SETTING UP TISSUE CULTURE TECHNIQUES FOR REGENERATION OF APRICOT TRANSGENIC SHOOTS IN A TRANSFORMATION PROGRAM FOR RESISTANCE TO PLUM POX VIRUS

Abstract

For recalcitrant woody species, such as apricot, one of the most critical steps of genetic transformation is inducing plant regeneration specifically from those explant cells that are (or become, in response to culture conditions) competent for stable transgene integration. In previous trials we were able to obtain regeneration from both mature tissues and zygotic embryos of various apricot cultivars with methods that, nevertheless, proved to be unsuitable for recovering transgenic plants, when applied to Agrobacterium-mediated transformation. Using this system, the shoots and somatic embryos that promptly differentiated from the explant tissues neither revealed GUS activity nor survived kanamycin selection, while callus masses proximally associated to them were eventually transformed. On the other hand, when we applied higher hormone levels than those required for direct shoot organogenesis to stimulate first proliferation of kanamycin-resistant cells, then their induction to morphogenesis, we achieved higher frequencies of stable transgene integration but recovered callus lines instead of normally developed shoots. The present work presents an update on the tissue culture methods under evaluation in our laboratory for indirect shoot regeneration. In order to shorten the callus proliferation phase with respect to our past experiments, we modified the in vitro culture conditions applied to leaf and stem explants of micropropagated cultivars ('Boreale', 'San Castrese', 'Sungiant', and 'Vitillo') inoculated with A. tumefaciens EHA101 pGA482GG/PPV-CP-33. Additionally, different substrates and subculture periods have been tested for shoot development from the transformed callus lines obtained from previous experiments. The newly-inoculated explants, consistent with our past results, yielded no transgenic shoots, but did produce kanamycin-resistant cell proliferation. Some parameters modified in the present tests promoted a slow callus hardening process and the production of bud-like structures from both the more recently obtained and the older transgenic material.