A New Genetic Transformation Technique Without Selection Marker Established By Dividing Treatment

The 21th century is the century of life science, and one who keeps ahead in the biotechnology would occupy the predominant position in the bio-economy ages in future. Transgenic technique, an important part of biotechnology, has a great and significant prospective for developing new and improving the existing commercial cultivars. But nowadays the most transgenic techniques depend on the selection markers. We want to establish a new transgenic technique without selection marker by dividing technique in order to eliminate the potential disadvantages of selection markers, such as influencing the gene expression, limiting the further transformation, disturbing the plant characteristics, endangering the environment and jeopardizing the community health. In recent years many researchers have put a lot of efforts to eliminate the selection markers from transgenic plants, but up to now all the results are unsatisfactory. So it is more important to establish a new genetic transformation technique without selection marker than to eliminate the selection markers from transgenic plants.In this study, the explants obtained from tobacco leaves were inoculated by Agrobacterium tumefaciens with GUS as reporter gene. The calli were grown and divided into 8, 16, 32 and 64 pieces for 2, 3, 4 and 5 times, respectively, and placed on media in original parental neighborhood sequence to provide an opportunity to transformed calli pieces to grow by weakening the competition from non-transformed cells. Every time the central calli were selected from the set of calli with positive GUS activity. Seedlings were regenerated from the finally obtained calli and their transformed status was confirmed through GUS or PCR activity.This research primarily includes three parts: Improving the calli pieces culture technique, improving the DNA transfer technique by Agrobacterium and ascertaining the necessary parameters of dividing technique."B5+0.6 mg/L 2ip+0.03 mg/L IAA+584 mg/l Glutamine" was used for small piece of callus culture for tobacco. "B5+ 584 mg/1 Glutamine+117.2mg/l betaine+0.12mg/l proline+400mg/l inositol (pH5.5)" was used for bacterial infection and co-culture. The agro-bacteria was diluted to OD (Optical Degree) 600=0.5 with subsequent addition ofacetosyrimgone at concentration of 5 mg/l, and infected the explants for 30min, followed by 3days co-culture in the dark. When the diameter of callus reached about 0.5 cm, the next division was conducted. That the number of division and division degree(numbre of division X times) at first and second times were more than 32 and 128, respectively, was time saving and cost effective with more than 50% chances to get higher transformed plants. As a whole, the Dividing Degree and percentage of transformed plants are linearly correlated. How many pieces the callus should be divided relies on the difficulty of callus-culturing and operation too. We think, if possible, the more pieces we divide the callus, the better it will be, especially in the earlier stage, In this way we can decrease the ratio of non-transgenic cells and obtain the transgenic plants as early as possible. When there are enough transgenic cells in callus, we can decrease the number of pieces in the latter stage.Using the transgenic technique by dividing treatment without selection marker, we have transferred chitinase and B-1,3-glucanase genes to SRI tobacco, and transferred B-1,3-glucanase gene to tomato cultivar (Mao T-5) successfully.