Functional expression of miraculin, a taste modifying protein, in Citrus unshiu Marc. and characterization of three novel citrus ERF genes

Abstract

Miyagawa Wase, a common cultivar of the Satsuma mandarin (Citrus unshiu Marc.), is characterized by its tender peel and seedless nature. In this study, Agrobacterium-mediated transformation of Miyagawa Wase was performd using embryogenic calluses from unfertilized ovules. Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1300 that contained the miraculin gene (a taste-modifying protein) and hygromycin as a selection marker were used. After 5 days of co-culture in a medium containing 100 pM acetosyringone, calluses were transferred to the liquid half EME medium with 15 mg/L hygromycin and 250 mg/L cefotaxime and then cultured for 2 weeks. Subsequently, the calluses were grown on a solid selection medium with 20 mg/L hygromycin for 4 weeks, followed by selection with 25 mg/L hygromycin for 4 more weeks. Total 168 resistant embryos were selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart-shaped embryos were transferred to MT medium containing 1 mg/L GA3, 20 ml/L coconut water, 20 pg/L NAA, and 14.6 pg/L coumarin for embryo germination. Finally, 135 germinated embryos were cultured on MT medium containing 30 g/L sucrose and 8 g/L agar and recovered 115 normal plants. This transformation procedure yielded 37 trangenic plants containing miraculin genes as verified by PCR amplification. Southern blot analyse of 5 randomly selected plants further confirmed the miraculin transgene was stably integrated into the Miyagawa Wase genome.