Production of Transgenic Lines of Wheat Containing Rice Chitinase Gene

Abstract

Wheat is the world 's major food crop. This crop needs attention when it comes to biotic and abiotic stresses. Rusts, which include str ipe rust, leaf rust and stem rust, are the main biotic stresses and can create a big problem in near future. Through conventional breeding resistant genes can be made avai lable but gene pool is limited and also these pathotypes are constantly transforming, making any cult ivar history by breaking its resistance. Molecular techniques can answer these questions, and genes from other related or unre lated species can be identified from their expression analysis. Pathogenic related proteins are proteins that are synthesized against funga l diseases in many species. Chitinases are pathogenic proteins that can enhance the resistance of wheat against different rusts. Rice Chitinase Gene was used for this research. Transformation is tissue culture dependent and tissue culture is genotypic dependent in wheat. So, for each variety, tissue culture protocols were optimized first. Seven varieties were chosen i.e. Inqilab 91, Chakwal 97, Manthar, Tatara, Kohsar, Khyber 87 and GA 2002. Callus induction was the first thing that was improved. 2mg/l was found to be optimum for Tatara where, 97.18% of the seeds produced calli . Inqilab 91, Chakwal 97, Manthar, Kohsar and Khyber 87 gave good response at 3mg/1 and 83.25%, 77.75%, 95.83%, 83.3% and 48.8% of the seeds produced embryogenic calli respective ly. In GA 2002, maximum number of calli were induced at 5mg/1 of 2,4-D and callus induction frequency of 81.15% was observed. For regeneration different combinations of four hormones, Indole Acetic Acid, Benzyl Amino Purine, Kinetin and 6-y-y-dimethylallylaminopurine, were used. For Inqilab 91, Chab.val 97, Manthar and GA 2002 best combination was O.lmg/l IAA, 0.4mg/1 Kinetin and O.5mg/1 of 2iP and 87.25%, 81.75%, 68.75 and 84.47% of the calli produced shoots respectively. Tatara showed poor regeneration response, and no significant difference was observed when IAA and BAP or IAA and Kinetin were used and on a average of 12% calli produced shoots. Best combination of hormones for Kohsar and Khyber 87 were found to be O.lmg/1 of IAA, 0.5mg/1 of BAP and 0.5mg/1 of Kinetin where Kohsar produced shoots in 80.5% of the calli and regeneration frequency of Khyber 87 was 62.2%.