NEET Biology Biotechnology and its Applications Notes

Biotechnology and its applications is a high-yield chapter for NEET Biology. These notes on Chapter 12 cover biotechnological applications in agriculture such as Bt cotton, herbicide-tolerant and pest-resistant crops and Flavr Savr tomatoes, the therapeutic applications including human insulin, interferons, hirudin and vaccines, medical diagnosis with PCR and ELISA, gene therapy for SCID, transgenic animals, DNA fingerprinting, and the ethical issues, patents and biological warfare concerns linked with genetic engineering.

Terms and Definitions

1. RNA interference
2. Bt toxin
3. Molecular farming
4. Xenotransplantation
5. Molecular probe
6. Biopiracy
7. Biopatent
8. Hirudin
9. Gene therapy

The chapter also covers transgenic animals and their useful applications, transgenic plants and their useful characters, and DNA fingerprinting.

The applications of biotechnology include therapeutics, diagnostics, genetically modified crops for agriculture, transgenic animals, processed food, bioremediation, waste treatment and energy production.

BIOTECHNOLOGICAL APPLICATIONS IN AGRICULTURE

Traditional methods of crop improvement, such as selection and hybridisation, involve the whole genome. These methods are tedious and time consuming, and the results are not always satisfactory. The latest biotechnological methods involve the transfer of one or more beneficial genes from one plant to another.

Plants, bacteria, fungi and animals whose genes have been altered are called Genetically Modified Organisms (GMO).

Genetic modifications have:
- made crops more tolerant to abiotic stresses (cold, drought, salt and heat);
- reduced reliance on chemical pesticides (pest-resistant crops);
- helped to reduce post-harvest losses;
- increased the efficiency of mineral usage by plants, to prevent early exhaustion of soil fertility;
- enhanced the nutritional value of food, for example vitamin A enriched golden rice.

A genetically modified crop, or transgenic crop, is prepared by transferring genes from one plant to another. Such genes are known as transgenes, and the plant in which foreign genes are introduced is known as a transgenic crop.

Biotechnological techniques improve crop yield in the following ways:

Increase in photosynthetic yield: Genetic manipulation of RUBISCO (ribulose 1,5-bisphosphate carboxylase/oxygenase), an important enzyme of photosynthesis, is used to increase crop yield. RUBISCO acts as both a carboxylase and an oxygenase. About 25% of the fixed CO₂ is photorespired. A genetic alteration in the RUBISCO gene inhibits its oxygenase activity. One such gene controlling RUBISCO has been transferred from the pea plant to the tobacco plant to increase the photosynthetic process and crop yield.

Herbicide tolerance: Herbicides are chemicals that kill weeds. When these chemicals are sprayed in the field, they not only kill the weeds but also damage the crop plants. By biotechnological techniques, transgenic crop plants have been developed which are resistant to herbicides and are not damaged on exposure to them. Some herbicide-resistant crops produced by genetic engineering are tomato, potato, tobacco, soyabean, cotton, corn, oilseed rape, sugarbeet and petunia.

Herbicide tolerance in these plants can be achieved by:
(a) overproduction of those enzymes which are inactivated by herbicides;
(b) inducing mutations that result in the production of enzymes which are less sensitive to the herbicide;
(c) introducing enzymes which cause detoxification of herbicides. Several such enzymes have been identified in plants as well as in microbes and transferred into crop plants to produce herbicide-resistant transgenic plants.

Pest resistance: Bt cotton is the first genetically modified crop of the country. It is a pest-resistant plant which resists the attack of Lepidopteran insects that cause the bollworm disease in cotton.

Bt toxin is produced biologically by the bacterium Bacillus thuringiensis (Bt). This toxin is an insecticidal protein (Cry protein) produced in bacteria during a particular phase of growth.
- The inactive protein (protoxin) is converted into the active form of the toxin in the alkaline pH of the insect gut, which solubilises the protein crystals.
- The activated toxin binds to the surface of the midgut epithelial cells and creates pores that cause cell swelling and lysis, and eventually the death of the insect.
- The Bt toxin gene causes the death of insect larvae by cell lysis and swelling of the epithelium of the midgut.

Bt toxin is coded by genes called CRY genes, which are of different types, such as cry IAc, cry IIAb, cry IAb and cry IIAc. These Bt toxin genes are isolated from the bacteria and incorporated into several crop plants, such as cotton, which has Lepidopteran insects as pests.

