REGENERATION-PROMOTING GENES IMPROVE TRANSFORMATION EFFICIENCY IN APRICOT

Abstract

Efficient selection of transformed plants is one of the limiting steps in the transformation of mature tissue of most woody fruit trees. The transformation with regeneration-promoting genes (i.e. ipt gene) may improve results in recalcitrant species. Transformation strategies with 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 include a recom-binase and specific sequences for the enzyme flanking the marker genes and the ipt. In this work, results from the utilisation of these constructions to transform apricot leaves are presented. Since ipt expression is supposed to induce regeneration of adventitious shoots in the absence of growth regulators, our standard transformation procedure was modified eliminating all growth regulators from the culture media. However, explants died in these conditions. When an auxin (NAA) was added to the regeneration medium at different concentrations, only production of adventitious roots was obtained from the transformed leaves. Therefore, addition of a cytokinin was necessary to avoid production of roots and to induce adventitious budding. The NAA concentration used in our standard procedure was combined with different TDZ concentrations during the co-culture period. These cytokinin concentrations were kept or lowered in the regeneration medium after co-culture. All regenerated buds were cultured in a medium specifically developed to grow apricot meristems into shoots that can be micropropagated. Kanamycin 60 μM was added to this medium to screen for those that were transformed. Transformation efficiencies obtained with the ipt gene have been three-fold those obtained with our standard transformation procedure and also selection efficiencies are much higher and, in some of the treatments with lower transformation efficiencies (3-5.7%), were as high as 50%. Moreover, the constructions used should allow elimination of marker genes and production of marker-free transformed apricot plants, which is the next aim of our research.