Transformation of perennial ryegrass with disease resistance genes from rice.

摘要

Pyricularia oryzae causes gray leaf spot on perennial ryegrass (Lolium perenne) grown in the Unites States. During the past two decades, gray leaf spot disease has emerged as a serious problem threatening the cultivation of perennial ryegrass on golf courses. All cultivars of perennial ryegrass are susceptible to gray leaf spot disease to a certain degree. Recently, a few cultivars with improved gray leaf spot resistance have been released. However, the resistance reported in these cultivars is quantitative in nature and the durability is not known. The goal of this study was to transfer major resistance genes identified from rice into perennial ryegrass in order to obtain resistance to gray leaf spot isolates.; Major resistance gene Pi-CO39(t) from rice corresponding to avirulence gene AVR1-CO39 from P. oryzae isolates infecting rice was recently mapped. In this study, a homolog of AVR1-CO39 (AVR1-CO39Lp) was identified in P. oryzae isolates pathogenic on perennial ryegrass. The function of AVR1-CO39Lp was determined by cloning this gene into a virulent rice isolate ML33 lacking AVR1-CO39 and inoculating the transformants on rice cultivar CO39 carrying the resistance gene Pi-CO39(t). The transformants became avirulent on CO39, however, their ability to infect rice cultivar 51583 lacking Pi-CO39(t) was unaffected indicating that AVR1-CO39 Lp was able to trigger a resistance response on rice cultivar CO39 carrying Pi-CO39(t) Hence, attempts were made to transfer Pi-CO39(t) into perennial ryegrass using biolistics.; In order to introduce genes of interest into perennial ryegrass, an efficient callus culture and regeneration system was developed. The concentration of 2,4-D necessary to obtain highly regenerative callus culture was determined. In order to further improve callus regeneration, various concentrations of kinetin were added to the callus culture media and the optimal conditions determined empirically. The effect of callus age on regeneration was studied over a period of one year in order to optimize the best time for transformation.; Factors affecting transformation of perennial ryegrass using the reporter gene GUS and the selectable marker gene, hygromycin B phospho-transferase (hph), were evaluated. Intrinsic GUS activity was shown to be present in perennial ryegrass calli. Sensitivity of transformed and untransformed calli to the selective agent, hygromycin B showed that the sensitivity was dependent on the genotype of the calli as well as several undetermined factors.; Two resistance gene candidates pCOR6 and pCOR8 occurring within the 230 kb DNA of the rice genome to which Pi-CO39(t) was mapped were biolistically transferred into perennial ryegrass cultivars Calypso II and Manhattan III. One plant recovered on the selection medium was shown to contain the hygB gene using PCR analysis. Southern analysis on the same plant confirmed that there may be two copies of pCOR8 gene.; Keywords. Magnaporthe oryzae, perennial ryegrass, rice, AVR1-CO39Lp, Pi-CO39(t)