Agrobacterium mediated callus based transformation protocol for transferring DREB1A gene in wheat (Triticum aestivum L.)

Abstract

The present study was designed to optimize protocol for transferring DREB1A gene in wheat and
selection of one suitable cultivar among Lasani-08, Inqlab-91, Chakwal-97 and GA-02 for the development of
transgenic plants. The already optimized callus induction and regeneration media was used for tissue culture.
Different treatments of optical density (O.D.), hygromycin, cefotaxime and acetosyringone concentrations were
optimized. After optimization of these parameters, the four wheat cultivars were compared with different treatments
of infection and co-cultivation time. The Lasani-08 was found to be one of the best cultivars among four cultivars
based on the maximum hygromycin resistant calli (18.36%). Its calli were shifted on regeneration media for the
development of transgenic plants. The transformed gene was confirmed using gene specific primers through
conventional PCR. A lethal dose (50 mg/l) of hygromycin for selection of transformed calli, 300 μM of
acetosyringone to enhance transformation process and 500 mg/l cefotaxime to eliminate Agrobacterium after cocultivation, were optimized. The infection time (5 min) and co-cultivation time (48 h) were found to be the most
suitable parameters for the maximum transformation efficiency. PCR confirmed that 0.38% of regenerated plants
were transgenic. This study will provide a way for transferring other agronomically important genes in wheat
cultivars for their improvement.