Development of agrobacterium-mediated transformation protocol for mature seed-derived callus tissues of citrus cultivar ‘gailiangcheng orange x weizhang satsuma mandarin’

The current study was conducted to develop Agrobacterium-mediated transformation protocol for mature seed-derived callus tissues of Citrus cultivar ‘GWZ’ (Gailiangcheng orange x Weizhang Satsuma mandarin) using gfp as reporter. Transformation experiments were performed with Agrobacterium tumefaciens strain ‘EHA-105’, harboring a binary vector ‘pBin19’. Some important transformation factors including Kanamycin, and Acetosyringone concentrations, optical density of Agrobacterium culture, pre-culturing period, immersing time, and co-culturing period were investigated based on gfp fluorescence. The gfp fluorescence enabled us in early detection and recovery of transformants. Explants pre-cultured for three days before immersing in bacterial suspension for 20 min at OD600: 0.6 significantly improved the transformation efficiency. By combining the best combinations of the transformation factors, we achieved highest transformation efficiency (43.56%). The integration of gfp gene in transgenic plantlets was confirmed by PCR analysis. The protocol reported herein would help in transformation of useful genes in tree fruit species particularly in the Citrus cultivar under study.