Tissue culture is used extensively in plant biotechnology, both for direct application in the clonal multiplication of plants and indirectly in providing target cells for genetic engineering. It is of paramount importance that the plants derived from culture in either of these two processes are true-to-type, genotypically and phenotypically. However, uncontrolled instability can occur when plant cells are cultured in vitro. Attempts have been made to identify DNA markers that would be diagnostic of cells with altered genomes using restriction fragment length polymorphisms, random amplified polymorphic DNAs and amplified fragment length polymorphisms. Each of these methods only surveys a small fraction of the genome at a time and has not yielded useful markers. The proposed program is directed at generating useful markers for somaclonal variation through the use of representational difference analysis (RDA). This technique allows a significant fraction of the genome (up to 15%) to be compared between closely related lines. The use of RDA to isolate DNA differences between true-to-type plants and somaclonal variants will result in the development of markers for genomic instability that can be utilized throughout the in vitro propagation process. These markers will also be useful to ensure that genetically-modified transgenic plants only differ from the starting material in terms of the added information and not in any unassociated regions of the genome. ANTICIPATED RESULTS & POTENTIAL COMMERCIAL APPLICATIONS OF RESEARCH Success in the development of markers for genomic integrity would provide an enhanced method of quality assurance of in vitro propagated plants. The use of these markers would remove the damage due to somaclonal variation only discovered after plants had been released to producers. The markers would also allow the monitoring of the regenerating cultures for genomic stability, further reducing the losses due to unstable cultures, and the discarding of productive, stable cultures from fear that they may become unstable. They will also have a use in allowing a more complete description of the genome of genetically-modified crop plants.
