Studies On Genetic Transformation Of Fungi-resistant Genes In Grapevine

Viticulture is one of the most important parts of the agriculture production in our country. The viticulture area growths quickly in these years, following with the manifold of plant diseases and insect pests, especially epiphyte diseases. Plant bioengineering has already become one of the most effective means to cultivate new cultivars which are resistant plant diseases and insect pests. There are many useful objective genes can be used in grape genetic transformation, but it is still hard to transform genes into grape, the main reasons are listed as follows:1. The low regeneration frequency is the key factor in grape genetic transformation. Organ genesis method tends to produce table unit and the induction of somatic embryos is hard and is restricted by gene types. For only a small part of cultivars succeed until today.2. It is hard to transform genes into grape, for a small number of transgenic plants were obtained until today and most of the genes are report genes.3. The tendency of being dead of recipients during and after co-cultivation period restricts the studies of agrobecterium-mediated transformation.4. Kanamycin is not an effective antibiotic to be used during selecting period.For resolving theses problems, a Europe cultivar (Semillion) was tested and a high effective regeneration system was established, moreover, the Fungi-resistant genes were transformed into grape via Agrobecterium-mediated transformation and particle bombardment-mediated transformation. Some main factors affecting the transform frequency were discussed and the genetic transform conditions were optimized. Positive plants were obtained by PCR amplification. All these studies established a stable base of getting Fungi-resistant transform plants. The main contents are listed as follows:1. A plant regeneration system via somatic embryogenesis method was established. Semillion- a Europe cultivar was used to induce embryogenic calli and somatic embryos. The medium-Nitscrn-BAo.2m6'L+NAAo.o2mg/L was regarded as the most suitable medium for improving the bourgeon of somatic embryos after three factors were considered - the percentage of bourgeon of somatic embryos, the percentage of whole plantlets in all plantlets and the percentage of abnormal plantlets. The medium-Nitsch+0.04mg/LNAA was effective for improving roots growth.2. A liquid culture system was established well to improve the effective somatic embryogenesis and plant regeneration. The medium-Nitsch basic medium with lumol/L 2,4-D was suitable for the growth of somatic cells. The trimness degree of somatic embryogenesis was more improved after the treatments of cells to be sifted with 1mm diameter screen in 4 days of subculture. For instance, the percentage of cotyledon embryos is 92.1% after 5 weeks of culture in Nitsch medium. The medium- Nitsch+ Ig/L Active charcoal is good for plantregeneration and the Active charcoal is an effective factor to reduce the formation of abnormal plantlets.3. The cell growth curve was appeared as S shape and the cells began their rapid growth period from the forth day and their growth were gradually slow down from the tenth day till were stop at last. Transformation can be conducted during the rapid growth period of the cells that is good for improving the transform frequency.4. The suitable antibiotics and their concentration were studied for the selecting of transformants. Neomycin is the most effective one in some antibiotics for the cells can't growth in the medium that was added with 80mg/L Neomycin. The most effective selection method is to select the cells in solid medium with 80mg/L Neomycin or the selection can be conducted as increasing the Neomycin concentration from 30mg/L to 50mg/L to 80mg/L gradually in solid or liquid medium.5. Some main factors infecting the genetic transformation were discussed.a. The influence of different strain. The ability of invasion of EHA105 was much better than LBA4404 when comparing the number of resistant buds. Embryogenic calli was more s...