Antisense inhibition of Waxy genes in hexaploid oat

Loss of function mutations are easily identified in diploids; they allow researchers to study gene function, and to modify plants. Recessive mutations in polyploid crops, like wheat and oat, are rarely found because mutations are masked by functional genes on homoeologous chromosomes. Antisense mechanisms may be used in polyploids to dominantly simulate loss of function mutations. The goal of this research was to investigate an antisense mechanism for gene silencing in oat. Oat plants were cotransformed with an unselected plasmid containing antisense Waxy cDNA and a second plasmid containing a selectable marker gene, nptII. None of 82 transgenic lines completely lacked amylose in seeds. Ten of the 82 lines were selected for detailed characterization. Southern blot analysis showed that six of the 10 lines contained the antisense construct as a complete unit. In addition as shown by RT-PCR, five of the 10 lines expressed the antisense Waxy cDNA. Seeds from the 10 lines had wild type levels of amylose except one line had reduced amylose in one environment out of two environments tested. The antisense constructs used in this experiment appear to not effectively silence the endogenous Waxy genes in allohexaploid oat.; The same 10 lines were further characterized in an effort to determine if current models explaining the process of integration also apply to the integration of two different plasmids into the genome of allohexaploid oat. DNA from the 10 lines was digested with a variety of restriction enzymes and Southern blots were made and probed with plasmid-specific probes. Across eight co-transformed lines, 12 integration sites contained the Waxy transgene while 19 integration sites contained at least one copy of the nptII gene. Nine of the 12 sites containing a copy of the Waxy gene also contained at least one copy of the nptII gene. Hybridization patterns showed that the structure of the integrated plasmids was random except for one line that had a head-to-tail concatemer of the selected plasmid. The random structures of the integration sites suggests that exogenous DNA is linked together by illegitimate recombination.