The choice of gene depends on the targeted crop and insect species, as most Bt toxins are insect-group specific.
- cry IAc and cry IIAb control the cotton bollworm.
- cry IAb and cry IIAc control the corn borer.

A pest-resistant plant crop is less susceptible to pest damage when compared with other crops.

Nematodes: Nematodes are a group of organisms which parasitise a large number of plants and animals, including human beings. One common nematode, Meloidogyne incognitia, infects the roots of tobacco plants and causes a reduction in yield. Prevention of parasitism of tobacco roots by this nematode is done by RNA interference (RNAi).

RNA interference (RNAi): RNAi is a method of cellular defence in all eukaryotes. It is a system within living cells that helps to control the activity of specific genes. This method involves the silencing of mRNA by complementary double-stranded RNA (dsRNA), which prevents translation of the target gene or mRNA. The source of dsRNA is a retrovirus (having an RNA genome) or transposons (mobile genetic material).

Using Agrobacterium as a vector, nematode-specific DNA is introduced into the host plant, which produces dsRNA. The dsRNA initiates RNAi and silences the specific mRNA in the nematode. Thus, the parasite cannot survive in a transgenic host expressing the specific dsRNA that acts as RNAi.

Flavr Savr tomato: The genetically modified tomato Flavr Savr produces much less of the cell wall degrading enzyme polygalacturonase, which is responsible for fruit ripening. Therefore, the fruits of this variety have a much longer and more favourable shelf life.

Miscellaneous uses: Transgenic/genetically engineered plants can also:
- produce pharmaceutically important compounds such as human insulin, interferons and hormones;
- synthesise antibodies and vaccines in fruits;
- bear flowers with new colours and long life, for better ornamental purposes.

Examples of transgenic plants

- Golden rice: Rich in vitamin A (beta-carotene). The rice grains are golden-yellow in colour, so it is called golden rice. It contains the beta-carotene gene from the daffodil plant and also genes from some bacteria. Golden rice will prevent child blindness caused by the deficiency of vitamin A.
- Wheat: Resistant against the herbicide.
- Brassica napus: Transgenic plants are male sterile, because the transferred gene prevented normal pollen development. In addition, a gene encoding hirudin (a protein that prevents blood clotting) is synthesised chemically and transferred into Brassica napus. This gene starts synthesising hirudin, which accumulates in the seeds. The hirudin is then extracted and purified for use in medicine.
- Tomato: Resistant tomatoes which show delayed ripening have been produced by using a gene for ACC deaminase. These transgenic tomatoes are called Flavr Savr tomatoes. They carry an artificial gene that gets transcribed into an antisense RNA. This inhibits ethylene production, so ripening of tomatoes is delayed, preventing rotting and spoilage.
- Potato: Starch content has been increased by using a bacterial ADP glucose pyrophosphorylase gene.
- Bt cotton: Transgenic cotton carrying the Bt toxin gene, isolated from Bacillus thuringiensis, is resistant against bollworms.
- Tobacco: A foreign gene has been introduced in tobacco to show resistance against pests.

THERAPEUTIC APPLICATIONS

Many pharmaceutical products have been produced by using DNA technology. These products are mostly proteins. The gene for a desired protein is transferred into a bacterium, yeast or another kind of cell which is easy to grow in culture. Such host cells produce large quantities of the protein in a short time. Thus, proteins that naturally occur in minute amounts can be obtained in large quantities.

Hormones: Mammalian hormones were among the first products prepared in bacteria by r-DNA technology. Human insulin and human growth hormone are the earliest examples.

Insulin production (connecting concepts): Human insulin consists of 51 amino acids arranged in two polypeptide chains. Chain A has 21 amino acids, and chain B has 30 amino acids. Insulin is a hormone used by diabetic patients. It regulates sugar metabolism, and an insufficiency or lack of insulin leads to a high blood sugar level.

The gene for insulin is introduced into bacteria, and these genetically engineered bacteria produce insulin in bulk, as they express the gene for insulin along with their genome. Insulin is synthesised first as a prohormone, an immature form with two chains, A and B, joined by a C-peptide. Removal of the C-peptide makes it mature.

Insulin chains A and B are produced separately in the plasmid of E. coli, extracted and combined by creating disulphide bonds to make human insulin (the active form), called humulin.

