Tranformation Of Watermelon With Chitinase Gene And Chitinase--β-1, 3-Glucanase Gene

The watermelon (Citrullus lanatus (Thunb.) Mansfeld (syn. C. vulgaris Schrad) is a kind of worldwide fruit with very high economic value, but its disease is various in styles. Soil-transmitted diseases caused by fungi endanger most serious, for instance wilt, and are the main problems existing in the production of watermelon. Because the genetic base of watermelon is narrow; the disease-resistant kind of germplasm resources is scarce; the hereditary law of disease resistance is complicated, there are limited progress in the traditional disease-resistant breeding. In addition, it is a reason that strong polymorphism and extensive variation of microorganism, too. The disease-resistant genetic engineering of plant makes sure progress in the improvement of some important species at present. But its application study of melon crop was later than others. The tissue culture of melon crops is more difficult than other crops in the process of culture, the rate of success, the plant regeneration, especially for watermelon. The optimization of genetic transformation system is difficult also. This entire make the research in this respect is comparatively backward.This experiment carried on systematic research to watermelon tissue culture system; optimized Agrobacterium tumefaciens mediated genetic transformation system of watermelon. Two antifungal gene plant expression vectors , one contained chitinase gene Chi3, another contained chitinase gene Chi3 and beta-l,3-glucanase gene Glu-Ac, were constructed and used in the genetic transformation of watermelon. The two antifungal genes were cloned from tomato. The transgenic plants were obtained and carried on relevant molecular biology analysis and disease resistance identification. The disease-resistance species or the breeding materials for further study were expected to obtain from the transgenic plants. At the same time these results should give useful references for application of this technology in other crops too. Main result and conclusion are the following:1 The foundation of watermelon tissue culture system1) Adventitious bud can regenerated directly from stem apex and cotyledon section. Adventitious bud can be obtained also from callus, but the frequency was very low. It was extremely difficult to obtain adventitious bud from leaf and stem segments.32 ) A direct high frequency regeneration system of watermelon was established successfully, which used cotyledon segment and stem apex as explants. Using the MS medium as the basic medium, optimum adventitious bud regeneration medium for cotyledon segment section was supplemented with 1.0 mg ?L 6-BA and 0.2 mg ?L" IBA, and for stem apex, 1.0 mg ?L" 6-BA and 0.5 mg ?, giving a regeneration frequency of 81.4% and 92.3%, respectively.3) Although some differences were present among genotypes, but for all genotypes used in this experiment 6-BA was the key to the induction of adventitious buds, while low concentration of IBA were favorable to the induction. Bud regeneration was not obtained from any explants on the culture medium without hormone.4) When the cotyledon of sterile seedlings was light green, the stem apex and cotyledon segment were most suitable to induce the adventitious bud directly and the regeneration frequency was highest.5) Adventitious root can be obtained easily from regenerated plants of watermelon. The low concentration of IAA (0.2 mg ?L) can induce normal adventitious roots with root hair, while the result of NAA was not good. The transplanting of regenerated plant was very difficult.2 The optimization of Agrobacterium tumefaciens-mediated genetic transformation of watermelon.1) The watermelon cotyledon segment was sensitive to hygromycin, and 15 mg ?L was the suitable screening concentration. Kanamycin inhibited the growth and division of explants obviously, the vitrification of adventitious bud was serious. Only with high concentration, cefotaxine and carbenicillin slowed down the growth rate of explant slightly, no other obvious influe...