An important aspect of the new approach to genetic modification is that it omits the use of bacterial selectable marker genes (see also Chap. 3). Initially developed procedures simply exclude a selection step, yielding potato transformation frequencies below 0.1%. These frequencies were increased to 2% by applying supervirulent Agrobacterium strains such as AGL0 (de Vetten et al. 2003). An alternative method was developed by employing two different transfer DNAs, one carrying a positive and negative selectable marker gene and the other one comprising the DNA of interest. A transient positive selection step for marker gene expression followed by a negative selection step against marker gene integration yielded plants containing only the desired DNA with frequencies of about 15% (Rommens et al. 2004; Kondrak et al. 2006). The newest and most preferred method for markerfree transformation includes a quality-control mechanism that also selects for backbone-free DNA transfer (Richael et al. 2008). This method employs vectors containing the bacterial isopentenyltransferase (ipt) gene as backbone integration marker. Agrobacterium strains carrying the resulting ipt gene-containing vectors were used to infect explants of various solanaceous plant species. Upon transfer to hormone-free media, 1.8-6.0% of the infected explants produced shoots that contained a marker-free P-DNA while lacking the backbone integration marker. Because of the very high frequency of left border skipping in potato, these frequencies equal those for backbone-free conventional transformation.
The absence of antibiotic or herbicide tolerance genes facilitates the regulatory approval process and may also alleviate some consumer concerns about the permanent introduction of foreign DNA into the food supply. Another advantage of the use of cytokinin vectors is that the temporary formation of the natural cytokinin isopentenyl adenosine in Agrobacterium-infected explants is highly effective in inducing regeneration. In fact, regeneration frequencies are tenfold higher for explants on hormone-free media that were infected with an Agrobacterium strain carrying a cytokinin vector than for conventionally infected explants on hormone-containing media. Interestingly, the new method is immediately applicable to other dicotyledonous plant species and may represent a new step towards the development of genotype-independent transformation methods (Richael et al. 2008). The utility of ipt-based transformation has already been demonstrated for nightshades and canola and may likely be extended to other crops, such as rice (Oryza sativa), sunflower (Helianthus annuus), and pineapple (Citrus sinensis), that are known to respond to ipt gene overexpressing by producing adventitious shoots (Endo et al. 2002; Molinier et al. 2002; Ballester et al. 2007). In future studies we will further extend the applicability of the cytokinin vector method, not only by using hormonefree media but also by testing the utility of media containing small amounts of auxins. This modification may be required for some genotypes that need exoge-nously applied auxins for the regeneration of proliferated cells.
The second characteristic of the intragenic approach is, as mentioned above, that the employed transfer-DNAs are derived from within the target crop itself. The potato sequence used to support DNA transfer was isolated from pooled DNA of wild potato species that are sexually compatible with potato (Rommens et al. 2004). Its original size of 1.6 kb was reduced, through deletion of an internal fragment, to obtain a 0.4-kb P-DNA with several unique restriction sites. The 25-base pair (bp) St01 border-like elements that delineate this P-DNA were more effective than conventional T-DNA borders in mediating plant transformation
(GenBank accession AY566555; Rommens et al. 2004). Efficient initiation of DNA transfer could also be accomplished by linking a second border-like element from potato, St02, to a GC-rich region derived from DQ235183 (the inverse-complement of nucleotides 256-329; Rommens et al. 2005). Vector pSIM781, which carries this right border region, promotes similar frequencies of tobacco transformation as either a T-DNA vector or a vector containing the original P-DNA from potato. The same border-like element facilitates the termination of DNA transfer as well if linked to the 79-bp AT-rich DNA region from AF216836 (nucleotides 3231-3310) in pSIM1141.
One of the most frequently used elements for tuber-specific gene expression is the promoter of the granule-bound starch synthase gene (Visser et al. 1991). This promoter has been used extensively in transgenic research, because it delivers high levels of gene expression. Other well-known tuber-specific promoters include the patatin promoter (Jefferson et al. 1990) and the promoter of the ADP glucose pyrophosphorylase (Agp) gene (Du Jardin et al. 1997). For most yield-associated traits, it may be important to express the associated target genes not just in the tuber but throughout the potato plant. Examples of such traits include salt, drought, and frost tolerance. The most effective near-constitutive promoters that can be used for these purposes are the ubiquitin-3 (Ubi3) and ubiquitin-7 (Ubi7) promoters (Garbarino and Belknap 1994; Garbarino et al. 1995). The standard terminator used for the construction of gene expression cassettes was isolated from the Ubi3 gene (Garbarino and Belknap 1994). This terminator is as effective as the frequently used bacterial terminator of the nopa-line synthase (nos) gene.
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