Effectiveness of genotype-based selection in the production of marker-free and genetically fixed transgenic lineages : ectopic expression of a pistil chitinase gene increases leaf-chitinase activity in transgenic rice plants without hygromycin-resistance gene
An extra copy of a rice pistil chitinase gene, termed<i> chip</i>, which is expressed predominantly in floral organs just before anthesis, was introduced into an economically important cultivar of rice (<i>Oryza sativa</i> L. cv. Koshihikari) together with the hygromycin-resistance gene (<i>hph</i>) as a marker gene by co-transformation. Over 100 regenerated rice plants (R<sub>0</sub>) and their self-pollinated first and second generations (R<sub>1</sub> and R<sub>2</sub>) were genotypically characterized by PCR-based genomic DNA analysis of the two transgenes to obtain stable transgenic lines, in which <i>chip</i> was inserted into a single locus separately from <i>hph</i>. We obtained homozygous <i>chip</i>-transgenic R<sub>1</sub> lines free of <i>hph</i> that stably expressed the <i>chip</i> mRNA and protein in rice leaf tissue. Chitin-hydrolytic activity in the leaves of these transgenic lines was higher than in those of non-transgenic controls. The resistance of the transgenic lines to rice blast disease fungus (<i>Magnaporthe grisea</i>) was examined by smearing water agar mixed with overcrowded conidia (race 007.0) on mechanically injured leaves, showing tendency to blast-disease resistance. Thus, the genotype-based selection was demonstrated to be effective at obtaining genetically stable transgenic plant lines without marker genes.
- Plant biotechnology
Plant biotechnology 23(4), 349-356, 2006-09-01
Japanese Society for Plant Cell and Molecular Biology