| Grapes belong to the Vitaceae Vitis, which are very high value of fruit tree cultivation. However, grape freezing damage brings great loss to the production, so cultivate the cold-resistant varieties imminently. The grape is a perennial fruit trees with a complex genetic background, long life cycle, and so on, that conventional breeding methods have been extremely limited in breeding, and the application of genetic engineering has opened up a new way for the improvement of grape breeding. ICE a similar activator of genes encoding bHLH transcription factor MYC, under the low-temperature-induced, can make a series of cold induced genes COR, as well as in work the expression of other genes in plant cold adaptation, thus increasing cold-resistance of transgenic plants. In this study, rootstock varieties’1202’(Mourvedre*Rupestris) and cultivated varieties ’Manicure Finger’ grape (Vitis vinifera L.’Manicure Finger’) as materials, establishing ’1202’ grape rootstock adventitious bud regeneration system; Agrobacterium-mediated transformation was adopted to introduce the ICE gene into ’Manicure Finger’ grapevine, and the ICE gene into the ’1202’ that is difficult transformation of grape rootstock was used by pollen-tube pathway method, in order to obtain cold-resistant transgenic grapevine and cultivate the new germplasm of grape. The experimental results of the study are as follows:1. ICE gene was cloned from the V. amurensis,’Manicure Finger’ grape transformation was mediated by the optimization of Agrobacterium-mediated transformation system, received a total of5resistant lines, after GUS detection, tested by PCR,5lines contained the target band; further detected by RT-PCR, in which3lines through RT-PCR detection. Preliminary confirmed that, ICE gene has been integrated into the ’Manicure Finger’ grape genome.2. By molecular detection of3lines of transgenic Manicure Finger Grape were in low temperature to cold resistance, choosing the most cold line of malondialdehyde (MDA), the content of soluble sugar, proline, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) was measured. The results showed that the transgenic line3can stand-8℃low temperature and recovery normal growth under room temperature. Further study found that importing ICE transcription factor can decrease MDA accumulation in plants, increase the contents of free proline and enzyme activity, thereby improving the cold resistance of transgenic grapevine.3. The effects of different explants types, different plant regulator concentration and ratio, the way of explants placement on regeneration of grape rootstock’1202’ were studied in the experiment. The results showed that the regeneration frequency of leaf was the highest, the highest regeneration frequencies were68.78%,.The optimal medium was MS+TDZ1.0mg/L+IBA O.Olmg/L+sucrose30g/L+agar5.5g/L, pH5.8; and the optimal way of leaf touching medium was inaxial surface. Under these conditions, establishing a grape rootstock’1202’ shoot regeneration in vitro technology, the regeneration of adventitious buds was placed in the medium3/4MS+0.35mg L-1BA to induce rooting, the rooting rate was80%. The regeneration plants were cultured35to50days later, transplanted to acclimatization, the survival rate was90%.4.’1202’rootstock inflorescence as ICE gene transformation receptor, using pollen-tube pathway to transform’1202’ grape, embryo culturing received a total of20plants. By GUS histochemical staining, PCR and RT-PCR methods for detection, results showed that,3lines of GUS positive plants by PCR detection, but only1line by RT-PCR. Preliminary ICE gene has been integrated in the genome of’1202’ grape. |
PDF Full Text Request