Banting and Best (1921) isolated insulin from the pancreas of a dog and demonstrated its efficiency in curing diabetes in humans. Earlier, insulin was extracted from the pancreas of cattle and pigs in limited quantity.

[Figure: Fig. 12.1, Maturation of pro-insulin into insulin after removal of the C-peptide.]

[Figure: Fig. 12.2, Steps involved in gene transfer for the production of human insulin. A plasmid and a human cell containing the insulin gene are cut by a restriction enzyme; the insulin gene is inserted into the cut plasmid with sticky ends, joined by DNA ligase to form recombinant DNA; the plasmid-free E. coli is transformed with the recombinant DNA; the engineered bacteria multiply to form clones in a fermenter in a containing medium; and the bacteria produce recombinant insulin.]

Human growth hormone (HGH): HGH is useful in hypopituitarism, which causes a form of dwarfism due to undersecretion of HGH by the anterior pituitary. HGH may also have other uses, such as healing injuries.

Milch cows are injected with bovine growth hormone (BGH), formed in genetically engineered E. coli, to raise milk production. BGH also improves beef yield in cattle.

Steroids: Steroids are complex lipids of high molecular weight found in both plants and animals. It was observed that the fungus Rhizopus stolonifer could bring about hydroxylation of steroids. Such microbial transformations of naturally available steroids are applied in the synthesis of chemotherapeutically important steroids.

Several microorganisms, such as fungi, bacteria, actinomycetes, yeasts, protozoa and algae, are known to transform steroids. During transformation, the microorganisms specifically add hydroxyl groups to almost all of the carbon atoms of the steroid ring. Some microorganisms modify steroids by removing hydrogens from specific carbons. The best example of microbial transformation of steroids is the commercial conversion of cortexolone to prednisolone.

Cholesterol is one of the most important steroids in animals and humans. It is an essential component of animal cell membranes. Cholesterol acts as the starting point for the synthesis of many steroid hormones of the body, including the male sex hormone testosterone and the female sex hormones oestrogen and progesterone.

Uses of steroids:
(i) Steroid hormones regulate metabolism in the animal or human body.
(ii) Cortisone and its derivatives (prednisone and prednisolone) are effective in the treatment of rheumatoid arthritis.
(iii) Oestrogens and progesterones are used in the preparation of oral contraceptives (birth-control pills).
(iv) Steroid treatment is given to suppress immune responses in patients with autoimmune diseases or persons who have had organ transplants.
(v) Prednisolone is used as an anti-inflammatory drug.

Antisense nucleic acids: Single-stranded molecules of DNA or RNA have been prepared that base-pair with mRNA molecules and block their translation. Inactivation of the specific mRNA involved in viral replication, or in the transformation of cells into a cancerous state, could check the spread of the disease.

Recombinant proteins: A recombinant protein mimics the receptor protein that HIV binds to while entering white blood cells. HIV binds to this protein molecule and fails to enter the white blood cell. Recombinant DNA technology can also be used to produce large amounts of a specific protein molecule normally present on the surface of a pathogen.

Vaccines: Vaccines are either attenuated (live but weak) or dead (inert) agents of a disease which, when administered to a healthy person, provide temporary or permanent immunity to that particular disease.

Recently, many new vaccines of immense importance have been produced with the help of genetic engineering. These are called second-generation vaccines. They are more uniform in quality and produce fewer side effects than first-generation vaccines produced by conventional methods. Examples of second-generation vaccines are those produced for hepatitis B and herpes virus.

Interferons (IFNs): Interferons are a class of antiviral proteins that function as immune regulators. They are produced by infected cells in response to viral infection. These proteins are produced by most body cells on exposure to viruses. They diffuse to neighbouring cells and trigger a reaction that neutralises the particular virus which induced their formation, and thus prevent viral infection. If these proteins are isolated and introduced into other cells, they develop antiviral properties.

Nowadays interferons are produced in large amounts by recombinant DNA technology. Interferons (particularly IFN-α) are used on a significant scale for the treatment of hepatitis B. They are also being tested for the treatment of cancer and other viral diseases, including AIDS.

Hirudin: Hirudin is a protein that stops blood clotting. The gene encoding hirudin was chemically synthesised, and then transferred into Brassica napus, where hirudin accumulates in the seeds. The hirudin is purified and used as a medicine.

