Studies On The Regeneration System And Transformation DAD1 Gene By Agrobacterium Tumefaciens In Chrysanthemum

The objective of this study is to transfer the DAD1 gene to the chrysanthemumcultivar "guohuaguangdi" in order to establish regeneration system andtransformation system of chrysanthemum mediated by Agrobacterium tumefaciensand obtain new types of chrysanthemum with characteristic of inhibiting or promotingcell death at last. Conclusions as follows:1. The best media for inducing callus, shoot and root were developed via theresearch on effects of different concentration of plant growth regulators on callus,shoot and root induction of chrysanthemum. The results showed that the optimalmedium for inducing callus from leaf disc is: MS+6-BA (2.0mg/L)+NAA(1.0mg/L). On the medium containing MS+6-BA (2.0mg/L)+NAA (0.2mg/L),callus can differentiated into tissues with efficiency of 95%, and the propagationcoefficient was 2.57. Roots were obtained when shoots were transferred onto themedium 1/2MS+NAA(0.2mg/L) after 9 days, and the rooting percentage was 100%.2. On the basis of establishing chrysanthemum regeneration system, the totalsequence of DAD1 gene was ampilified with two pairs of specific primers and wereinserted into the binary expression vector pWM101 at sense and anti-sense directionunder control by promoter CaMV 35S, and the recombinated vector was named aspWM101-DAD1 and pWM101-antisense DAD1 respectively. Double enzymesdigestion and PCR confirmed we have got the sense and antisense constitutionalexpression vectors.3. Genetic transformation was studied with leaf discs by co-cultivation withAgrobacterium tumefaciens. An optimal transformation system was obtained. Theresults showed that the genetic transformation rate was highest when preculture for 1 day,OD₆₀₀ is 0.6,infection for 10 minutes,co-culturation for 4 days and delayedscreening for 2 days.4. PCR detection of transgenic chrysanthemum. We detected the transgenicchrysanthemum plants which were screened with antibiotic Hyg by PCR. The PCRresults indicated that sense and antisense DAD1 gene has integrated into genome ofchrysanthemum because we found the sense and antisense DAD1 gene existed in partof transgenic chrysanthemum plants via PCR and gel electrophoresis. Thetransformation rate of sense and antisense DAD1 gene is 14.3% and 7.1%respectively.