Study On Conversion Of Disease Resistance Gene CcRIP Ⅱin’yuanhong’ Jujube

As the origin and cultivation center of the jujube tree, China is very rich in germplasm resources. The jujube is the seventh largest fruit trees and big fruit trees, especially fresh fruit has high nutritional and medicinal value, which is called "the ecological and economic species of21st century". Jujube Witches Broom is a devastating infectious disease and almost distributes in all the domestic jujube cultivation area and there is no effective treatments to Jujube Witches Broom. Screening and cultivating species or plants of anti-jujube witches broom is one of the important ways to solve the jujube witches broom. The traditional cross-breeding is very difficult to get resistant varieties quickly. Last ten years it has made rapid development in transgenic technology. It has been widely used in fruit breeding at home and abroad. Transgenic has become one of the most promising methods to improve fruit varieties. Now Agro bacterium has been successfully used in crops, but rarely in fruit, which is reason to the long breeding cycle in fruit. So it is very meaningful to find effective methods in breeding. This study use Yuanhong jujube transfer the disease resistance gene (CcRIP II) from the anti-virus Cinnamomum camphora (L.) Presl tree by Agro bacterium and then determine the physiological indexes of the seedling cultivation. We have gain results as followed.1. Establishment of Regeneration System. BA is the most important impact on callus color and new lateral bud while NAA is that of new lateral bud growth and callus, and KT has effect on lateral bud while new lateral bud elongation has the minimal impact on the size of the callus; the MS the+BA1.0mg/L the+KT1.5mg/L+NAA0.01mg/L culture medium is the most conducive to the proliferation of newborn lateral bud; MS+BA1.0mg/L+KT1.0mg/L+NAA0.01mg/L medium is conducive to increased callus; MS+BA3.0mg/L,+KT1.0mg/L+NAA0.05mg/L of culture medium is the most conducive to lateral bud elongation.2. Optimization of the system of transferring the CcRIP II gene by Agro bacterium. When the stem segments are parts, DNA broth liquid or1/2concentration of original solution and dip10min or20min, cultured average differentiation and numerical are the maximums, those are2.4shoots and1.7buds; when the stem segment are shoots, select parts of the shoot tip DNA broth1/2liquid or liquid concentration, dip15mins-20min, and a higher average seedling height is1.31cm and1.15cm respectively; green seedling rate was also higher by 62.5%and75.5%respectively.3. Three most important factors affecting transformation efficiency (different infection parts, infection time and DNA concentration) were analyzed by using L9(34) orthogonal experimental design. Result indicated that the best transformation efficiency was obtained by using bacterium liquid culture of1/3concentration of primary culture liquid to infect the shoot-tip for20min.4. We get some transgenic seedlings of Yuanhong jujube in this study,10tested positive for PCR. It showed that exogenous CcRIP II gene were successfully transformed into Yuanhong j uj ube.5. Inoculated with pathogens transgenic plants and the control group compared to the not found the source of the disease infection of the typical symptoms and strong growth potential, PCR, molecular test results of transgenic plants not found in jujube infected plant primordial mark the product of the positive expression, untransformed plants there are three witches broom positive expression, infection rates reached15.0%, indicating that the transgenic plants than the transgenic plants of witches’ broom my co plasma showed higher resistance.6. By the disease resistance contrast detection between transgenic plants and non-transgenic plants, it showed that the activity of SOD, POD activities were enhanced, the activity of SOD, CAT were significantly enhanced; The content of Pr and Free Praline showed a general up trend and were higher than the contrast.