| Using biolistic mediated method and Agrobacterium-mediated method for genetic transformation of wheat,provides an important approach for the directional genetic improvement of wheat,but compared to other crops,its low transformation efficiency limits the study of wheat gene function and its application in breeding.Therefore,it is very critical to establish a highly efficient,stable and perfect genetic transformation system for wheat transformation.The factors such as the time of hypertonic culture-treated before and after the particle bombardment in biolistic mediated method and the type of osmotic agent are important factors affecting the transformation efficiency of biolistic mediated method.The concentration of Agrobacterium tumefaciens infection solution,time of infection and type of co-culture medium will affect the transformation efficiency in Agrobacterium-mediated method.Therefore,it is very critical to establish a highly efficient,stable and perfect genetic transformation system for wheat transformation.In this thesis,the wheat variety Kenong 199 immature embryos is used as explants,and optimization of the key factors in genetic transformation system of biolistic and Agrobacterium-mediated method we carried out.Based on the results of GUS report gene,we can identify the influence of the period of collecting immature embryo,the time of hypertonic medium treatment,the type of and concentration of osmotic agents,the concentration of 2,4-D in hypertonic medium,induction medium in biolistic mediated method.We also identify the influence of the time of collecting immature embryo,the concentration of Agrobacterium tumefaciens solution,time of infection,time and types of co-culture in Agrobacterium-mediated method.In addition,mature embryos are also studied because of their convenience and similar physiological state.The findings are as follows:1.GUS staining is performed after biolistic genetic transformation of Kenong 199 immature embryos,the results indicate that GUS instantaneous expression rate of immature embryos collecting at 16 days after anthesis was significantly higher than that of 14 days after bombardment,GUS transient expression is also significantly higher when pre-hypertonic culturing for 6 hours,contined hypertonic treating for 18 hours after bombardment,treating with 0.6 mol·L-1 sucrose or 2 mg-L-1 2,4-D in hypertonic medium than other treatments.2.Using Kenong 199 immature embryo as the receptor for the parameters of optimization in Agrobacterium-mediated method,under the same conditions,GUS instantaneous expression rate of immature embryo with G2(1/10 MS)co-culture medium treatment is observably higher than that of G1(MS).When co-culture medium is Gl,the most suitable concentration of Agrobacterium tumefaciens(A660)was 0.8,the most suitable time of infection is 15 minutes.When co-culture medium is G2,the most suitable concentration of Agrobacterium tumefaciens(P660)is 1.2,the most suitable time of infection is 10 minutes or the most suitable days of co-culture are 3 days.3.In this thesis,the effects of pretreatment mode,different induction medium and different differentiation medium on the callus induction and differentiation of wheat mature embryos were studied with wheat cultivars Lunxuan 987 and CB037 as materials,aiming at optimizing the regeneration of mature embryo tissue.The induction rate,differentiation rate and plant regeneration rate of Lunxuan 987 are significantly better than those of CB037.The physiological condition of mature embryos can be changed after the wheat seeds are sterilized and placed in a sterile water dish for 4℃ hours.When induction medium is MSA,its abilities to induce mature embryos to form callus are better than MSB.The differentiation medium for MSC and MSD on callus differentiation have obvious difference,the differentiation culture of MSC is better than that of MSD for improving the ability of induction to callus and regeneration to plants,which can be used in the genetic transformation system dependent on wheat mature embryos. |
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