Mustard flowers contain both male and female organs and the crop is pre-dominantly self-pollinating. Therefore, a pollination control mechanism is required to disallow self-pollination and encourage cross-pollination for hybrid seed production. For this, one of the two parental lines of a hybrid has to be made male sterile so that it receives pollen from the other parent to set seed. Seeds harvested from the male sterile line are hybrid seeds which can be provided to the farmers, who can reap the benefit of higher productivity of the hybrids.
Male sterile lines can be developed by using cytoplasmic male sterility (CMS) through conventional breeding or by genetic engineering using transgenes. A number of CMS systems have been tested in mustard. However, CMS/ restorer systems have been found to be inadequate for large scale hybrid seed production with high purity. CMS systems are either unstable or their restoration to fertility is inadequate. A more versatile hybrid seed production system is based on the use of transgenes – barnaseandbarstar as explained below.
A novel way to developing male sterile (MS) lines through genetic engineering was developed by scientists in Belgium in early 1990s through the use of two genes – barnase and barstar from soil bacterium Bacillus amyloliquefaciens. In nature, bacterium excretes a defense protein called Barnase (a type of ribonuclease) which degrades the RNA of competing bacteria in an ecological niche. To protect itself from Barnase, the bacterium produces another protein called Barstar which tightly binds with Barnase and renders it ineffective. Bacterial genes can only express in plants if these are expressed under plant promoters. Both Barnase and Barstar encoding genes were expressed under a tapetum specific promoter. Tapetum is a layer of cells in the male organs called anthers present in the flower. Tapetum produces metabolites which are essential for the development of mature pollen. In the barnasegene containing lines, the tapetum tissue ablates (dies)as a consequence developing pollen degenerate, providing MS lines. The other parental line, called restorer of fertility (RF) line, contains barstargene that also expresses in the tapetum cells. The MS line receives pollen from the RF line through wind pollination or bee pollination, resulting in the production of hybrid seed that has both the barnase and the barstar genes. When hybrids are grown by the farmer these are fully fertile. Thus the MS/ RF system ensures that the MS line will only produce hybrid seeds by outcrossing with RF lines thereby providing an efficient system of pollination control for production of hybrid seed. The system hereafter is referred to as barnase-barstar system.