Molecular farming: Mass production of drugs and proteins in transgenic animals or crops, which are used as bioreactors, is called molecular farming or molecular pharming. By introducing useful genes, plants can serve as bioreactors to produce compounds such as amino acids, proteins, vitamins, pharmaceuticals, drugs and enzymes.

MEDICAL DIAGNOSIS OF DISEASES

Some of the techniques used in molecular diagnosis are recombinant DNA technology, PCR and ELISA.

Infectious diseases can be diagnosed using a probe consisting of a short segment of ds DNA attached to a fluorescent marker. These are used to identify food poisoning (Salmonella), pus-forming bacteria (Staphylococcus), the hepatitis virus and the HIV of AIDS, using ELISA.

PCR (polymerase chain reaction) is a gene amplification technique that has found widespread use in medicine and molecular biology. The PCR assay was developed in 1985, and one of its earliest and most important clinical applications has been the diagnosis of HIV infection. PCR is used to detect HIV in suspected AIDS patients by amplifying the defective genes and observing them along with the normal ones.

ELISA (enzyme-linked immunosorbent assay) is a laboratory test based on antigen-antibody interaction. It quickly determines antigen or antibody levels in blood or other fluids.

Molecular probe: A single-stranded DNA or RNA attached to a radioactive molecule is called a molecular probe. Molecular probes are used for the detection of genes by complementary hybridisation in cloned cells, followed by autoradiography.

GENE THERAPY

Gene therapy is the alteration of specific genes to modify the patient's genotype for the treatment of hereditary diseases such as SCID, haemophilia and phenylketonuria. During gene therapy, DNA that codes for specific genes is delivered to individual cells in the body.

Gene therapy is used in many ways, for example to:
- replace a missing or defective gene;
- deliver genes that speed the destruction of cancer cells;
- supply genes that cause cancer cells to revert back to normal cells;
- deliver bacterial or viral genes as a form of vaccination;
- provide genes that promote or impede the growth of new tissue;
- deliver genes that stimulate the healing of damaged tissues.

A gene cannot be directly inserted into a person's cell. It must be delivered to the cell using a carrier, or vector. The vectors most commonly used in gene therapy are viruses. The first clinical gene therapy was given to a 4-year-old in 1990 for ADA deficiency.

A patient with severe combined immunodeficiency disease (SCID) has a defective gene for the enzyme adenosine deaminase (ADA). Such a patient does not have T lymphocytes and cannot mount an immune response against pathogens. In gene therapy:
- white blood cells are extracted from the bone marrow of the patient and cultured;
- a good copy of the human gene encoding ADA is introduced into them by a retrovirus used as a vector;
- this leads to white blood cells with a good copy of the gene, in addition to the existing faulty copy;
- the treated cells are reinjected into the patient's bone marrow, where they play a normal role in the immune system.

[Figure: Fig. 12.3, An example of gene therapy. (1) Normal allele inserted into retrovirus. (2) Retrovirus infects lymphocyte extracted from the bone marrow of the patient and cultured. (3) Retrovirus makes a DNA copy of its RNA; this DNA carrying the normal allele inserts into the chromosome of the host cell. (4) Engineered cells injected into the patient's bone marrow. A patient of SCID starts developing an immune response after receiving a normal functioning gene.]

TRANSGENIC ANIMALS

Animals which carry foreign genes are called transgenic animals. They receive foreign genes to produce highly valuable material. Foreign genes that alter the host cell genetically are called transgenes.

Introducing useful genes into animals has the following benefits:
(i) increased milk production in cattle;
(ii) increased growth rate of useful animals;
(iii) large-scale production of valuable proteins in the milk, urine and blood of beneficial animals, making use of transgenic animals as bioreactors for molecular farming;
(iv) improvement of wool production through transgenic sheep.

In 1997, the first transgenic cow, 'Rosie', produced human protein-enriched milk (2.4 g per litre). The milk contained human alpha-lactalbumin and was nutritionally a more balanced product for human babies than natural cow's milk.

Transgenic sheep produce a clotting factor for haemophilics, which can be separated from the milk. Transgenic goats produce a protein that dissolves clots, which is a cure for coronary thrombosis.

