High efficiency transformation protocol for two Indian cotton (Gossypium hirsutum) varieties via pollentube pathway

Abstract

Genetically engineered resistance has been actively investigated in recent years as an alternative for genes from wild relatives. The development of a method to obtain transformants, which is independent of problems inherent to tissue culture procedures, has been the ultimate goal of plant breeders. The alternative method, which involves Agrobacterium, but does not require tissue culture steps, is in planta tansformation. Although use of pollen tube pathway to deliver foreign DNA in to embryo sacs first reported in cotton was encouraging the overall quality of the data was very weak. Therefore, our attempts to modify this method yielded fruitful results. Out of 5 619 flowers, which were treated with Agrobacterium, where in the stigmatic surface was treated with 5% sucrose along with 20 ppm boric acid prior to application of Agrobacterium resulted in a boll set of 32.5%. Stringent in solium kanamycin screening of the treated plants in T₁ and T₂ generations yielded 7 PCR positive transformants, which was also confirmed by insect bioassay studies and Southern blots. Transformation efficiency of Pollen Tube Pathway (PTP) mediated transformation was 0.30%.