It is estimated that about 15% of the world crops yield is lost due to insects. The damage to crops is mainly caused by insect larvae and to some extent by adult insects. Majority of insects that damage crops belong to the following orders:
• Lepidoptera (bollworm)
• Coleoptera (beetles)
• Orthoptera (grasshoppers)
• Homoptera (aphids)
Initially, only the chemical pesticides are used to control the insect pests. But there are various drawbacks of using these pesticides, which includes:
1) About 95% of the pesticides sprayed is washed away from the plant surface and accumulate in the soil.
2) It is very difficult to deliver pesticides to the vulnerable parts of plants such as roots, stem
3) Chemical pesticides cause environmental pollution as not easily degraded.
4) These are toxic to humans and animals.
So keeping all these facts in view, scientists are working in search of new biotech alternatives to chemical pesticides, thereby providing insect resistance to crop plants.
Resistance gene from microorganism
Bacillus thuringiensis (Bt) toxin: - B. thuringiensis is a gram-positive soil bacterium, which produces a parasporal crystalline proteinous toxin with insecticidal activity. This protein is called as insecticidal crystalline protein (ICP). ICP is the endotoxin produced by sporulating bacteria and was called Î´- endotoxin.
Bt toxin genes:- Bt toxin genes were also called cry genes because they produce the crystal protein. Cry genes were classified into large no. of distinct families (about 40) designated as cry I, cry 2, cry 3,cry 4..............cry 40 based on their similar size and sequence and within each family, there may be sub- families. Thus the total number of genes producing Bt toxins (cry proteins) are more than 100. Despite the difference in the cry protein, they share a common active core of 3 domains.
Structure of cry protein
Cry protein has 3 domains in 3-D structure: - first domain A is made up of 6Î± helices of approximately 250 amino acids. Second domain B called as receptor binding domain is made up of Î²-sheets of 200 amino acids, while the third domain C is made up of Î² sandwich of 150 amino acids.
Mode of action of cry proteins
Most of the Bt toxins are active against Lepidopteron larvae while some of them are specific against Dipterans and Coleopteran insects. Protoxin of cry 1 protein has molecular weight of 130KDa, when this parasporal crystal protein is ingested by the target insect; the protoxin gets activated within the gut of the insect by the combination of alkaline pH (7.5-8.5) and proteolytic enzymes. This results in the conversion of protoxin into an active toxin with a molecular weight of 68 KDa.
Now, Domain A is inserted into the epithelial cell membrane of insect gut, resulting in the formation of pores (around 6 subunit of this domain insert). Domain C protects the toxin from protease thus helps to maintain the structural integrity of the protein. Domain B helps in the binding of epithelium membrane of mid gut. Through the pores, there occurs loss of cellular ATP. This result in disturbance of osmotic equilibrium, cellular metabolism ceases, insect stop feeding and become dehydrated and finally dies. Bt toxin is not toxic to animals and humans.
Bt toxin as biopesticide: Preparation of Bt spores or isolated crystals have been used as organic biopesticides. But this approach has several limitations.
1) Bt toxin cannot effectively penetrate into various parts of plants like roots.
2) Cost production is high
3) Low persistence and stability.
Bt based genetic transformation of plants
Now it has been possible to genetically modify plants by inserting Bt genes and provide pest resistance to these transformed plants. For effective pest resistance, bacterial gene must possess high level of expression. For this cry 1A and cry 3A were expressed under the control of CaMV35 S promoter or Agrobacterium mediated T-DNA transfer in tobacco, tomato and potato plants. But genes show very low expression. So, for high level of expression, nucleotide sequence of this gene was modified: - 1) G + C content altered 2) Several polyadenylation signals removed, 3) ATTTA sequences deleted.
With these appropriate changes, an enormous increase in the Bt toxin production was observed. Transgenic Bt crops were given approval for commercial planting in USA. Bt Maize and cotton are used and controversy is still there for brinjal.
Advantages of Bt genes
1) toxic protein is produced within the plants and hence environmental friendly.
2) Bt genes could be expressed in all parts of the plants including roots.
3) Bt toxin is rapidly degraded in the environment.
Problems of insect resistance Bt crops
1) Major limitation is the development of Bt resistant insects. Bt toxin is a protein and membrane receptor through which the toxin mediates its effect is also a protein. So it is possible that certain mutations in the insect gene coding for receptor protein may reduce toxin binding making it ineffective.
Possible approaches to avoid this
1) Rotating Bt crops with non Bt crops
2) Introduction of two different Bt genes for the same target
3) Development of transgenic plants with two different types of insect resistance genes. For example use of Bt gene and proteinase inhibitor gene.
About Author / Additional Info: