Lecture 21

Biotechnology in IPM – genetic engineering – transgenic crops – Constraints in using transgenic crops. Sterile
male technique and gamma radiation in IPM

I. BIOTECHNOLOGY IN IPM
Tools of molecular biology and genetic engineering have provided humankind with
unprecedented power to manipulate and develop novel crop genotypes towards a safe and sustainable
agriculture in the 21st century. Insecticidal proteins present in Bacillus thuringiensis (Bt), which have
shown efficacy as spray formulations in agriculture over the past five decades, have been expressed in
many crop species with positive results. Bt-crop containing resistance to insect pests, particularly
Helicoverpa armigera, has been released for commercial cultivation in the farmers’ fields.
Genetic engineering techniques available for pest management
a. Protease inhibitors - The protease inhibitors gene have been introduced into a variety of different
transgenic plants like apple, pea, potato, rice, tobacco, tomato etc. The cowpea trypsin inhibitor (CpTi)
has been found an ideal candidate for genetic transformation and found to import resistance against
Heliothis, Spodaptera etc.,
b. Bean α-Amylase Inhibitors and Stored Product Pests - The α-amylase inhibitors from some legume
seeds, which are similar to legume lectins in sequence, have been shown to be causative factors in the
resistance of specific varieties of legumes to coleopteran seed weevils. The bean (Phaseolus vulgaris) α-
amylase inhibitor gene was expressed in seeds of transgenic garden pea (Pisum sativum) and other grain
legumes, using a strong seed-specific promoter.
c. Photorhabdus luminescens Insecticidal Proteins - Nematodes of Heterorhabditis species that
contain symbiotic enterobacteria are widely used for small-scale biological control of insect pests.
When nematodes enter an insect host, bacterial cells from the nematode gut are released into the insect
circulatory system. Toxins secreted by the bacteria cause cell death in the insect host, leading to a lethal
septicemia. P. luminescens, the most well-investigated bacterial species of this type, contains a large
number of potentially insecticidal components
d. Cholesterol Oxidase - Bacterial cholesterol oxidase has an insecticidal activity comparable
to Bt toxins, dependent on its enzyme activity, which is thought to promote membrane destabilization.
Expression constructs containing part or all of the coding sequence of the protein, or the coding
sequence fused to a chloroplast-targeting peptide, resulted in production of active enzyme in transgenic
tobacco.
e. Avidin - has a strong insecticidal effect on many insects, although susceptibility varies widely
between different insect species (apparently based on biotin requirements). Expression of avidin in
transgenic maize initially aimed to produce the protein as a high-value product, but maize seed
containing more than 0.1% avidin (of total protein) was fully resistant to larvae of three different
coleopteran storage pests.
f. RNAi - Disrupting gene function by the use of RNAi is a well-established technique in insect
genetics based on delivery by injection into insect cells or tissues. The observation that RNAi could
also be effective in reducing gene expression, measured by mRNA level, when fed to insects (Turner
et al., 2006) has led to two recent articles in which transgenic plants producing double-stranded RNAs
(dsRNAs) are shown to exhibit partial resistance to insect pests.
GENE TRANSFER METHODS - Genetically engineered crops have genes added or removed
using genetic engineering techniques, originally including gene guns,
electroporation, microinjection and agrobacterium. More recently, CRISPR and TALEN offered much
more precise and convenient editing techniques.
1. Gene guns - "shoot"target genes into plant cells. It is the most common method. DNA is bound to
tiny particles of gold or tungsten which are subsequently shot into plant tissue or single plant cells under
high pressure. The accelerated particles penetrate both the cell wall and membranes. The DNA
separates from the metal and is integrated into plant DNA inside the nucleus. Applied successfully for
many cultivated crops.
2. Agrobacterium tumefaciens-mediated transformation is another common technique. Agrobacteria
are natural plant parasites, and their natural ability to transfer genes provides another engineering
method. To create a suitable environment for them, these Agrobacteria insert their genes into plant
hosts, resulting in a proliferation of modified plant cells near the soil level (crown gall). The genetic
information for tumour growth is encoded on a mobile, circular DNA fragment (plasmid). When
Agrobacterium infects a plant, it transfers this T-DNA to a random site in the plant genome. When used
in genetic engineering the bacterial T-DNA is removed from the bacterial plasmid and replaced with
the desired foreign gene. The bacterium is a vector, enabling transportation of foreign genes into plants.
This method works especially well for dicotyledonous plants like potatoes, tomatoes, and tobacco.
Agrobacteria infection is less successful in crops like wheat and maize.
3. Electroporation is used when the plant tissue does not contain cell walls. In this technique, "DNA
enters the plant cells through miniature pores which are temporarily caused by electric pulses."
4. Microinjection directly injects the gene into the DNA.[50]
Transgenic plants
The application of transgenic plants through genetic engineering is the latest concept in IPM.
Transgenic plants have genes inserted into them that are derived from another species. The inserted
genes can come from species within the same kingdom (plant to plant) or between kingdoms (for
example, bacteria to plant). In many cases the inserted DNA has to be modified slightly in order to
correctly and efficiently express in the host organism. Transgenic plants are used to
express proteins like the cry toxins from B. thuringiensis, herbicide resistant
[55]
genes, antibodies and antigens for vaccinations.
These transgenic plants produce inseciticidal or anti-feedant proteins continuously in the plant
under field conditions. Bt endotoxin gene and cowpea protease inhibitor (Cp Ti) genes are the common
genes conferring insect resistance. The genome responsible for  endotoxin is isolated and introduced
into plants and thus the plants with expression of the introduced genes are called transgenic plants.
A. Bt endotoxins - Bt toxins are highly specific and sotomach poisons. Bt toxins are produced from
B.t. var. kystaki (Btk) for Lepidopteran pests and Bt var israeliensis (Bti) for dipteran pests.
In July 1987, a Belgian Biotechnology company (Plant Genetic systems) developed transgenic
plants of tobacco containing  endotoxins against Manduca sexta and Helicoverpa . Monsanta company
– developed transgenic tomato plant against tobacco hornworm M.sexta. Transgenic plants carrying
Bt genes have been produced in tobacco, tomato, potato, cotton, maize, rice, soybean, sugarcane, apple,
peanut, chick pea, and alfalfa with different crystal protein genes. Recently the Mahyco introduced
transgenic cotton into India for cultivation with resistance against boll worms especially Helicoverpa.
Transgenic plants in IPM
➢ Transgenic plants are compatible with all other tactics
➢ Transgenic plants do not need any insecticide application against target pests.
➢ Transgenic plants slow down the development of resistance
➢ Transgenic plants provide protection to these plants parts which are difficult to be treated with
insecticides
➢ No need for continuous monitoring of pests
➢ No environmental pollution or risk, sage to non-target species and human beings.

Constraints in using transgenic crops

The major biosafety concerns falls into these categories:
Bio-safety of human and animal health
1. Risk of toxicity, due to the nature of the product or the changes in the metabolism and the composition
of the organisms resulting from gene transfer.
2. Newer proteins in transgenic crops from the organisms, which have not been consumed as foods,
sometimes has the risk of these proteins becoming allergens.
3. Genes used for antibiotic resistance as selectable markers have also raised concerns regarding the transfer
of such genes to microorganisms and thereby aggravate the health problems due to antibiotic resistance
in the disease causing organisms.
Ecological concerns
1. Gene flow due to cross pollination for the traits involving resistance can result in development of tolerant
or resistant weeds that are difficult to eradicate.
2. GM crops could lead to erosion of biodiversity and pollute gene pools of endangered plant species.
3. Genetic erosion has occurred as the farmers have replaces the use of traditional varieties with
monocultures.
Environmental concerns
 1. Effect of transgenic plants on population dynamics of target and non-target pests, secondary pest
problems, insect sensitivity, evolution of new insect biotypes, environmental influence on gene
expression, development of resistance in insect population, development of resistance to herbicide
2. Gene escape into the environment- accidental cross breeding GMP plants and traditional varieties
through pollen transfer can contaminate the traditional local varities with GMO genes resulting in the
loss of traditional varieties of the farmers.

STERILITY METHOD/ STERILANTS

Sterility method envisages the use of insects to bring down the population. Insects are used against
members of their own species to reduce the populations and hence called as autocidal control. Autocidal
control received siginificance after E.F. Knipling, a USDA scientist in the 1950’s when the population
of screw wormfly Cochliomyia hominvorax, a parasite of cattle was eradicated in Curaca island in
United State.
Principles of Autocidal control
• Flooding a population with sterile males which mate with normal females
• Such mating result in inviable eggs
• With continued sterile male releases the population declines
• The ratio of sterile to normal males increases until virtually no normal males remain
• Population becomes extinct for lack of progeny
Release of sterile males in the ratio of 9:1 of the wild populations of male for successive
generations results in the population reaching zero in F4 generations (This is called male sterile
technique)
Methods of sterilization
A. Ionizing radiation
Electromagenetic radiation such as gamma rays and X rays cause sterilization in insects. At 200-
500 kilorads (k rads) ionizing radiation brings about complete death. At 100 krads ionizing radiation
causes sterilization and subsequent death. At 8-10 krads ionizing radiation causes sterilization
B. Chemosterilants
Chemicals which deprive insect species of their ability to reproduce chemosterilants are
dangerous and carcinogenic or mutagenic.
They are classified into
a. Alkylating agents. E.g. TEPA and Metapa. Tepa 0.025% in a protein hydrolysate trap is
used for sterilizing the Mexican fruit fly.
b. Antimetabolities. E.g Amethopterin and
c. Miscellaneous compounds. E.g. Hempa and Hemel. They are effective against housefly.
The chemosterilants could be applied in traps containing attractants, so that the lured insects
pick up the chemical and sterilized. Housefly, Mosquito, fruit fly, screw worm fly etc. are controlled
by this male sterile technique.