AGROBACTERIUM-MEDIATED TRANSFORMATION OF POLLEN EMBRYOS IN DATURA-INNOXIA AND NICOTIANA-TABACUM - PRODUCTION OF TRANSGENIC HAPLOID AND FERTILE HOMOZYGOUS DIHAPLOID PLANTS

We have successfully produced transgenic haploid plants with Agrobacte rium-mediated gene transfer to pollen-derived embryos. Isolated pollen and pollen embryos of Datura innoxia and Nicotiana tabacum were cocul tured with a disarmed Agrobacterium tumefaciens strain harbouring bina ry and cointegrate vectors. There was a direct correlation between dev elopmental stage and the competence of pollen embryos for transformati on. No transformants were obtained from infected pollen grains and pro embryos. In contrast, the transformation frequency was the highest(> 7 5%) with injured or cut cotyledonary-stage pollen embryos. Several hun dred haploid and/or spontaneous dihaploid transgenic plants were obtai ned from a Single experiment. The spontaneous dihaploids or colchicine -treated dihaploids were fertile, and set seeds. Analysis for kanamyci n-resistance and beta-glucuronidase (GUS) activity showed that the tra nsgenes were inherited as dominant Mendelian traits. It is possible to obtain homozygous, dihaploid transgenic plants with flowers and seeds with this technique within 14-16 weeks after inoculation with Agrobac terium. Therefore, this method should speed up the production of trans genic plants in breeding programs.