The PUTNHX Salt-tolerance Gene Transformation And The Construction Of Transformation System In Two Kinds Of Small Berries

In order to transfer the salt-tolerance PUTNHX gene into craneberry and blueberry, to culture new transgenic species, the tender stems and new-born shoots of cranberry and blueberry were chosen as materials. According to comparative testing with different types and concentrations of hormones, the best regeneration concentration of hormones for this two fruit trees were confirmed. The best regeneration system of the two small berries was constructed. Through the selection of kanamycin pressure, Agrobacterium concentration, infection time, total training time and methods of co-culture in genetic transformation, The best transformation systems were constructed. The salt-tolerance PUTNHX gene was transformed into the cells of cranberry and blueberry through Agrobacterium tumefaciens-mediated. The regenerate buds, roots and the whole transgenic mutants were acquired. The main results were as follows:1 .Establishing the cranberry regeneration systemThe new shoots were chosen as materials. The highest proliferation number was gotten on the medium of 1/2MS+KT0.5mg/L+IAA0.5mg/L cultured for 28d , the highest proliferation index was 4.8.Then the tender stems were chosen as materials to culture on the differentiation medium MS+KT0.25mg/L+IAA0.5mg/L for 28d. The highest regeneration rate was 100%.Then they were transferred on the medium 1/2MS+KT0.25mg/L+IAA 1.0mg/L to elongate for 28d. The length of shoot were about 3cm, then they were cultured on the root regeneration medium 1/2MS+NAA2.0mg/L for 35d, the highest root regeneration rate was 100%.2. The effective gene transformation system of cranberry stemsThe tender stems were selected as explants that infected by Agrobacterium with PUTNHX gene and PBI121 expression vector. The concentration of Agrobacterium was 0.4OD₅₅₀.After infection they were transferred into the regenerated media without kanamycin pressure. Then they were turned into the buds regeneration medium with 50mg/L kanamycin pressure (MS+KT0.25mg/L+IAA0.5mg/L+400mg/LCarbenicillin). After three subcultures, 60d later, the kanamycin resistant shoots were acquired. The highest transformation rate was 7.4%. Resistant buds were tranfered into elongation medium with selection pressure (1/2MS+ KT0.25mg/L+IAA1.0mg/L+400mg/LCarbenicillin). After 28d culture, they were transferred into rooting medium with kanamycin pressure (1/2MS+NAA2.0mg/L+ 400mg/L Carbenicil- lin ). 35d later, they were rooted. After rooting 2-3 weeks, the plants were transplanted. The PUTNHX gene was transformed into the cranberry for the first time.3 .Established the blueberry regeneration systemThe new shoots were chosen as materials. The highest proliferation number was gotten on the medium of 1/2MS+KT0.25mg/L+IAA0.2mg/L cultured for 28d , the highest proliferation index was 5.7. Then the tender stems were chosen as materials to culture on the differentiation medium 1/2MS+KT0.3mg/L+IAA0.5mg/L for 28d. The highest regeneration rate was 100%.Then they were transferred on the medium 1/2MS+6-BA0.25mg/L+IAA 1.0mg/L to elongate for 28d. The length of shoot were about 3cm, then they were cultured on the root regeneration medium 1/2MS+NAA2.0mg/L for 35d, the best root regeneration rate was 8%.4. The effective blueberry transformation systemThe H28 blueberry stalks were chosen as materials that infected by Agrobacterium with PUTNHX gene and PBI121 expression vector. The concentration of Agrobacterium was 0.4OD₅₅₀. After infection they were transferred into the buds regeneration medium (1/2MS+KT0.3mg/L+IAA0.5 mg/L+kanamycin50mg/L+Carbenicillin500 mg/L), After three subcultures, 60d later, the kanamycin resistant shoots were acquired. The highest transformation rate was 5.2%.Then they were transferred into the elongation media (1/2MS+0.25mg/L6-BA+1.0mg/LIAA+50 mg/L kanamycin+300mg/LCarbenicillin), After 28d culture to 3cm, the they were transferred into rooting medium (1/2MS+2.0mg/LNAA+50 mg/L kanamycin+ 100mg/L Carbeni- cillin). 35d later, some transgenic mutants were gotten. The PUTNHX gene was transformed into the blueberry for the first time.