Maqsood Ahmed, Nadra Khan, Ishfaq Ahmed Hafiz, Nadeem Akhtar Abbasi, Muhammad Akbar Anjum and Sajjad Hussain
Effect of Various Factors on the Efficiency of Agrobacterium-Mediated Transformation of Grape (Vitis vinifera L.)
The present investigation was conducted to study the effect of inoculation time, cocultivation period and various concentrations ofhygromycin and kanamycin on genetic transformation of grape (Vitis vinifera L.) cv. King’s Ruby through Agrobacterium tumefaciens. The Chitinase gene (for fungal resistance) and GUS gene (for phenotypic expression of transgenic) were introduced in the embryogenic calli developed from leaf discs, through Agrobacterium strain LBA4404 harboring plasmid pBI121 nptII as selectable marker for GUS gene and hptII for Chitinase gene. Regarding transformation efficiency rate, 10 minutes inoculation period with bacterial suspension showed the highest transformation efficiency rate i.e. 2.83% with Chitinase gene and 2.5% with GUS gene. Infected calli with Chitinase gene and GUS gene co-cultivated for 2 days showed the maximum transformation efficiency of 2.75% and 3.25%, respectively. For elimination of excess bacteria, cefotaxime treatment (300 mg L-l) showed the highest survival rate of 3.16% for Chitinase gene and 2.5% for GUS gene. The maximum i.e.2.83% transformation efficiency rate was achieved at 10 mg L-l of hygromycin for selection of transformed calli. The highest 2.25% transformation efficiency was yielded when kanamycin was used at the rate of 100 mg L-l. Five out of 8 calli showed positive expression with 62.5%transformation efficiency through histochemical GUS assay. Present results may be helpful in improving genetic transformation efficiency of grape through Agrobacterium tumefaciens against fungal diseases and reduce the use of fungicides.
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