This document discusses developing insect resistance in crops using biotechnological approaches. It describes how some plants have natural mechanisms of insect resistance by secreting toxic chemicals. Current strategies to develop insect resistance include using microbial proteins from Bacillus thuringiensis (Bt) toxins, which are inserted into plant genomes using recombinant DNA techniques. The mechanisms of Bt toxins are described, including how they bind to receptors in insect guts and cause cell death. Advantages of Bt crops include reduced need for insecticides and improved pest management. However, there is a risk that insects may develop resistance to Bt toxins over time from repeated exposure.

1. Biotechnological Approach to
Develop Insect Resistance Crops
Md. Nafizur Rahman
Dept. of Genetic Engineering & Biotechnology,
Shahjalal University of Science & Technology, Sylhet.

2. Introduction
• Insect resistance is the properties of a plants to resist the attack of
insects.
• Despite the use of wide array of insecticides, extent of damage seems to
be far reaching implications.
• Insects depends on the parts of plant for survival and also cause damage
to the plant.
• If a plant has insect resistance capability, it can resist the attack of
insects.
• Some plants have their own insect resistance mechanism by secreting
toxic chemicals which resist the insect.
• Another emerging area of study with potential to develop insect
resistance is the relationship between insect behavior and alterations in
the plant’s phytochemical profile.

3. Category and strategies
• Attack by insects in plants belongs to the overall stress tolerance
category.
• There are several types of insect or pest control strategies are currently
under investigation or implementation.
• Microbial Protein-Based Antibiotic Strategies
• Bacillus thuringiensis Toxins
• Vegetative Insecticidal Protein (VIP)
• Photorhabdus luminescens toxins
• Lipid Disrupting Proteins
• Plant Protein-Based Antibiotic Strategies
• Protease inhibitors
• Amylase inhibitors
• Lectins
• Chitinases

4. Overview of insect resistance mechanism in crops
• Plants are under constant threat of attack from pathogens as well as
vertebrate and invertebrate herbivores.
• Insect resistance in crops has long been a major objective in plant
breeding. Yet little on the specific mechanism conferring insect
resistance was understood till now.
• This is largely because insect resistance in crops is a "non-host specific
resistance", meaning that generally certain plant species are not
attractive to given species of insects which leads to immunity.
• In classical breeding, three systems of resistance are of particular
relevance:
• morphological barriers to insects
• the presence of insect-repellent or toxic substances
• toxins that have a repellent or deadly effect

5. Bacillus thuringiensis: A Weapon for Insect Control
• Bacillus thuringiensis, commonly known as Bt, is a bacterium that occurs
naturally in the soil.
• Some strains of Bt produce proteins that kill certain insects with alkaline
digestive tracts.
• When these insects ingest the protein produced by Bt, the function of
their digestive systems is disrupted, producing slow growth and
ultimately death.
• Bt is very selective - different strains of the bacterium kill different
insects and only those insects.
• Strains of Bt are effective against European corn borers and cotton
bollworms (Lepidoptera), Colorado potato beetles (Coleoptera), and
certain flies and mosquitos (Diptera).
• Bt is not harmful to humans, other mammals, birds, fish, or beneficial
insects.

6. i. Identify and Isolate the Bt toxin gene from Bacillus thuringiensis
ii. Insert the gene into a vector for multiplication
iii. Screen the vector for expression of Bt toxin
iv. Isolate the desired Bt toxin gene from the vector and insert into host
v. New host plant contains expressed Bt toxin gene
vi. Bt toxin (Cry protein) is enter the insect by attachment
vii. Solubilisation and proteolytic activation of Cry protein
viii. Binding of Cry proteins to target site into the insect gut
ix. Formation of toxic lesions into the gut which cause septicaemia
Steps of making Bt Crops with mechanism

7. Mechanism of Action of Bt Toxin
• Bacillus thuringiensis (Bt) produces crystals of endotoxin (Cry protein or
delta toxin) - toxic to insect mainly in their larval stage, thus they act as
insecticides.
• Insects stop feeding within two hours of a first bite and, if enough toxin
is eaten, insect may die within two or three days.
• Bt crystals, sometimes referred as insecticidal crystal proteins (ICP), are
protein crystals formed during sporulation in some Bt strains coded by
cry genes.
• It is an important biological insect control agent.
• Recombinant DNA technology makes it possible to locate the gene that
produces Bt proteins lethal to insects and transfer the gene into crop
plants.

