PRODUCTION OF MARKER-FREE TRANSGENIC PLANTS AFTER TRANSFORMATION OF APRICOT CULTIVARS

Efficient selection of transformed plants is one of the limiting steps in the transformation of mature tissue of most woody fruit trees. Transformation efficiencies are usually very low and procedures are largely genotype-dependent. Transformation with regeneration-promoting genes may improve results in recalcitrant species. A classical example of a regeneration-promoting gene is the ipt gene from Agrobacterium, which is involved in cytokinin synthesis. However, its constitutive expression in the plant genome produces, frequently, aberrant phenotypes due to an excessive accumulation of cytokinins. Transformation strategies to use this gene include the use of inducible promoters or the removal of the ipt gene from the transformed plant. The latter strategy is the basis of the MAT vector system, a construction that includes a recombinase and specific sequences for the enzyme flanking the marker genes and the ipt. This allows elimination of non-useful genes after they have fulfilled their role, including the ipt gene. In this work, results from the utilisation of one of these constructions to transform apricot leaves are presented. Expression of the ipt gene was not enough to induce regeneration of transformed buds in the absence of plant growth regulators (PGRs) and therefore a specific combination of PGRs was designed. After recovery and elongation of all the regenerated buds in a medium supplemented with antibiotic, those transformed were identified by rooting them in a medium supplemented also with the appropriate concentration of antibiotic. These putative transformed shoots have been confirmed by PCR analysis. Once confirmed, transformed lines have been examined periodically for gus expression. Since this marker gene is only expressed in the MAT construction used after excision of the cassette containing the marker genes, it is an indirect indication of the elimination of these genes. Excision of the cassette was checked with the appropriate PCRs. This strategy is discussed in terms of efficiency of production of marker-free transformed plants and comparison with our standard procedure.