C Kameswara Rao

Foundation for Biotechnology Awareness and Education, Bangalore, India

For reasons inherent in the reproductive biology, the highly inbred Brassicas are difficult to hybridize, using conventional breeding techniques.  The hybrids suffer from low vigour making them unsuitable for commercial cultivation.   Vigour can be greatly enhanced in the progeny from crosses of genetically distinct parents, to outperform the parental lines.  This phenomenon called ‘hybrid vigour’ has been a great boon in plant breeding.  


For a time, plant breeders tried to use male sterility, the absence of functional pollen while the female gametes were normal, to produce hybrids in highly inbred crops. 

Naturally occurring gene controlled male sterility occurs only sporadically among crop plants, as they were always selected for high levels of fertility.   Nevertheless, natural male sterile plants were exploited in hybrid seed production in such crops as cotton, tomato, sunflower, cucurbits, tobacco, rice, wheat, barely, maize, sorghum and pearl millet.

In hybrid production, several lines of male sterile (female fertile) plants are planted alternately with one or two lines of male fertile (pollinator) plants, which also have fertile female gametes.    The undesirable gene combinations formed from the female gametes of the male fertile plants, have to be identified and removed.  Such a procedure is difficult to be performed manually even in experimental situations and impossible in cultivated crop fields. 

Decades of research on canola and mustard led to the identification of very few male sterile lines and imperfect restorer female lines, making the natural system commercially unviable.   Robert Goldberg’s team developed the barnase/barstar genetic system over a decade ago to overcome this handicap.   The objective is to produce hybrids to exploit hybrid vigour ensuring higher crop performance.


Separate male sterile (MS) and fertility restorer (RF) lines developed through GE are used to emulate the natural phenomenon of hybrid vigour. Crosses of the MS line with the RF line ensure the production of fully fertile hybrids, which are employed in agricultural production.

Barnase-barstar genes:

The barnase gene, from the bacterium Bacillus amyloliquefaciens, encodes the enzyme barnase (ribonuclease), which is produced at a specific stage early in the development of the anthers (the pollen bearing parts of the flower) and in a specific cell layer (called tapetum) of the anthers.   Barnase prevents pollen production, conferring male sterility.

The barstar gene, also from Bacillus amyloliquefaciens, encodes an enzyme that inhibits barnase.  Expression of the barstar gene is also restricted to the anthers. 

The hybrid plants derived from crosses of MS and RF lines are fully fertile, as the expression of the barnase the barstar gene inhibits gene. 

Elimination of undesirable hybrids:

The bar gene from the bacterium Streptomyces hygroscopicus and pat gene from the bacterium Streptomyces viridichromogenes, encode for the enzyme phosphinothricin acetyl transferase, that detoxifies glufosinate ammonium and confers tolerance to herbicides with this active ingredient.   Genes linked to other herbicides may also be used. 

The herbicide tolerance trait is used as a selection tool for the barnase-barstar breeding system, to eliminate unwanted hybrid genotypes, by spraying an appropriate herbicide.   This trait also enables to control weeds in the canola crop, in conjunction with other measures.  

The bar and pat genes are good markers useful to detect gene flow, by spraying an herbicide.   Technology now exists to remove herbicide resistance genes at the time mass production of seed for cultivation purposes, if this was necessary for political or public concern reasons, though not for scientific reasons.

Selectable marker:


The nptII gene from the bacterium Escherichia coli confers resistance to the antibiotics neomycin and kanamycin. Such antibiotic resistance traits are used as selectable markers in the initial laboratory stages to screen genetically modified plants.  Now non-antibiotic selectable markers are employed.

The barnase/barstar technology:

The whole of the process of male sterility and selective removal of undesired hybrids from the male fertile plants, is performed elegantly by the barnase/barstar gene system, in which herbicide resistance is linked with male fertility, so that the herbicide will kill the male fertile lines, leaving the seed producing male sterile plants unharmed.   The system is used only to develop novel hybrids and the farmer is provided with highly viable seed of uniform quality that produces fully (male and female) fertile plants. 

The whole set of gene systems used in canola and mustard are a complicated but an ingenious development in rDNA technology that can confer nuclear male sterility to self-pollinating plants in a stable manner to produce hybrids using a female restorer line.   It helped to produce improved hybrids of such crucifer crops as canola and mustard, which is impossible without this gene system.  The barnase/barstar gene system is meant to produce hybrids, and not herbicide resistant/tolerant crops, as alleged by come activists.  

The World’s experience with GE Canola:


Aventis has successfully introduced a GE canola hybrid, using the barnase/barstar gene system in 1996.  Since then, high yielding GE hybrid canola cultivation has expanded to over a couple of million hectares in North America and Australia .  Regulatory agencies in such countries as Canada , USA , Mexico , Europe, Australia and Japan have approved consumption of this GE canola.   The health and environmental safety questions about the technology have been settled beyond any reasonable doubt.

Roundup Ready is a GE canola resistant to Monsanto’s herbicide Roundup and AgroEvo’s GE canola is resistant to their herbicide Liberty.

The same barnase/barstar gene system is being used in India to produce mustard hybrids.

December 2, 2006