PLANT TRANSFORMATION BY PARTICLE BOMBARDMENT OF EMBRYOGENIC POLLEN

Direct delivery of DNA into embryogenic pollen was used to produce tra nsgenic plants in tobacco. A plasmid bearing the beta-glucuronidase (G US) marker gene in fusion with the 35S-promoter was introduced by micr oprojectile bombardment into mid-binucleate pollen of Nicotiana tabacu m that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10(4) pollen grains were GUS( +). Visual selection by staining with a non-lethal substrate for GUS w as used to manually isolate transformed embryos. From the initial popu lation of embryogenic GUS(+) pollen, 1-5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transfor mants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and bac kcrosses of one transformant were tested for GUS expression and by Sou thern blots. All F1-plants were transgenic, in accordance with Mendeli an inheritance.