GENE-TRANSFER TO INFLORESCENCE AND FLOWER MERISTEMS USING BALLISTIC MICRO-TARGETING

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporoge nic tissues. Despite the wide potential of its applications, direct ge ne transfer to floral meristems has not been achieved so far because o f the lack of suitable technology. We show in this paper that ballisti c micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised whea t immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plas mid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (beta-glucuronidase) gene. The transient ex pression of these marker genes was observed in the different developme ntal stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0. 06 to -0.52M. At the highest concentration, 100% of the targeted spike s expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after micro-targeting, multicellular sectors showing tran sgene expression and containing up to 17 cells were found in 85% of th e shot immature inflorescences. This indicated that targeted cells sur vived particle bombardment. Sectors were found in primordia of both ve getative and reproductive organs.