Development of transgenics in Indian oilseed mustard (Brassica juncea) resistant to herbicide phosphinothricin

Abstract

Transgenic lines resistant to herbicide phosphinothricin (PPT) were developed in mustard (Brassica juncea), a major oilseed crop grown in more than 6 million hectares of land in North India. Seedling-derived hypocotyl explants were transformed with a disarmed Agrobacterium tumefaciens strain GV3101. The developed constructs contained the bar gene encoding the enzyme phosphinothricin-acetyl-transferase (PAT) which inactivates phosphinothricin (PPT) by acetylating it. The expression of the bar gene was controlled either by the double enhancer version of CaMV35S promoter (35Sdebar) or a CaMV35S promoter with a leader sequence from RNA4 of alfalfa mosaic virus introduced at the 5' end of the bar gene (35SAMVLbar) or without (35Sbar) it. Plant viral leader sequences have been shown to be translational enhancers. In vitro selections for transformed plants were carried out on a medium containing PPT. Transgenic shoots were recovered at a frequency of 23% with 35Sdebar gene construct and at a frequency of 16% with 35SAMVLbar containing construct. Transformation frequencies were low with 35Sbar construct. Individual transgenics with 35Sdebar and 35SAMVLbar constructs were tested for copy number on both the right and left border flanks of T-DNA by Southern hybridization. Single copy transgenic lines were further analysed for transcript levels of the bar gene by Northern blotting and for protein levels by PAT assays. Wide variation in expression levels were observed, particularly amongst the transgenics containing the 35Sdebar construct. Single copy transgenics were selfed to develop homozygous lines which could be used for the study of resistance to herbicide PPT at the field level and to correlate this protection with expression levels observed through molecular analysis. Herbicide- tolerant lines could be used for testing the possibility of low-till or no-till cultivation of mustard in the rain-fed areas where it is extensively grown.