**6. Biotechnology as a source of new diversity**

Many natural mutants discovered in cultivated tomato have been extensively studied and characterized (http://tgrc.ucdavis.edu/), but their amount is limited. Thus reverse genetic techniques were developed aiming to discover gene function by analyzing the phenotypic effects of specific variants of targeted gene sequence. This approach is complementary to classical ("forwards") approaches, as they allow silencing or promoting the expression of targeted gene. They can also be used to generate genetic diversity within DNA sequences.

A delayed ripening tomato, named *Flavr-savr*® tomato*,* with a reduced expression of a cell wall protein (a polygalacturonase), obtained by an antisense construction, was the first transgenic plant released on the fresh market (Kramer and Redenbaugh 1994; Sanders and Hiatt 2005). This transformation significantly improved fruit shelf life and storage quality. Nevertheless, it was a commercial failure. Few years later, consumer's concerns about GMO6,

<sup>6</sup> Genetically Modified Organism


Table 2. List of genes characterized through molecular techniques with their related function and germplasm origin

Table 2. List of genes characterized through molecular techniques with their related function

and germplasm origin

as well as the high engineering cost, stopped further commercial developments. In the research field, transformation with *Agrobacterium thumefaciens* is still widely used for the functional characterization of specific genes. For instance, transformed tomato plants were produced to enable study of endotoxins genes (Zhang, Buehner et al. 2006) plant disease resistance genes, abiotic stress genes or to produce molecules useful in human medicine (Sharma, Singh et al. 2008).

TILLING (Target Induced Local Lesion In Genomes), a mutagenesis technique, has experienced important development (Comai and Henikoff 2006). Early days of this technique were in the 50's (Rick 1991). It is now widely used for reverse genetics to generate and identify induced point mutations in genomes. A chemical reagent (Ethylmethane Sulphonate) is used to induce genetic mutations. Collections of tomatoes carrying artificially induced genetic variants, called mutant libraries are currently available (Menda, Semel et al. 2004; Minoia et al. 2010) or under development (Okabe, Asamizu et al. 2011). In contrast to transgenic methods, mutagenesis is random, cost effective and is not submitted to GMO regulation. TILLING allows generating variants in cultivated genetic background (Piron, Nicolaï et al. 2010) and thus transfer rapidly interesting mutations into cultivars (Gady, Hermans et al. 2009). Application of TILLING technique to screen for natural variation within tomato germplasm collection is now performed (Rigola, van Oeveren et al. 2009).
