Different serials of binary vectors such as pCAMBIA, pGA, pBIN, pGreen with regular insert capability, and BiBACs, pYLTACs with large insert capability have been reported for Agrobacterium-mediated transformation (AMT) of various plant species (Hellens et al., 2000b; Shibata and Liu 2000). Most of these binary vectors have nptII (neomycin phosphotransferase for resistance to kanamycin) as a selection gene and uidA (β-glucuronidase for GUS assay) as a reporter gene. They allow early selection by kanamycin and later verification of transformed plants by GUS assay. The GUS assay requires a part of the plantlet to be removed for X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glycopyranoside) staining. This will obviously result in damage to or removal of the plantlet if an early stage of screening is required. Since the green fluorescent protein (GFP) gene (gfp) was cloned and used as a visual marker for gene expression study (Chalfie et al., 1994), it has been applied in many organisms, including plants (Haseloff and Amos 1995; Haseloff et al., 1997). Expressed green fluorescent protein allows early detection and recovery of transgenic shoots without any damage to plantlets; thus, escape shoots, which were frequently observed in AMT (Domínguez et al., 2004; Peña et al., 1995), can be visually distinguished and categorized shortly after regeneration. One GFP binary vector, pBin19-sgfp (Chiu et al., 1996), was evaluated in citrus, indicating the advantage of GFP in the early and rapid in vivo screening of transgenic shoots (Ghorbel et al., 1999). Another nonbinary GFP vector was also used in citrus protoplast transformation (Fleming et al., 2000). These vectors usually are developed for specific use. Generally, it is very time-consuming to modify them to develop new constructs for other applications.
The pGreen is considered to be a new concept binary Ti vector (Hellens et al., 2000a). High-transformation efficiencies have been demonstrated in tomato and rice using some original pGreen vectors (Hellens et al., 2000a; Vain et al., 2003). The pGreen consists of a plasmid backbone plus a BglII-flanked T-DNA region containing two unique blunt restriction sites, HpaI and StuI, located internally to the left and right border sequences (LB and RB), respectively, which facilitate the cloning of any blunt selection and reporter gene cassettes. Two BglII sites are useful in cloning and posttransformation analysis. These special features allow new binary vectors to be developed optimally and quickly to meet user's different demands. Moreover, pGreen contains 18 unique cloning restriction sites within the pBlueScript polylinker maximizing the options to clone an alien gene for construct development. Compared with other binary vectors, the size of the vector is small because the origin of the replication element required by Agrobacterium is in a helper plasmid, pSoup, thus making the manipulation relatively easy.
In this article, we report the development of eight new pGreen-derived GFP vectors and preliminary studies to evaluate one of the vectors using citrus Agrobacterium-mediated transformation.
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Domínguez, A. , Cervera, M. , Pérez, R.M. , Romero, J. , Fagoaga, C. , Cubero, J. , López, M.M. , Juárez, J.A. , Navarro, L. & Peña, L. 2004 Characterization of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies Mol. Breed. 14 171 183
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