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