Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Abstract

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.

Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants

“…Transformation methods differ in their suitability for various purposes and plant species Repellin et al 2001), DNA integration patterns (Christou 1995;Birch 1997;Christou 1997) and their efficiency (Snape 1998). It has been shown by many authors that the development of any of transformation procedures may be much faster and more efficient if proper signal gene(s) are used throughout the study (Birch 1997;Baranski et al 2006). …”

“…Based on differences in GFP fluorescence, the effect of desiccation of co-cultivated explants on efficacy of transformation has also been analysed (Polin et al 2006). For example, Baranski et al (2006) successfully employed the GFP fluorescence for critical assessment of the whole transformation procedure of the Agrobacterium rhizogenes-mediated transformation of carrot. Based on the green fluorescence intensity they selected the most virulent Agrobacterium strain, effective acetosyringone concentration and the most suitable carrot genotype for transformation.…”

“…for the study of the expression patterns of promoters (Sheen et al 1995;Nagatani et al 1997), protein tagging (Chytilova et al 1999;Shiina et al 2000), disease tracking (Itaya et al 1997), developmental studies (Misteli and Spector 1997), expression studies and ecological monitoring of transgene spread (Halfhill et al 2001). GFP is being increasingly used for various purposes associated with the transformation of plants ( Baranski et al 2006;Yong et al 2006). Nowadays, many GFP homologues originating from various organisms are available, allowing for broad range of use in biology (Chudakov et al 2005) (Table 1).…”

“…The use of visual markers, which enable direct observation of transformation events, results in a more precise and easier evaluation of various treatments and procedures. They can increase transformation efficiency by reducing the time and amount of material to be handled and screened (Baranski et al 2006) allowing the most efficient, reliable and reproducible transformation protocol to be established. The ideal marker should possess the following desirable traits.…”

Recombinational Cloning with Plant Gateway Vectors

“…In some of these, the T-DNA also incorporates a visible reporter (Table I,no. 80), enabling the distinction between transformed and untransformed cells mixed in chimeric tissues, such as hairy roots formed upon A. rhizogenes-mediated transformation ( Van de Velde et al, 2003;Baranski et al, 2006).…”