8. Mechanism of Action of Bt Toxin

9. • When insects attack and eat the cotton plant, the Cry toxins are
dissolved. This is made possible due to the high pH level of the insect’s
stomach.
• The dissolved and activated Cry molecules bound to cadherin-like
proteins on cells comprising the brush border molecules.
• The Cry toxin molecules attach themselves to specific locations on the
cadherin-like proteins present on the epithelial cells of the midgut and
ion channels are formed which allow the flow of potassium.
• Due to the formation of Cry ion channels sufficient regulation of
potassium ions is lost and results in the death of epithelial cells.
• The death of such cells creates gaps in the brush border membrane. The
gaps then allow bacteria and (Bt) spores to enter the body cavity
resulting in the death of the organism.
Mechanism of Action of Bt Toxin

10. Mechanism of Action of Bt Toxin

11. Advantages of Bt Crops
• Improved pest management: Insect-protected Bt crops provide the farmer
with season-long protection against several damaging insect pests, and
reduce or eliminate the need for insecticide sprays.
• Reduction in insecticide use: A study by the US Department of Agriculture
reported that 8.2 million pounds of pesticide active ingredients were
eliminated by farmers who planted Bt crops in 1998.
• Greater net return: Lower input costs often contribute to a higher net return
compared to conventional crops. Bt-cotton farmers in the US earned an
incremental $99 million as a result of decreased pesticide costs and/or
increased yields.
• Improved conditions for non-target organisms: Since Bt crops are able to
defend themselves against pests, the use of chemical insecticides is
significantly reduced, thereby encouraging the proliferation of beneficial
organisms.
• Less mycotoxin in corn: Aside from being effective against insect pests, Bt-
crops have lower incidences of opportunistic microbial pathogens. This
fungus produces mycotoxins that can be deadly to livestock and also cause
cancer in humans.

12. The Insect Resistance Problem
• Transgenic Bt plants like corn, potatoes, and cotton have a possible
problem - what if insects build up a resistance to the lethal proteins?
• The crop defence might be a chemical or biological agent, a gene already
in the crop species and transferred to commercial plants by conventional
plant breeding methods, or a gene introduced by recombinant DNA
technology.
• Because more than 500 insects and mites already have acquired
resistance to a number of insecticides, there is concern that similar
resistance to Bt toxins could develop (McGaughey and Whalon).
• Several major pests, including the tobacco budworm, Colorado potato
beetle, Indianmeal moth, and diamondback moth, have demonstrated
the ability to adapt to Bt in the laboratory.
• It has been reported that the diamondback moth evolved high levels of
resistance in the field as a result of repeated use of Bt.

13. Reference
• N Carozzi, M Koziel. Advances in Insect Control: The Role of Transgenic Plants. London:
Taylor and Francis, 1997.
• Insect resistance in crops: A case study of Bacillus thuringiensis (Bt) and its transfer to
developing countries- Anatole F. Krattiger, Executive Director of ISAAA.
• Wrage, Karol. "Bt's: Flagship for 21st Century BioControls: Industry Execs, University
Researchers Say Future Promising and Profitable." Biotech Reporter. Vol 10, No. 5, May
1993. p. 8-9.
• Marois, James J.; Grieshop, James I.; and Butler, L. J. (Bees). "Environmental Risks and
Benefits of Agricultural Biotechnology." In Agricultural Biotechnology, Issues and Choices,
p. 67-79. Edited by Bill R. Baumgardt and Marshall A. Martin. West Lafayette, Indiana:
Purdue University Agricultural Experiment Station, 1991.
• McGaughey, William H. and Whalon, Mark E. "Managing Insect Resistance to Bacillus
thuringiensis Toxins." Science. Vol. 258, November 27, 1992. p. 1451-1455.
• Monsanto. "B.T. - The Inside Story"; "Insect-Reslistant Cotton Replaces Insecticides"; "Self
Defense for Potatoes" - News releases. The Agricultural Group, Public Affairs Department,
700 Chesterfield Parkway North, St. Louis, Missouri.

14. Thanks to All