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10 April 2008 An improved procedure for Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf.) via indirect organogenesis
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Abstract

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA) 1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.

Xiuping Zou, Demou Li, Xiaoying Luo, Keming Luo, and Yan Pei "An improved procedure for Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf.) via indirect organogenesis," In Vitro Cellular and Developmental Biology - Plant 44(3), 169-177, (10 April 2008). https://doi.org/10.1007/s11627-008-9106-5
Received: 10 May 2007; Accepted: 4 February 2008; Published: 10 April 2008
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