The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L.

Male-sterile lines were generated in oilseed mustard (Brassica juncea) with a cytotoxic gene (barnase) in conjunction with either of two tapetum-speci fic promoters, TA29 and A9. Several transformation vectors based on differe nt promoter and marker gene combinations were developed and tested for thei r efficacy in generating agronomically viable male-sterile lines. Use of st rong constitutive promoters (e.g. CaMV 35S or its double-enhancer variant) to express the marker gene (bar) in barnase constructs generated male-steri le plants at an extremely low frequency with most plants showing abnormalit ies in vegetative morphology, poor female fertility, low seed germination f requencies and/or distortion in segregation ratios of transgenes. Such abno rmalities were considerably reduced on using weaker promoters (e.g. nos) to drive the marker gene (nptII) in barnase constructs and could therefore be attributed to leaky expression of the barnase gene under enhancing effects of strong constitutive promoters. We show that the use of a Spacer DNA fra gment between the barnase gene (driven by a tapetum-specific promoter) and the CaMV 35S promoter-driven bar gene insulates tissue-specific expression of the barnase gene over all developmental stages of transgenic plants and significantly enhances recovery of agronomically viable male-sterile lines. All TA29-barnase male-sterile lines containing the Spacer DNA fragment exh ibited normal morphology, growth and seed set on backcrossing as observed f or wild-type plants. Around 75% of single-copy events tested further also s howed proper segregation of the marker gene/male-sterile phenotype among ba ckcross progeny. Constructs based on the use of Spacer DNA fragments as ins ulators could be successfully used to alleviate limitations associated with transformation of plant systems using cytotoxic genes for development of a gronomically viable male-sterile lines in crop plants and for cell/tissue a blation studies in general.