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-resista

摘要

An extra copy of a rice pistil chitinase gene, termed chip, which is expressed predominantly in floral organs just before anthesis, was introduced into an economically important cultivar of rice (Oryza saliva L. cv. Koshihikari) together with the hygromycin-resistance gene (hph) as a marker gene by co-transformation. Over 100 regenerated rice plants (R_0) and their self-pollinated first and second generations (R_1 and R_2) were genotypically characterized by PCR-based genomic DNA analysis of the twotransgenes to obtain stable transgenic lines, in which chip was inserted into a single locus separately from hph. We obtained homozygous chip-transgenic R_1 lines free of hph that stably expressed the chip 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 (Magnaporthe grisea) 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.