Establishment Of High Efficient Regeneration System And Research On Genetic Transformation Of Lactuca Sativa L

Leaf lettuce(Lactuca sativa L.)production on the Mediterranean coast,occupy an important position in the leafy vegetable supply,the annual supply but its poor thermal resistance,the temperature is too high to normal cultivation.So through genetic transformation technology to improve the tolerance of the purpose,and efficient tissue culture regeneration system is the important guarantee of variety improvement and innovation and the foundation.The establishment of the efficient regeneration system and genetic transformation were investigated,screening out the selective pressure of antibiotics,in order to ensure the normal growth of transformation cells for subsequent transgenic research lay the foundation and guarantee.The results were as follows:1.Leaf lettuce PS-11 regeneration system(1)The best disinfection method of seed for the concentration of 75%alcohol disinfection of the surface of the 30s,sterile water repeated washing three times,with 0.2%concentration of sodium hypochlorite disinfection of the depth of 15 min,sterile water repeated washing five times.(2)Lettuce leaves PS-11 callus induction.The 28 d induced with the leaf as material with MS as the basic culture medium added different concentration ratio of exogenous cytokinins(6-BA)and auxin(NAA).The optimum culture medium of inducing differentiation of formula for MS+ 0.2 mg/L 6-BA +0.4 mg/L NAA,to cultivate after 28 d,the callus induction rate of 100%.through the surface treatment,the callus induction rate is 2～4 days in advance.(3)Lettuce leaf PS-11bud induction of adventitious.There is no 28 d take induced leaf as material,budding rate reached 78.88%,the average number of budding is 6.89.By optimizing the,materials will be transferred to the induction of cotyledon in the middle part,the budding rate up to 87.77%,the average number of budding is 7.76.(4)Leaf lettuce PS-11 adventitious bud induction.The results showed that:the best inducing medium for rooting was 1/2 MS medium,rooting rate was 80.00%,the average root number was 7.73 per hill,the root is much and thick.2.Screening of genetic transformation in the process of concentration of antibioticsThe concentration of Kanamycin screening.Preliminary selection pressure range of Kanamycin in the 70 mg/L-90 mg/L for induction screening concentration.Under the concentration,explant materials in wither and white phenomenon at around 85 percent,no budding formation of callus and differentiation.