High frequency regeneration of Brassica napus varieties and genetic transformation of stocks containing fertility restorer genes for two cytoplasmic male sterility systems

Abstract

Transformation and regeneration protocols for Brassica napus varieties adapted to agronomic conditions prevailing in India were standardized. Five different varieties of B. napus (BO-15, GSL-1, HNS-8, H-18, ISN706) and a hybrid of ISN706 and GSL-1 were tested for regeneration response. B. napus var GSL-1 showed a very high frequency of regeneration (98%). Regeneration response of some of the varieties was improved by alterations in the composition of media. Efficient transformation protocol was developed for the variety GSL-1 with a binary vector containing nptll and bar genes conferring resistance to kanamycin and herbicide phosphinothricin as selection marker and a gus-lnt gene as a reporter. A new construct that contained Ds border, bar gene as integration marker and a mutant csr-1 gene conferring resistance to chlorsulfuron as an excision marker was developed for transposon-mediated gene tagging. This Ds-construct was introduced into B. napus genetic stock (ISN706 GSL-1 Rft/-, Rfp/-) harboring fertility restorer (Rf) genes for two different CMS systems namely 'tour' (Rft) and 'polima' (Rfp).