High efficient transgenic plant regeneration from embryogenic calluses of <Emphasis Type="Italic">Citrus sinensis</Emphasis> |
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Authors: | Y X Duan W W Guo H J Meng N G Tao D D Li X X Deng |
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Institution: | 1.National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding,Huazhong Agricultural University,Wuhan,P.R. China |
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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. |
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