Study On The Development Of Regeneration Systems And Genetic Transformation For Hami Zyziphus Jujube And Munage Grapevine

Cold/freezing is an important abiotic stress injuries on plant growth. Xinjiang is one of the main producing areas ofChinese date, but occasional freezing in Xinjiang, has become an important factor restricting the development of the fruitindustry in Xinjiang. In order to obtain transgenic Chinese jujube with traits of high cold tolerance by transforming CBF gene.The aim is to provide a theory to solve Xinjiang jujube frost damage. The regeneration system of adventitious shoots fromleaves has been established and the Agrobacterium-mediated transformation of wheat CBF has been achieved in Chinesejujube.The main results are as follows:1. The results show, MS, NN69, WPM three media compared, NN69more suitable as the basic medium leaf regeneration;The efficiency of TDZ was significantly higher than BA in the induction of adventitious bud from leaf. AgNO3addition couldgreatly increased the regeneration frequency and the dark culture in the first two week was much more conducive toincreasing the regeneration frequency; prevention of explant browning: the use of10mg/L Vc soak15min to preventbrowning, and can improve shoot regeneration rate; medium supplemented with PVP, or Vc shoot regeneration was notsignificantly improved; medium supplemented with AC inhibits differentiation of explants.The adventitious buds weresuccessively cultured in medium NN69+1mg/L TDZ+0.2mg/L IBA+AgNO31mg/L, culture two weeks in dark, afterculture thirty30days, MS+1mg/L6-BA+0.2mg/L IBA until they grew3cm long roots and then they were transferred andcultured in the1/2MS medium to which0.4mg/L IBA was added, and this could better induce them to root.2. Effects of Agrobacterium transformation of Hami jujube factors found suitable conditions for the conversion of Hamijujube leaves: Leaves disseminated pre-culture before Agrobacterium2d, infecting bacteria concentration OD600of0.5,infection time was20min, infection after co-culture medium NN69+TDZ1mg/L+IBA0.2mg/L in the dark for3d,transferred to selective medium NN69+TDZ1mg/L+IBA0.2mg/L+AgNO31mg/L+Kan20mg/L+light trainingafter Cef300mg/L in the first dark culture9d,30d, then transferred to selection medium containing MS+6-BA1mg/L+IBA0.2mg/L+Kan20mg/L+Cef300mg/L continues to light training, photoperiod of14h light/10h darkness. For thefirst time in CBF gene into Hami, initially identified by PCR to detect positive transgenic plants7.3. In order to induce multiple shoots from ‘Munage’ grape （Vitis vinifera）explants, the effects of explants, media,hormone concentrations and dark treatment on the induction of shoots were studied. Plant regeneration were induced fromleaf, petiole and stem explant of grape cultivar Munage. The shoots were induced only from the leaf and petiole explants. Theresults indicated that the regenerating rate of adventitious buds were obtained when petiole explants were incubated on NN69media with TDZ1mg/L+IBA0.2mg/L, it reached16.3%. The optimum media for inducing adventitious buds from leafexplants were NN69+TDZ4.0mg/L+IBA0.1mg/L. The optimum media for elongating adventitious buds were MS+6-BA1mg/L+IBA0.2mg/L.4. The CBF gene was introduced into grapes through Agrobactrium tumefaciens with using leaf and petiole as the targettissue. The results showed that Kanamycin critical lethal mass concentration of leaf and petiole was5mg/L, optimalconcentration of Cef was300mg/L. It could reduce restraining of Kan on prape that adding Kan firstly with2.5mg/L andgradually increasing to5mg/L. Twelve Kan-resistant plants of ‘Munage’ grape were obtained by optimizedAgrobacterium-mediated transformation. PCR analysis on these resistant plants showed that there were five PCR positive plants, and confirmed that CBF gene was integrated into the ‘Munage’ grape genome.