A High Efficient Genetic Transformation System For Cherry (Prunus Cerasus L.) Leaves Mediated By Agrobacterium Tumefaciens

The drupes fruit tree mainly includes the peach, Li, the apricot, the almond, the cherry and so on, belongs to the rose family plum plant in the plant classification, holds the quite important status in the fruit tree production. Since long, the drupes fruit tree has been recognized by the domestic and foreign colleagues for has been most difficult to carry on a kind of tree seed which the exsomatize (asexual material) regenerates, everybody attempted in this aspect has done the massive experimental study, but result really micro. The drupes fruit tree as a result of questions and so on its own exsomatize regeneration difficulty, the genetic engineering technology's research also obviously relatively falls behind other fruit tree's development. Therefore, the establishment and the consummation effective genetic transformation regeneration system becomes the development drupes fruit tree in this area research key. Since long, the domestic and foreign several laboratories have devoted to this aspect research, and has obtained certain research results. All sorts of signs indicated that the drupes fruit tree (asexual material) the genetic transformation highly efficient regeneration technology will make the breakthrough hopefully in the near future.The cherry is middle one year most in the morning the city fruit, has spring the fruit first fine reputation vulgarly. Its nutritional value is high, the use is broad. Besides the fresh food may also make the dessert, the wine-making, to be used as medicine and so on functions. The sour cherry also has moves benefits was mad that raises fine stops flows swiftly and so on nutritious function. Its, the nucleus, the fruit all may be used as medicine, all through not abandoned. Because the multi-dimensional phenol compound content is high, therefore has the obvious anticancer treatment, the anti-arteriosclerosis and other cardiovascular disease's effect. However easy to infect the root cancer in the middle of transplanting, has limited this fine tree seed's promotion greatly, thus enhances its resistance is the goal which the present will develop. The traditional breeding method cycle is long, but genetic engineering to speed up its breeding advancement to provide the new method. But lacks the highly effective genetic transformation system, as the establishment cherry fine genetic transformation system, the present paper take the sour cherry as the material, takes outside with the exsomatize leaf blade plants the body, has established the exsomatize leaf blade high frequency regeneration system; And lies between through root-cancer agriculture bacillus leads from optimization genetic transformation each parameter, has established the exsomatize leaf blade highly effective genetic transformation system, its result is as follows:1. determines the sour cherry exsomatize leaf blade the regeneration systemFrom the regeneration culture medium, the hormone allocated proportion, the leaf blade physiology condition, the culture medium the agar-agar amount used, the different raise vessel and so on several aspects to affected the exsomatize leaf blade highly efficient regeneration the key aspect to conduct the research. Had determined the CAB-6p regeneration culture medium is WPM, the best hormone allocated proportion is BA 2mg/L + IAA 2mg/L, leaf blade's best physiological condition sends out newly for the test tube seedling crown extremely young tender, and is closes up the condition young leaf, the agar-agar use density is 4.5g/L, the exsomatize leaf blade induction regeneration raise vessel regenerates the effect with the wide mouth bottle to be best.2. in sour cherry genetic transformation to antibiotic resistance sensitivity test researchExamines the research to indicate that the card that mildew element's screening best density should be 40mg/l. Thought that the spore zuo lin sodium is the CAB-6p best bacteriostasis antibiotic, and is suitable the density scope is 500mg/l-600mg/l.3. sour cherry genetic transformation system's establishmentTakes the acceptor with the aseptic CAB-6p test tube seedling's exsomatize leaf blade, optimized the agricultural bacillus to lie between leads transformed system's many parameters: As 0.5-1.0 agricultural bacillus vaccine invades dyes 10min altogether raises after OD600 for 3 days, can obtain GUS rate reaches as high as above 80%.4. this experiment also further studied has screened, detention screening, the gradient to screen three kind of selector mode antagonism bud immediately the percentage and the conversion rate influence, obtained the gradient screening conversion rate is 1.2%. Is this transformed system best screening way. Moreover, AS is also a genetic transformation key influencing factor. This experimental study indicated that in altogether raises the culture medium and in the agricultural bacillus suspending liquid simultaneously joins the density is 20mg/l AS may enhance the resistant bud rate.5. . increases the analysis through PCR, proved initially the exogenous gene already changed over to sour cherry CAB-6p.