adult citrus tissues. A series of experiments was performed to solve these problems. ‘Newhall’, ‘Succari’ and ‘Bingtang’ sweet orange cultivars were tested. Adult shoots from the 3 cultivars were grafted and re-grafted on Poncirus trifoliata rootstocks in the greenhouse. Internodal stem segments of about 1 cm in length from 30-day old mature shoots of were taken as explants. Through re-grafting mature shoots, the contamination rate of explants was much lower, even 0%. The adventitious bud regeneration rate was obviously improved, approximately 80%, 67.5% and 55% in ‘Succari’, ‘Bingtang’ and ‘Newhall’ sweet oranges, respectively. After Agrobacterium infection, the highest adventitious bud regeneration rate of ‘Bingtang’ sweet orange reached 25%. The transformation rate was 3.8%, 4.6% and 3.2% in ‘Succari’, ‘Bingtang’ and ‘Newhall’ sweet orange cultivars, respectively. The ‘gene deletor’ technology is an efficient system to remove all the functional transgenes from the pollen and seeds of the transgenic plants, activated by specific promoters. Two ‘gene-deletor’ constructs containing pLF_polB-FLP and pLF_polL-Cre boxes, respectively, were transferred into mature explants of sweet orange by the improved procedures, and 10 transformed plants were confirmed to be positive by GUS expression and PCR analysis. The transformed plants will be tested for the gene-deletor function later on.