A Study On Introducing The Blast Resistance Gene Pi-d2 Into The Main Parents Of Hybrid Rice Shuhui527 Mediated By Agrobacterium Tumefaciens

Rice(Oryza sativa L.)is one of the most important food crops and almost half of the world population depends on it as the major source of nutritional calories.Rice blast,which is caused by the fungus Magnaporthe grisea,is one of the most devastating diseases occurred in most rice growing areas worldwide.The high variability in pathogenicity of M.grisea isolates makes the control and management of rice blast difficult.Therefore, breeding rice cultivars resistant to the blast disease has been a major goal over several decades.Genetic engineering has emerged as an alternative approach to create blast-resistant rice.The technology introduces the foreign resistant genes into cultivar and develops the gene source,which provides a new way to crop resistant breeding.Indica rice is leading cultivar form.However,the majority of indica rice cultivars are recalcitrant in both tissue culture and attempt of transformation,which has restricted severely application of transferring breeding in indica rice.Pi-d2 gene expresses constitutively in root,stem and leaf respectively,and this Ser/Thr receptor-like kinase gene contains other domains such as B-Lectin and TM,suggesting that this gene belongs to a novel resistance gene in plants.In the research,we firstly introduce the Pi-d2 gene into indica rice Shuhui527 and japonica rice Zhonghua11 with agrobacterium-mediated transformation.A highly efficient regeneration system of Shuhui527 suitable for genetic transformation was established. After the molecular test,GUS histochemical assay and resistance test,some plants with high resistance to rice blast have been initially obtained,which establish the foundation of molecular breeding for rice and other gramineous plants.The main results were as follows:1.The induction ratio was different with different varieties and different explants. The callus induction efficiency from immature embryos of Shuhui527,Shuhui498, Zhonghua11 and Taibei309 could be as high as 88.7,87.5,91.4 and 96.0 percent respectively,and the callus induction efficiency from mature embryos could be as high as 78.5,75.3,85.1 and 86.3 percent respectively.In conclusion,the ratio of immature embryos is higher than which of mature embryos,and the ratio of japonica rice is higher than which of indica rice.2.A highly efficient regeneration system of Shuhui527 suitable for genetic transformation was established.The results indicate that NMB medium with 2.0 mg/L 2,4-D was suitable for callus induction and subculture of indica rice Shuhui527. Meanwhile,the MS + 6-BA 2.0 mg/L + KT 2.0 mg/L + NAA 0.25 mg/L medium was advantageous to callus differentiation.Callus after two subculture passages was more effective in inducing plant regeneration.Partial desiccation of callus before transfer to regeneration medium have been found that incease the frequency of explant regeneration of indica rice.3.HygB-resistant calli rate and differentiation rate mainly influenced the transformation efficiency,especially the latter did more.It was found in the test,Shuhui498 can form a great deal of resistant calli,but most of the calli can't differentiate.4.Through agrobactetium-mediated transformation with the callus from immature embryos and mature embryos of different rice varieties,three difference expression vectors including pCB6.3kb,pCB5.3kb and pZH01-2.72kb were introduced into rice.As a result, totally 292 regenerated plants were obtained.PCR assay was carried out for the 292 regenerated rice plant,and 176 showed positive,87 of which were Shuhui 527,1 of which was Shuhui498,37 of which were Zhonghua11 and 51 of which were Taibei309.RT-PCR assay for Pi-d2 gene showed that it had been expressed in transcription level.5.The hygromycin resistance analysis of the T₁ lines showed that 64.6 percent of Shuhui527 and 68.2 percent of TP309 displayed the segregation ratio of 3:1,indicating that these lines were segregated in accordance with Mendel's single gene inheritance rule.It suggested that the transgenes were integrated in a single locus in most of transgenic plants.6.The results of the inoculation with the rice blast race ZB15 indoors showed that the transgenic plants from T₁ enhanced the resistance to rice strain ZB15,the size and number of the lesion on leaf decreased,some populations showed the resistant and susceptible separation with Mendelian segregation or non-Mendelian segregation.The resistant expression of foreign gene from three difference expression vectors to ZB15 showed no significant difference.7.The results of investigation of the agronomic traits of transgenic plants showed that the introducing exogenic genes and the tissue culturing courses had some effects on the agronomic traits of their recipient plants,but most of these variations could not be delivered to their progenies.