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Bean tissue culture and genetic transformation with Agrobacterium
Published February 23, 2000

In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.

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In vitro regeneration from cotyledons of watermelon
Published August 23, 2000

Cotyledonary segments of five different genotypes of watermelon were used to induce organogenesis. Five different hormone combinations were applied to enhance the induction of shoot formation on the surface of the segments. The phases of organogenesis were followed with light and scanning electron microscope. Shoots were obtained after four wee...ks, then the shoots were transferred to hormone free medium for root induction.

This method of regeneration can be applied in transformation experiments. GUS histochemical assay was made to check the expected success of using Agrobacterium for the transformation.

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The Effects of Some Parameters on Agrobacterium-Mediated Transformation in Muskmelon
Published September 13, 1999

Some parameters involved in Agrobacterium-mediated transformation in muskmelon Hales best (HBS) were studied. Cotyledon explants excised from 3.5-day-old seedlings were co-cultivated with Agrobacterium tumefaciens harbouring binary vectors which contained GUS and BAR genes. After co-cultivation on a low pH medium, explants were transfe...rred to selective medium, with higher pH, containing Claforan and Finale. The medium was changed every two weeks till shoots were induced. All shoots rooted on MS medium supplemented with 0.3 mg/L IBA. These parameters combined as a whole led to successful transformation. The expression of the introduced gene construct was confirmed by GUS staining of shoot segments.


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Genetic transformation of bean callus via Agrobacterium- mediated DNA transfer
Published October 20, 2003

Callus cultures were induced from hypocotyl of young bean seedlings. Callus developed and maintenaned on B5 medium supplemented with 2mg/1 2,4-D and 1 mg/1 kinetin. The results demonstrate that A. tumefacins-mediated transformation is a convenient method to obtain transient gene expression in callus of bean. The results have shown that... the bean callus co-cultivated with A. tumefaciens can be transformed to get heibicide Finale (glufosinate-ammonium) resistant GUS positive tissues. Southern blot analysis of transformed calli showed integration of gusA marker gene carried by a binary vector. Transformed calli were selected on herbicide containing media. Data of molecular analysis (Southern blotting) confirmed the insertion of gusA gene in the genome of herbicide resistant calli with bar gene. There are three evidences that calli are stable transformants: (1) herbicide resistance, (2) GUS activity which is indicative since the coding region containing an intron, (3) the results of Southern hybridization technique.

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