What is this application related to GM Mustard?

This application relates to Environmental release of Genetically Engineered Mustard (Brassica juncea) hybrid DMH-11 and use of parental events (Varuna bn3.6 and EH2 modbs2.99) for development of new generation hybrids

What is this technology about?

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.

What are the issues of concern for the environment?

Issues of concern include the capability of the GE Plant to escape and potentially introduce the engineered genes into wild populations; the persistence of the gene after the GE Plant has been harvested; the susceptibility of non-target organisms (e.g. insects which are not pests) to the gene product; the stability of the gene; the reduction in the spectrum of other plants including loss of biodiversity; and increased use of chemicals in agriculture.

What safety studies were done for ensuring that GM Mustard is safe and were they done according to relevant guidelines

The following safety studies were done for GM Mustard and they were as per prescribed guidelines developed by Government of India from time to time


Tests Done

Relevant Guidelines

Molecular characterization ·      Gene sequences, constructs and molecular characterization

·      Expression studies of the three inserted genes – bar, barnaseand barstar

·      Cloning, expression, purification and production of three expressed proteins

·   Recombinant DNA Safety Guidelines, 1990


·   Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants, 2008 prepared by Indian Council of Medical Research (ICMR), Ministry of Health and Family Welfare, Government of India


Food safety


·      Equivalence of the Bar, Barnase and Barstar recombinant proteins produced in bacteria with that expressed in GE plants

·      Bioinformatics analysis of the three proteins

·      Pepsin digestibility of the three proteins

·      Heat stability of the three proteins

·      Acute oral toxicity of the three proteins in mice

·      Sub-chronic toxicity of leaves and seeds containing the three proteins in rats

·      Compositional analysis


·   Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants, 2008 prepared by Indian Council of Medical Research (ICMR), Ministry of Health and Family Welfare, Government of India



safety studies

·           Research & Development Phase including primary field trials 2004-2007.

·           BRLI trials for two growing seasons (2010-11, 2011-12)

·           BRLII trials for one growing season (2014-15)

·           Weediness potential and aggressiveness parameters

·           Impact on soil microflora during BRLI and BRLII trials

·           Crossability and pollen flow studies

·           Pollination behavior, pollen morphology and physiology

·   Revised Guidelines for Research in Transgenic Plants, 1998 (DBT)


·   Guidelines for the monitoring of Confined Field Trials of Regulated, Genetically Engineered (GE) Plants, 2008 (DBT & MoEF&CC)



· Protocol for testing at a level of detection (LOD) of 0.01{b9cca8aede1995d450ecbb7cabdd9c806c66ba8f7afe5985dcdbcb8cb5055004}

· Development of ELISA kits for Bar, Barnase and Barstar

·      Tests and Detection methods developed

Were any Public Sector Institutions engaged in conducting safety studies related to this technology?

Yes, the following Public Sector Institutions were engaged in conducting safety studies related to this technology

  • ICAR-Directorate of Rapeseed Mustard Research (DRMR), Bharatpur was engaged for conduct and monitoring of confined field trials (CFTs) and generation of data related to environmental safety studies
  • ICMR-National Institute of Nutrition (NIN), Hyderabad was engaged for undertaking all food safety related studies
  • CSIR-Institute of Microbial Technology (IMTECH), Chandigarh was engaged in conduct of soil micro flora studies.

Are GM foods safe?

Different GM organisms include different genes inserted in different ways. This means that individual GM foods and their safety should be assessed on a case-by-case basis and that it is not possible to make general statements on the safety of all GM foods. GM foods currently available on the international market have passed safety assessments and are not likely to present risks for human health. In addition, no effects on human health have been shown as a result of the consumption of such foods by the general population in the countries where they have been approved.

What kind of GM foods are on the market internationally?

GM crops available on the international market today have been designed using one of the following basic traits: resistance to insect damage; resistance to viral infections; and tolerance towards certain herbicides. GM crops for hybrid seed production for heterosis breeding (e.g. rapeseed/canola) and with higher nutrient content (e.g. soybeans increased oleic acid) have been also studied recently.