Volume 51, Number 2, 212-216, DOI: 10.1007/s10535-007-0043-7

High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis

Y. X. Duan, W. W. Guo, H. J. Meng, N. G. Tao, D. D. Li and X. X. Deng

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Abstract

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.

Additional key words  Agrobacterium tumefaciens - Bingtang sweet orange - GUS - kanamycin - PCR - plantlet regeneration - Southern blot - transformation

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