Mice are the most preferred mammal for studies of gene transfer, because of their many favourable features: short gestation period, relatively short generation time, production of several offspring per pregnancy, convenient in vitro fertilisation and successful culture of embryos in vitro. Recently, rats and rabbits are also being used for research on gene transfer.

Rabbits are quite successful in gene farming or molecular farming, which aims at the production of pharmaceutically or biologically important proteins encoded by transgenes. The following human genes encoding valuable proteins have been transferred into rabbits: interleukin 2, growth hormone, tissue plasminogen activator and α1 antitrypsin. These genes were expressed in the mammary tissues, and their proteins were harvested from the milk.

Transgenic cattle, sheep and fish grow most quickly and produce more meat. Sheep containing bacterial genes which code for the enzymes needed for the synthesis of the amino acid cysteine have been produced. They give a higher wool yield, because cysteine is an important component of the keratin protein of wool. Sheep with the gene for growth hormone have also been produced, and they yield more meat.

Table: Transgenic animals and their useful applications

Transplantation of an organ from one species to another is called xenotransplantation.

Transgenic microbes: Transgenic bacteria and yeast are used to produce human proteins such as insulin, TPA (tissue plasminogen activator), interferon, interleukin and tumour necrosis factor (TNF). E. coli has been made to produce the enzyme cellulase, which hydrolyses cellulose and enables animals to digest their entire food.

Several bacterial genomes have been modified by introducing foreign genes to control: (i) insects, by production of endotoxins; (ii) fungal diseases, by production of chitinases, which suppress the fungal flora in the soil; and (iii) pathogens, by production of an antibiotic that degrades the toxin produced by the pathogen.

The nitrogen-fixing efficiency of the bacteria Rhizobium has also been increased by introducing useful nif (nitrogen fixation) genes into their genome.

Bioremediation: Bacteria are genetically manipulated for the degradation of chemicals and waste material. For example, Pseudomonas bacteria possess genes for the degradation of waste material. But in natural conditions this is not very efficient, whereas multiple genes integrated by genetic engineering make them efficient biodegraders.

Utility of transgenic animals:
- They can be specifically designed to study specific functions of genes.
- They serve as models for the study of human diseases, to find novel treatments.
- They allow mass production of pharmaceutical products.
- They are used to test the safety of various vaccines.
- They are used for toxicity or safety testing. Transgenic animals are made to carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are then exposed to the toxic substance, and its effect is studied in less time.

DNA FINGERPRINTING

Sir Alec Jeffreys (1984) invented the DNA fingerprinting technique. Its applications are:
(i) Paternity disputes can be solved by DNA fingerprinting.
(ii) It can help to solve problems of evolution.
(iii) It can be used to study the breeding patterns of animals facing the danger of extinction.
(iv) It is useful in restoring the health of patients suffering from leukemia (blood cancer). It is very useful in the detection of crime and in legal pursuits.

DISADVANTAGES OF BIOTECHNOLOGICAL APPLICATIONS

1. Manipulation of living organisms by the human race is unethical and should not go unregulated.
2. GM organisms can have unpredictable results when they are introduced into ecosystems.
3. Modification or use of living organisms for food and medicine production creates patent problems.

The Indian government has set up the Genetic Engineering Approval Committee (GEAC), which takes decisions regarding the validity of GM research and the safety of introducing GMOs into the ecosystem. GEAC works under the Ministry of Environment and Forests, to regulate the research, testing and commercial release of GM crops, foods and organisms. Its aims and objectives are:
(i) to give approval for the use of GM organisms and their products for commercial applications;
(ii) to adopt strategies for restriction, production, import, export and applications of GM organisms;
(iii) to give permission for the conduct of large-scale field trials and release of transgenic crops in the environment;
(iv) to authorise agencies or persons to have large-scale production and release of GM organisms into the environment.

The committee gave approval for the cultivation of Bt cotton, the only commercially grown GM crop in India, in 2002.

Biopiracy is the unethical and illegal use of the biodiversity resources of other nations without proper authorisation from the countries concerned.

Biopatent is government protection given to an inventor of biological resources, to prevent others from the commercial use of the invention. Mainly the USA, Japan and the members of the European Union grant biopatents. Transnational companies rush to get patents on new products derived from biological resources, so as to prevent competitors from using them. They can then earn larger profits by raising the prices of the products or by charging royalties to other firms wishing to use the technology.

