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• Co-transformation of bean callus using high-velocity microprojectiles- mediated DNA transfer

  76-78.

  E. Eissa Ahmed

  Gy. Bisztray

  I. Velich

  Views:

  122

  We have found that 50 mg/I kanamycin and 0.8 Mo1/1 mannitol concentration was sufficient to kill the control callus of bean (Phaseolus vulgaris L.) and differentiate transgenic from the non-transgenic cells. The GeneBooster particle delivery system was used for the bombardment of bean callus. The kanamycin resistance gene was used as a selectable marker. The test was made by transferring the healthy white callus, subcultured for three months on selective and non-selective medium. After selection on kanamycin containing media, several kanamycin resistant calli had been obtained, survived and grew. After selection on mannitol containing media no drought resistant calli had been obtained. Resistance of the selected calli were verified by their ability to grow repeatedly on selective medium containing 150 mg/I kanamycin. Selective pressure was maintained over a period of 8 months.

• Production of transgenic bean callus via genetic transformation by DNA-coated tungsten particles

  43-47.

  E. Eissa Ahmed

  Views:

  106

  Callus cultures were induced from hypocotyl of young bean seedlings. The B5 medium completed with 1 mg/1 KIN and 2mg/1 2,4-D proved the best. Callus developed and maintenaned on B5 medium supplemented with 1mg/1 kinetin and 2mg/I 2,4-D. The B5 medium supplemented with 1mg/1 KIN and 2mg/1 2,4-D induced much more callus than half strength MS medium supplemented with 0.5 or 0.75mg/1 BA and 0.1 mg/1 NAA. The results demonstrate that GeneboosterTM is convenient method to obtain transient gene expression in callus of bean. The results have shown that the bean callus shot by GeneboosterTM can be transformed to get (kanamycin-resistant and stress mannitol­tolerant) calli. The presence of mannitol-dehydrogenase gene (mt/) was verified by PCR, showing the integration of mt/ gene carried by two plasmids. Co-transformed calli were selected after bombardment on kanamycin, mannitol and (kanamycin+mannitop-containing media. Data of molecular analysis (PCR) confirmed the insertion of mtl gene in the genome of mannitol-tolerant callus lines.

• High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

  99-102.

  E. Eissa Ahmed

  Gy. Bisztray

  I. Velich

  Views:

  112

  We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.