期刊
BIOLOGIA PLANTARUM
卷 51, 期 2, 页码 212-216出版社
SPRINGER
DOI: 10.1007/s10535-007-0043-7
关键词
Agrobacterium tumefaciens; Bingtang sweet orange; GUS; kanamycin; PCR; plantlet regeneration; Southern blot; transformation
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 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 degrees 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.
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