Grant: Research Tools and Instruments - Category 1 (<$150,000)

Successful generation of transgenic plants is dependent on the availability of efficient system for DNA delivery. Integration of DNA is a random and uncontrolled process frequently leading to multiple and complex integration events. Recently we found that enrichment of the plant growth media with certain salts drastically increases the homologous recombination (HR) frequency. Since the involvement of HR in the mechanism of the transgene integration can not be excluded, we transformed Arabidopsis and tobacco plants grown on the enriched medium with Agrobacterium. We found that the transient and stable transformation frequency was increased by 10- and 2-fold, respectively. Similar media composition also improved particle-mediated transformation of wheat scutella; a 50% increase in the number of stable integrations was noticed.   In this project we propose to use the improved media composition in order to increase monocot transformation rates. We will use two different transformation approaches, agrotransformation and particle bombardment. We will analyze multiple parameters, such as transformation rate, average number of integrated transgenes per plant genome and per single locus as well as the precision of integration (potential loss of transgene or genomic sequences). The HR plays a crucial role in gene targeting. The low frequency, with which plants use HR to repair breaks prevents gene targeting from being efficient. The ability of the transgene to integrate into a particular locus together with the ability of replacing the less active genes with more active (i.e. to target certain metabolism) genes, would have a tremendous impact on plant biotechnology. The increase in HR frequency will result in higher chances of obtaining true gene targeting events, better percept by scientific community. This approach will allow to save time and money required for production of transgenics.