biologia plantarum

International journal on Plant Life established by Bohumil Nìmec in 1959

Biologia plantarum 51:212-216, 2007 | DOI: 10.1007/s10535-007-0043-7

High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis

Y. X. Duan1, W. W. Guo1,*, H. J. Meng1, N. G. Tao1, D. D. Li1, X. X. Deng1
1 National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan, P.R. China

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.

Keywords: Agrobacterium tumefaciens; Bingtang sweet orange; GUS; kanamycin; PCR; plantlet regeneration; Southern blot; transformation
Subjects: Agrobacterium tumefaciens; auxins; Citrus sinensis; glutamic acid, glutathione; in vitro culture, callus growth and duration; in vitro culture, embryogenesis, somatic embryogenesis; in vitro culture, regeneration, proliferation, differentiation; kanamycin; nutrient medium, Lucia-Bertany (LB); nutrient medium, Murashige Tucker (MT); polymerase chain reaction (PCR); Southern blot analysis; sweet orange; transgenic plants

Received: April 29, 2005; Accepted: January 7, 2006; Published: June 1, 2007  Show citation

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Duan, Y.X., Guo, W.W., Meng, H.J., Tao, N.G., Li, D.D., & Deng, X.X. (2007). High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis. Biologia plantarum, 51(2), 212-216. doi: 10.1007/s10535-007-0043-7
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