|
Pak. J. Bot., 47(SI): 193-198, 2015. |
Back to Contents | ||||
|
|
Updated: 31-12-15 | ||||
|
|
AGROBACTERIUM-MEDIATED TRANSFORMATION OF SPINACIA OLERACEA L. THROUGH A SYNTHETIC CHITINASE GENE INDUCING RESISTANCE TO FUNGAL PATHOGENS
JAWERIA GUL1,2, IQBAL MUNIR1, HASEENA GULZAR2 AND GUL HASSAN2*
1Institute of Biotechnology and Genetic Engineering (IBGE), Faculty of Crop Production Sciences, University of Agriculture, Peshawar, Pakistan 2Shaheed Benazeer Bhutto University, Sheringal, Dir Upper, Pakistan *Corresponding author’s e-mail: hassanpk_2000pk@yahoo.com
Abstract: Agrobacterium-mediated transformation of spinach (Spinacia oleracea L.) Cv. Desi was carried out with a synthetic chitinase gene, NIC. The plasmid, pEKH/NIC, harboring the NIC gene, encoding chitin-hydrolyzing enzyme; the gene for neomycin phosphotransferase II (nptII), which confers kanamycin resistance and the hpt gene conferring resistance to hygromycin, was transformed into Agrobacterium tumefaciens strain EHA101. Hypocotyls and cotyledons from the 6-7 days old in vitro germinated seedlings and leaf discs from 3-4 weeks old green house grown plants of spinach were infected with overnight culture of the transformed Agrobacterium. Conditions such as co-cultivation period, bacterial concentration and concentration of plant growth regulators were optimized. Co-cultivation of explants with a bacterial concentration of1 x 109 cells/ml for 72 hours in dark, followed by culturing on a medium containing 20 mgl-1and 200 mgl-1cefotaxime resulted in an overall transformation efficiency of 23% for all the three explants. Transformation of spinach plants was confirmed by callus formation on selection medium, and PCR analysis using the primers for NIC gene. The results showed that T-DNA containing the NIC gene is integrated into genome of the spinach. Shoot regeneration, rooting, further molecular analysis and disease resistance tests of the transgenic plants are suggested to be carried out in future.
Key words: Agrobacterium tumefaciens, NIC, Synthetic chitinase, Transformation efficiency, Spinach.
|
||||
|
|
|||||
| Back to Contents |
|
Back to Contents | |||
|
|
|||||