Biotechnology Research Center, Zhongshan University, Guangzhou 510275, China

     Blast and sheath blight diseases are prevalent in all rice-growing countries and cause significant yield losses. Magnaporthe grisea, the causal agent of blast, has a variety of physiological races, which mutate easily. No single rice variety shows resistance to all isolates or races. Naturally resistant germplasm for sheath blight has not been found in cultivated rice plants nor in its wild relatives. Resistance breeding to blast and sheath blight is not feasible. Therefore, biotechnology approaches are valuable in introducing genes which encode antifungal proteins against Magnaporthe grisea and Rhizoctonia solani (Lin et al. 1995). Plants possess a variety of mechanisms to protect themselves from pathogen attack and abiotic stress, suggesting that different protection mechanisms may have complementary roles in the overall expression of resistance. Transgenic tobacco plants co-expressing chitinase/glucanase or chitinase/ribosome inhibitor protein revealed significantly enhanced protection compared to plant lines expressing a single transgene (Zhu eta!. 1994; Jach et a!. 1995).

     To evaluate the potential effect of a “multi-transgene” tolerance strategy, we co-introduced a rice basic chitinase gene, RC24, an alfalfa glucanase gene, beta-Glu, and a barley ribosome-inactivating protein gene, B-RIP, into an indica rice variety, Qisiruanzhan, by the biolistic method. Fertile independent transgenic lines were obtained with variable levels of chitinase and glucanase gene expression. Southern blot analysis showed that the inheritance of RC24 and (beta-Glu transgenes in Tl, T2 generation behaved as a monogene with a ratio close to 3:1. However, B-RIP transgenes in other plasmids were not always genetically linked with the RC24 and beta-Glu transgenes. Ten different homozygous lines containing RC24/beta-Glu and ten different homozygous lines containing RC24/beta-Glu/BRIP were obtained in their T2 generations.

     These transgenic rice plants were assayed for resistance to fungal pathogen attack with Magnaporthe grisea and Rhizoctonia solani. The data showed an increased protection against infection by both pathogens, and the degree of resistance displayed correlated with the level of foreign gene expression. Blast resistance test results for part of the transgenic rice plants are shown in Table 1. Eighteen isolates belonging to ten dominant races of Magnaporthe grisea in the Guangdong province were tested. Our results indicate that 44.4% (R%) of the control showed resistance, while 55.6-.88.9% of T3 homozygous transgenic lines containing RC24/beta-Glu/B-RIP transgenes displayed resistance, and 55.6-100% of T3 homozygous transgenic lines containing RC24/beta-G1u transgenes displayed resistance. Among these plants, transgenic line Q3-3-9 showed more resistance than all three control rice varieties, including the resistance control rice variety, Sanhuangzhan. The results indicate synergistic protective interaction against Magnaphorthe grisea and Rhizactonia solani of the co-expressed antifungal proteins in transgenic rice plants. Following field tests, several resistant transgenic rice plants have now been selected.
 

 

disease scale 0-3. MR represents moderate resistance, disease scale 4.

in Guangdong. MR% represents the frequencies of moderate resistance to eighteen different isolates of M. grisea in Guangdong. R%=HR%+MR%.

tain the RC24/beta-Glu transgenes. Among these, lines Q3-3-9 showed stronger resistance when compared to the resistance control (RCK), Sanhuangzhan.