Studies On Introduction Of Lysozyme Gene Into Indica Rice And Selection Of Blast-Resistance Hybridization Combination

Rice blast is responsible for large yield loss in epidemically favorable areas and crop seasons.Lysozyme can decompose the cell wall of bacteria and fungi,resist the invasion of pathogens.Therefore,obtaining new germplasm with broad-spectrum blast-resistance by introduction of lysozyme gene into indica rice has been considered as an effective and economical strategy to control the disease.In this thesis,the Lysozyme gene was introduced into indica parents PA64s,R640, 9311 and Minghui63 by Agrobacterium-mediated,plasmid earstem injecting and Agrobacterium earstem injecting transformation systems.The factors influencing the transformation efficiency were investigated.Genetic stability analysis and blast-resistance identification of transgenic progenies were performed.And new hybridization combinations from the transgenic lines and other hybrid rice parents were selected.The main research contents and results were as follows:1 Optimization of culture medium of in vitro regeneration system of indica riceAnalyzing the organic maters,macro elements,micro elements-â… (manganese, zinc,boron) and micro elements-â…¡(copper,molybdenum,cobalt,iodine) of CC,NB and MS basic media,an orthogonal experiment of L₉(3⁴) was carried out to optimize the compose of media on calli induction,subculture and buds differentiation of indica rice.The result showed:Micro elements-â… of NB,organic maters of NB or MS contributed to callus induction,and the calli induction ratio on the medium with 2,4-D alone was higher than that of the combination of 2,4-D and BA or BA alone.Organic maters and macro elements of MS,micro elements-â… of NB contributed to the growth of callus.Macro elements and micro elements-â… of NB acted significant role on differentiation,10 times of micro elements-â…¡of CC was appropriated to differentiation. On the optimized media,the average calli induction ratio of 4 indica rice was 86.6%,the average calli subculture proliferation ratio after sub-cultured 25 d was 3.85, the adventitious buds induction ratio was 88.1%.2 Optimization of Agrobacterium-mediated transformation system for indica riceWhen OD₆₀₀ was 0.4-0.6,the inoculum with 120μM AS was very strong vitality. 20 min to 30 min of infection time,callus dealt with negative pressure of-0.8MPa for 10 min,co-cultured 2-4 d before screening,covered with a filter paper on the co-culture medium,were suitable for the infection.Increasing gradually the screening concentration of G418(150 mg/L at first,then 165 mg/L) after 2 weeks' resumption culture can increase the resistant calli ratio.As a result,the average resistant callus rate of 4 varieties was 17.77%,and average resistant plant rate was 3.94%by G418 screening.3 Optimization of the earstem injection(1) Earstem injection with plasmidOn the development stage of mature embryo sacs,the distance between pulvinus longer than 7 cm,plasmid DNA of 500μg/ml dissolved with TE was injected into the first internode under the neck-panicle node.Transgenic lines were obtained,and the transformation rate of the three indica rice(R640,9311,MH63) was reached 7%. There was no significant differences on the transformation rate of the three indica rice, which showed that the optimized method was suitable to different indica varieties.(2) Earstem injection with AgrobacteriumNine transgenic lines were obtained by Agrobacterium earstem injection with lysozyme gene,the transformation ratio was only 0.03%.The transgenic lines were confirmed by NPTâ…¡gene and lysozyme gene PCR detection.The result of Southern blotting and the segregation ratios of progenies showed that the lysozyme gene was integrated as a single copy.With Agrobacterium integration mode,taking germ lines as receptor and combined with the advantages of Agrobacterium transformation system and earstem injection,this transformation system is a new transformation system worthy to be explored.4 Transgenic plants with blast resistance were obtained 55 transgenic plants were obtained by Agrobacterium transformation system,98 by plasmid earstem injecting and 9 by Agrobacterium earstem injecting.The lysozyme gene integrated into rice genome was confirmed by PCR and Southern blotting analysis.Segregation ratios analysis of transgenic progenies showed,the exogenous gene were integrated with sigle copy in the transgenic plants by Agrobacterium transformation and Agrobacterium earstem injecting,and the segregation ratios in T1 and T2 progenies were agreement with Mendelian genetic law expected from a single dominant gene segregating.But part of the transgenic plants by plasmid earstem injecting were confirmed integrated with single copy,part with multi-copy.21 moderate resistance,positive,homozygous T2 plants were selected by resistance identification,PCR detection and identification of homozygous of T1 and T2 plants.5 Superior hybridization combinations with good blast resistance were obtainedThe 21 homozygous lines were hybridized with corresponding parents. Identification of disease resistance showed,the blast-resistance was improved significantly in all the hybridization combinations compared with the controls (resistance degree were susceptible),3 combinations showed resistance,26 combinations showed moderate resistance,4 combinations showed moderate susceptible.Investigation of agronomic traits showed,there was no significant differences among 22 combinations and the controls,the yield per plant of 11 hybridization combinations was significantly lower than that of the control.Combining the identification of disease resistance and investigation of agronomic traits,18 combinations with blast resistance were obtained,and further study of the 18 new combinations was in process.