High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis
Duan, Y.X.,Huazhong Agricultural Univ., Wuhan (China). National Key Lab. of Crop Genetic Improvement | Guo, W.W.,Huazhong Agricultural Univ., Wuhan (China). National Key Lab. of Crop Genetic Improvement | Meng, H.J.,Huazhong Agricultural Univ., Wuhan (China). National Key Lab. of Crop Genetic Improvement | Tao, N.G.,Huazhong Agricultural Univ., Wuhan (China). National Key Lab. of Crop Genetic Improvement | Li, D.D.,Huazhong Agricultural Univ., Wuhan (China). National Key Lab. of Crop Genetic Improvement | Deng, X.X.,Huazhong Agricultural Univ., Wuhan (China). National Key Lab. of Crop Genetic Improvement
Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harbouring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron beta-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 deg C and supplementation of the co-culture medium with 2 mg/cubic dm acetosyringone (AS). Kanamycin (50 mg/cubic dm) 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.
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