Gene targeting

In plants there is a preference for random integration of the introduced DNA, which frequently leads to the accidental inactivation of important genes and to variable and unpredictable expression of the transgene itself. In some plants, over 90% of T-DNA insertions may disrupt transcriptional units leading to transformants with visible mutant phenotypes. [74] These observations, together with the silencing phenomena described above, sound an alarm for direct production of improved cultivars in highly selected crops, where most phenotypic changes from random mutations are likely to be adverse. Therefore, there is an urgent need to develop techniques for the directed integration of transgenes at specific locations in the genome.

Homologous genetic recombination is the transfer of genetic information between regions of similar sequence composition. Gene targeting, that is the directed integration of introduced DNA into the genome via homologous recombination, can be a valuable tool to solve the problems of genetic damage and gene silencing in genetically engineered plants. As an alternative tool to antisense strategies, gene targeting can be also a valuable tool in both fundamental and applied research to down-regulate gene expression by reverse genetics approaches. Nevertheless, the main route used by somatic plant cells for integration of transgenes is via non-homologous recombination, irrespective of the transformation procedure used for the introduction of the genes. [75–7] The efficiency of homologous recombination is in the range of 10^-3 to 10^-5 compared with non-homologous recombination. In contrast to the case of mammalian cells in which several factors have been shown to influence homologous recombination frequencies [78–80] factors such as vector type, homology and isogenicity of the delivered DNA, do not affect gene targeting in plants. However, analysis of the recombination enzymes and mechanisms operating in plant cells, and their possibly different prevalence in different cell types, will hopefully shed more light on the different recombination events that take place in plants. [81]

Knowledge of the enzymes participating in recombination reactions may favorably contribute to the development of strategies for gene targeting. Most of such enzymes have been purified directly or have been identified through the molecular analysis of recombination mutants in E. coli and S. cerevisiae. In E. coli the RecA single-stranded DNA binding protein plays a key role in homologous recombination. Remarkably, a plant homolog of the E. coli recA gene has been isolated from Arabidopsis thaliana on the basis of sequence conservation. [82] In yeast, Rad51 has a role in recombinational repair of DSBs whereas Dmc1 has a function in DSB repair and formation of synaptonemal complexes. Recently, in lily (Lilium longiflorum), as well as in Arabidopsis thaliana, plant homologs of the yeast Dmc1 and Rad51 proteins were identified. [83–5] Further progress in plant recombination is envisaged by the isolation of interesting mutants with altered recombinational behavior.

In plants, homologous recombination is performed in tissues or cells that are highly competent for non-homologous recombination, which is not necessarily the best choice. It would also be very interesting to test the capacity of meiotic or meristematic cells for homologous recombination of foreign DNA in plants.