PJB-2009-349
FACTORS AFFECTING AGROBACTERIUM TUMEFACIENS MEDIATED TRANSFORMATION OF ARTEMISIA ABSINTHIUM L.
ABDUL MANNAN1,2, TOOBA NOOR SYED1 AND BUSHRA MIRZA1*
Abstract
The present work was aimed to study various factors affecting Agrobacterium tumefaciens mediated transformation of Artemisia absinthium L. Agrobacterium strain C58C1 harboring binary vector p35SGUSINT having kanamycin resistance gene (NPTII) as selectable marker and β-glucuronidase (GUS) as a reporter gene was used for transformation. Factors affecting transformation efficiency, such as age and type of explants, presence of ampicillin and kanamycin in Agrobacterium inoculum, concentration of Agrobacterium, infection and co-cultivation time period of Agrobacterium, effects of 2,4-D, pH of co-cultivation medium and effects of cefotaxime in regeneration medium were studied. An amount of 50mg/L each of kanamycin and ampicillin was used to select the pure Agrobacterium cultures and 500mg/L cefotaxime along with 20mg/L kanamycin was used to select transformed cells. Results concluded that one week old leaf and root ex-plants of A. absinthium inoculated with A. tumefaciens for 5 minutes and co-cultivated for 3-4 days in B5 medium with 0.5mg/L of 2, 4-D at media pH 5.8 showed 100% transformation efficiency.
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