- A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques.
- The first genetically modified mouse was created in 1974 by Rudolf Jaenisch and the first plant was produced in 1983.
- Bt cotton is a genetically modified variety of cotton that produce an insecticide.
- Bacillus thuringiensis, was first discovered by Ishiwatari in 1901.
- B.thuringiensis is a gram positive, soil bacterium.
- This bacterium produces a parasporal crystalline proteinous toxin with insecticidal activity.
- Strains of the bacterium B. thuringiensis produce over 200 different Bt toxin, each harmful to different insects.
- Bt toxin are insecticidal to the larvae of moths and butterflies, beetles, cotton bollworms but are also harmful to other form of life.
- The protein produce by bacterium is referred to as insecticidal crystalline protein (ICP). ICPs are among the endotoxins produced by sporulating bacteria and were originally classified as δ-endotoxins.
- The gene coding for Bt toxin has been inserted into cotton as a transgene, causing it to produce this natural insecticide in its tissues.
- The main pests in commercial cotton are lepidopteran larvae, which are killed by the Bt protein in the genetically modified cotton when they eat.
Bt toxin genes
- Several strains of a B. thuringiensis producing a wide range of crystal (cry) proteins have been identified. Further , the structure of cry genes and their corresponding toxin (δ-endotoxin) products have been characterized.
- The cry genes are classified into large number of distinct families (about40) designated as cry 1 cry 40, based on their size and sequence similarities.
- And within each family, there may be sub-families. Thus, the total number of genes producing Bt toxins (cry protein) is more than 100.
- There are difference in the structure of different cry protein, besides certain sequence similarities.
- The molecular weight of cry protein may be either large( ~130 k Da) or small (~70 k Da).they share a common active core of three domains.
Mode of action of cry proteins
- Most of the Bt toxins (cry protein) are active against lepidopteran larvae, while some of them are specific against dipteran and coleopteran insects.
- Bt cotton was created through the addition of genes encoding toxin crystal in the cry group of endotoxin.
- When this parasporal crystal is ingested by the target insect, the protein gets activated within its gut by a combination of alkaline pH (7.5 to 8.5) and proteolytic enzymes. Thus result in the conversion of protoxin into an active toxin with a molecular weight of 68 kDa.
- The dissolved and activated cry molecules bond to cadherin-like protein on cells comprising the brush border molecules. The epithelium of the brush border membrane separates the body cavity from the gut while allowing access for nutrients.
- This result in the formation of ion channels through which there occurs an excessive loss of cellular ATP and allow the flow of potassium.
- As a consequence, cellular metabolism ceases, insects stops feeding and becomes dehydrated and finally dies. The death of such cells creates gaps in the brush border membrane.
Bt toxin as biopesticide
- Preparation of Bt spores or isolated crystals have been used as organic biopesticide for about 50 years. This approach has not met with much success for the following reason-
1.Low persistence and stability (sunlight degrades toxin) of the toxins on the surface of plants.
2.The Bt toxin cannot effectively penetrate into various parts of plants, particularly roots.
3.Cost of production is high.
Bt based genetic transformation of plants
- It has been possible to genetically modify plants by inserting Bt genes and provides pest resistance to these transformed plants. For an effective pest resistance the bacterial gene in transgenic plants must possess high level expression.
- The wild type transgene Bt cry 1A was found to express at a very low levels in transgenic plants. The nucleotide sequence of this gene was modified. With appropriate sequence changes, an enormous increase in the Bt toxin product formation was observed.
Bt cotton has several advantages over non-Bt cotton. The important advantage of Bt are briefly:
- Increases yield of cotton due to effective control of 3 types of bollworms, viz American, Spotted and pink bollworms.
- Insects belonged to lepidoptera (bollworm) are sensitive to crystalline endotoxic protein produced by Bt gene which in turn protects cotton from bollworms.
- Reduction in insecticide use in the cultivation of Bt cotton in which bollworms are major pests.
- Potential reduction in the cost of cultivation ( depending on seed cost versus insecticide costs).
- Bt genes could be expressed in all parts of the plants, including the roots and internal regions of stems and fruits.
- Bt toxin are rapidly degraded in the environment.
Bt cotton has some limitations:
- High cost of Bt cotton seeds as compared to non Bt cotton seeds.
- Effectiveness up to 120 days, after that the toxin producing efficiency of the Bt gene drastically reduces.
- Ineffective against sucking pests like jassids, aphids, whitefly.
- The major limitations of Bt-gene possessing transgene plants is the development of Bt-resistant insects.