| The Welsh Onion(Allium fistulosum L.), are biennial herbaceous plants, belong to Allium and Liiaceae. We use Zhangqiu Welsh Onion, special local products of Shandong, as materials for tissue culture in vitro and Agrobacterium tumefaciens mediated Chitinase transformation in this study, the main results are summarized as follows:1. The establishment of the regeneration and transformation system of Zhangqiu Welsh Onion Calli. The different organs of welsh onion-seeds, immature leaves and roots were taken as explants to induce Calli and regeneration. It was indicated that the seeds were the best, Calli induction rate can reach to 90.28%; MS+1.0 mg/L 2,4-D+2.0 mg/L BA is the subculture medium, ager concentration is 9g/L, the interval of subculture time is 15 days, all can reduce Calli vitrification effectively; MS+2.0mg/L BA is the optium shoot regeneration medium, and MS medium without any hormone content is best for shoot-extension induction.2. The selection agent concentration was confirmed. The suitable concentration of Cef was 300 mg/L (Calli catching Agrobacterium tumefaciens rate was 8.75% and browning rate was 6.88%); and Hyg was screening agent, when its concentration was 50 mg/L, Calli growth stopped and weak Calli die, when its concentration was 75 mg/L, all the Calli were death. Therefore, we chose the concentration of Hyg was 50 mg/L for the first screening, and Calli differentiated for 10 days, then the Hyg was 75 mg/L for the second screening.3. The optimization of transformation conditions. The Calli were soaked in the Agrobacterium tumefaciens suspension, which OD600 value of concentration was 0.6, besides, the better infecting time was 15~25 minutes, especially 20 minutes was the best, after directly infecting the Calli were transferred onto the solid co-cultivation medium with pH 5.6, and co-cultured 3 days under 25℃, dark conditions, the high ratio of resistance Calli were obtained, and the biggest ratio could reach to 5.35%.4. The histochemical analysis and molecular assay. The histochemical analysis of gus gene activity indicated that Calli of transient expression at 53.08% to show blue, the Chi gene and reporter gene gus are in the same open reading frame, accordingly we deduced that chi gene could express also. We got 48 resistant plants in this experiment, and in all of whom there were 3 chi-PCR positive plants, the positive ratio was 6.25%.5. The express detection of purpose gene. The crude chitinase liquid was extracted from transgenic plants and non-transferred plants, the result of chemical methods examination manifested that minimum absorption value of OD420 in transgenic plants was -0.404, lower than the non-transferred plants distinctly (-0.173), so the chitinase activity in transformants is higher than those in control.6. The inhibition effect of Hyg on Zhangqiu Welsh Onion. The resistant plants obtained from experiment were slow-moving both in differentiation and the next growth. When to study and research the dissolved protein in Calli, we found Hyg have an influence on growth and differentiation of Welsh Onion tissues, like effectiveness of some plant hormone.
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