Weeds are unwanted and useless plants that grow along with the crop plants. Weeds compete with the crops for light and nutrients besides harboring various pathogens. It is estimated that the world's crop yield is reduced by 10-15% due to the presence of weeds. So, herbicides are used to kill the weeds. Ideal herbicide is expected to possess the following characteristics:-
1. Capable of killing weeds without affecting crops.
2. Non-toxic to animals and microorganisms.
3. Rapidly translocated within the target plant.
4. Rapidly degraded in the soil.
None of the commercially available herbicides fulfils all the above criteria. Major limitation is that they cannot discriminate weeds from the crop plants. So, scientists are working with different strategies to develop herbicide resistant plants. Few strategies are:-
1. Over expression of target protein: - Target protein on which the herbicide acts can be produced in large quantities so that the effect of herbicide becomes insignificant. This can be achieved by integrating multiple copies of the gene or by using strong promoter.
2. Improved plant detoxification: - Plants also possess natural defense system against toxic compounds (herbicide). Detoxification involves the conversion of toxic herbicide to non-toxic or less toxic compound. By enhancing the plant detoxification system, effect of herbicide can be reduced.
3. Detoxification of herbicide by using forgein gene: - By introducing a foreign gene into crop plant, the herbicide can be effectively detoxified.
4. Mutation of target gene: - Target protein which is being affected by the herbicide can be suitably modified. Changed protein should be able to perform normal functions but is resistant to inhibition by herbicide. Once the gene encoding such protein is identified, it can be introduced into plant genome to develop herbicide resistance.
Glyphosate is a glycine derivative and acts as a broad spectrum herbicide and is effective against 76 of the world's worst 78 weeds. It is less toxic to animal and is rapidly degraded by microorganisms. Also, it has short half life. Monsanto markets glyphosate as Round Up.
Mechanism of action:-
Glyphosate is rapidly transported to the growing points of plants. It is capable of killing the plants even at low concentration. It acts as a competitive inhibitor of enzyme 5-enoyl-pyruvylshikimate 3'-phosphate synthase (EPSPS). This is a key enzyme in shikimic acid pathway that results in the formation of aromatic amino acids (tryptophan, tyrosine and phenylalanine), phenols and certain secondary metabolite. Enzyme EPSPS catalyses the synthesis of 5' enoylpyruvylshikimate-3-phosphate from shikimate-3-phosphate and phosphoenoylpyruvate. Glyphosate have structural similarity with the substrate phosphoenol pyruvate. Consequently, glyphosate binds more tightly with EPSPS and blocks the normal shikimic acid pathway. Thus, herbicide inhibits the biosynthesis of aromatic amino acid and other important products. This results in inhibition of protein synthesis due to lack of aromatic amino acid. As a result, cell division and plant growth are blocked, resulting in the death of plants. Glyphosate is also toxic to microorganisms as they also possess shikimate pathway, but non-toxic to animals including humans.
Strategies for glyphosate resistance:-
1. Over expression of plant EPSPS gene:- This was facilitated by isolation of Petunia complementary DNA (cDNA) from glyphosate tolerant tissue culture and stepwise selection of petunia cells capable of growing in the presence of increasing amount of glyphosate that led to the isolation of culture in which the level of EPSPS enzyme were much higher than normal. This was not because of increased expression of EPSPS gene but result of gene amplification. The tolerance was due to increased amount of EPSPS enzyme.
2. Use of mutant EPSPS gene: - An EPSPS mutant gene aroA that conferred resistance to glyphosate was first detected in bacterium salmonella typhimurium. It was found that single base substitution in aroA (C to T) resulted in change of an amino acid from proline to serine in EPSPS. This modified enzyme cannot bind to glyphosate. This gene was used to transformed tobacco plant but a little success was met. It was later found that shikimate pathway occur in chloroplast. Years later, mutant EPSPS gene was tagged with chloroplast specific transit peptide sequence. Now the enzyme freely entered the chloroplast. Later, a gene from herbicide resistant A. tumifaciens strain encoding an EPSPS enzyme was isolated. This gene in conjugation with an enhanced CaMV35S promoter and a chloroplast transit peptide sequence from Arabidopsis or Petunia is incorporated into plants. These plants shows high resistant to glyphosate.
3. Detoxification of glyphosate by heterologous genes: - In soil microbes glyphosate can be degraded by cleavage of C-N bond, catalyzed by an oxidoreductase to form aminomethylphosphonic acid (AMPA) and glyoxylate. A gene encoding enzyme glyphosate oxidase (GOX) has been isolated from a soil microorganism, Ochrobactrum anthropi strain LBAA and modified by addition of transit peptide. Transgenic crops such as oil seed rape transformed with this gene shows very good glyphosate tolerance in field.
4. Use of combined strategy: - More efficient resistance of plants against glyphosate can be provided by employing a combined strategy. Thus both the resistant Agro bacterium CP4 EPSPS gene and GOX gene are introduced into plants. In addition to enhanced glyphosate tolerance, this approach avoids the accumulation of herbicide in the tolerant plant because glyphosate is broken down into harmless products. Canola (a variety of oilseed rape grown in USA) crop is transformed using this approach.
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