High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis

作者：Yanxin Duan, Wen‐Wu Guo, Haijun Meng, Nengguo Tao, Dongzhi Li, X. X. Deng · 发表于：Biologia Plantarum · 年份：2007 · DOI：10.1007/s10535-007-0043-7 · 被引用次数：41 · 研究领域：Plant tissue culture and regeneration、Transgenic Plants and Applications、Plant Reproductive Biology

Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange [Citrus sinensis (L.) Osbeck] was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harboring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron β-glucuronidase (GUS) gene and the Arabidopsis APETALA1 (AP1) gene. Transformation treatment was with inoculation time of 30 min, co-culture of 3 d at 23 °C and supplementation of the co-culture medium with 2 mg dm-3 acetosyringone (AS). Kanamycin (50 mg dm-3) was effective to inhibit the growth of non-transformed calluses while it did not affect the transformed ones. The total number of transformed callus lines was 7 with 100 % embryo induction. High efficient regeneration of the transgenic embryos (88 % with 4-5 shoots per embryoid) was realized within 3 months. Integration of the transgene into the citrus genome was confirmed by histochemical GUS staining, polymerase chain reaction (PCR) analysis with AP1-specific primer and Southern blot hybridization with a 712 bp PCR fragment of AP1 as the probe.