Gene therapy in plants

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Abstract
Genetics allows the elucidation of gene function through the analysis of gene malfunction. Modern genetics and genomics require ways for in situ modification of genes, by means of point mutations, deletions, and additions. The availability of sequence information of many organisms dictates rapid development of reverse genetics procedures. Until recently, targeting of genes with the help of introduced homologous sequences, here referred to as homologous recombination-dependent gene targeting (hrdGT), was the method of choice, at least for mammalian systems. However, in higher eukaryotic organisms such as mammals and plants, exogenously introduced DNA preferably integrates in random positions in the genome, by the process of illegitimate recombination, and only infrequently can targeted integration events be detected. Recently an alternative strategy became available for precise reverse genetics. Specific chimeric oligonucleotides, COs, consisting of DNA and RNA stretches, were found to induce point mutations in several mammalian genes tested (see below). This technique, here referred to as chimeric oligonucleotide-dependent mismatch repair, cdMMR, has now been used for plants: this issue of the Proceedings includes two reports describing stable changes in the genomes of tobacco and maize after treatment with chimeric oligonucleotides (1, 2). In the mouse system hrdGT is (almost) routine. Since early pioneering work (3, 4), gene targeting in embryonic stem cells led to the generation of several thousands of “knock-out” mice. Gene targeting frequencies of 10−2 greatly facilitated the work (5) and led to the establishment of extremely interesting loss-of-function phenotypes and animal models for human diseases. In contrast, despite an urgent need for academia and agriculture, hrdGT in plants suffers from a deplorable inefficiency, which has not significantly changed since it was first accomplished (6). Homologous recombination in plants was studied in classical work as well as by using molecular markers (reviewed in refs. 7 and …