DEVELOPMENT OF A POLLEN-MEDIATED TRANSFORMATION METHOD FOR NICOTIANA-GLUTINOSA

The development is described of a new procedure to genetically transfo rm plant species using the male gametophyte as a natural transformatio n vector. Our system avoids the need for complicated regeneration proc edures thus making it broadly applicable. Naked plasmid DNA encoding k anamycin resistance and GUS activity was introduced by particle gun bo mbardment into mature pollen grains of Nicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, d esignated VIP A and VIP B, were obtained in two independent experiment s. In VIP A, TR2'-driven GUS activity was observed in vascular bundles , trichomes and in a small number of pollen grains. DNA gel blot analy sis indicated that the introduced DNA was integrated independently int o the genome of VIP A and VIP B. It was shown that male and female gam etophyte development and seed set were highly aberrant in both VIP A a nd VIP B and that the offspring of self- and cross-pollinations did no t contain the transgenes. This might be caused by a recombination even t during the integration of the naked DNA resulting in a deletion of p art of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VI P B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.