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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Abbreviations
- AS:
-
acetosyringone
- BA:
-
6-benzylaminopurine
- Cef:
-
cefotaxime
- Cm:
-
chloromycetin
- GUS:
-
β-glucuronidase
- Kin:
-
kinetin
- Km:
-
kanamycin
- LB medium:
-
Luria-Bertani medium
- MT:
-
Murashige and Tucker
- NAA:
-
α-naphthaleneacetic acid
- NPTII:
-
neomycin phosphotransferase II
- PCR:
-
polymerase chain reaction
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Duan, Y.X., Guo, W.W., Meng, H.J. et al. High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis . Biol Plant 51, 212–216 (2007). https://doi.org/10.1007/s10535-007-0043-7
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DOI: https://doi.org/10.1007/s10535-007-0043-7