The use of karela (bitter gourd) and jamun for the control of diabetes has been known in India for generations, and their antidiabetic properties are documented in ancient literature. Claiming the use of karela, jamun and bengan for antidiabetic treatment as an invention is false, because this fact has been known and documented in India since ancient times.

Intellectual property rights and patent laws can save resources such as tamarind from exploitation by multinational companies.
- The entire germplasm of Basmati rice of Indian origin has been patented by an American company.
- Basmati rice was patented by an American company by crossing an Indian variety with a new dwarf variety and claiming it as a novel invention, due to a lack of awareness and strong patent legislation.
- Similar attempts have been made for turmeric.
- Patent laws are desirable to solve the ethical issues related to GMOs and new varieties, to prevent exploitation, and to protect the rich bioresource heritage of India.

The Indian Parliament has cleared the second amendment of the Indian Patent Bill, which takes such issues into consideration, including emergency provisions and research and development initiatives.

Practical problems associated with genetic engineering:

Biological warfare: Biowar, or biological war, is fought with biological weapons against humans and/or their crops and animals. Viruses, bacteria and other harmful organisms can be used as biological weapons. Biological weapons are low in cost compared with conventional weapons, and they can cause more casualties than conventional weapons. They are invisible and easy to handle. The most common bioweapons cause:
(i) anthrax; (ii) smallpox; (iii) plague; (iv) Q fever (fever, chills, fatigue, etc.); (v) viral encephalitis; (vi) botulinum toxin (skeletal muscle paralysis).

Other problems include:
- production of drug-resistant pathogens;
- the ethical problem of examining someone's genetic map;
- the introduction of super weeds due to the introduction of a faulty gene;
- harmless intestinal bacteria turning into cancer-causing bacterial forms.

CHECK POINT

Fill in the blanks

(i) Mass production of drugs and proteins in transgenic animals or crops, which are used as bioreactors, are called ____________.

Answer: Molecular farming (or molecular pharming).

(ii) The first clinical gene therapy was given to cure ____________.

Answer: ADA (adenosine deaminase) deficiency in a 4-year-old child, a form of SCID.

(iii) The first transgenic cow produced milk enriched in ____________.

Answer: Human alpha-lactalbumin (a human protein).

(iv) Fungal flora in the soil can be suppressed by using ____________.

Answer: Chitinase, produced by transgenic bacteria carrying the chitinase gene.

(v) ____________ bacteria possess genes for degradation of waste materials.

Answer: Pseudomonas.

Check Point, State true or false

(i) Insulin chains A and B are produced separately in the plasmid of E. coli, extracted and combined by creating disulphide bonds.

Answer: True.

(ii) Human growth hormone is also used in healing of injuries.

Answer: True.

(iii) RNA is the method of vaccine production.

Answer: False. Vaccines are produced by recombinant DNA technology or conventional methods. RNA interference is a gene-silencing method and is not a method of vaccine production.

(iv) Cry IAc and cry IIAb control cotton bacterium.

Answer: False as written. Cry IAc and cry IIAb control the cotton bollworm, which is an insect pest, not a bacterium.

(v) Steroids are complex lipids of high molecular weight which are found in both plants and animals.

Answer: True.

Check Point, State true or false (uses of steroids)

(i) Steroid hormones are known to be regulators of metabolism in the animal or human body.

Answer: True.

(ii) Cortisone and its derivatives (prednisone and prednisolone) are effective in the treatment of rheumatoid arthritis.

Answer: True.

(iii) Oestrogens and progesterones are employed in the preparation of oral contraceptives.

Answer: True.

(iv) Steroid treatment is given to suppress immune responses in patients with autoimmune diseases or persons who have had organ transplants.

Answer: True.

(v) Prednisolone is used as an anti-inflammatory drug.

Answer: True.

Check Point, Fill in the blanks (agriculture)

(i) The gene which has been transferred from one organism to another is called ____________.

Answer: A transgene.

(ii) ____________ is the transgenic variety of rice, rich in ____________.

Answer: Golden rice, rich in vitamin A (beta-carotene).

(iii) Herbicides are the chemicals which kill ____________.

Answer: Weeds.

(iv) Bt cotton is the first genetically modified crop, which resists attack of ____________.

Answer: Lepidopteran insects, which cause the bollworm disease of cotton.

(v) ____________ nematode infects the roots of tobacco plants.

Answer: Meloidogyne incognitia.