USING MAT VECTOR SYSTEM TO PRODUCE MARKER-FREE TRANSFORMED APRICOT PLANTS

Abstract

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. The 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 cytokine 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 later strategy is the base 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 constructs 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. Transformation efficiencies using this construct have ranged from 3 to 12% depending on the PGR combination used. These putative transformed shoots have been confirmed by PCR analysis. More than one year after the beginning of the experiments all shoots that proliferate in medium with antibiotic were examined for GUS expression and 24% of shoots were found positive. Since this marker gene is only expressed in the MAT construct after excision of the cassette containing the marker genes, it is an indirect indication for 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 also by comparing with our standard procedure.