Biotechnology and its Applications | CBSE Class 12 Biology Notes

This note covers NCERT Class 12 Biology Chapter 10, Biotechnology and its Applications: tissue culture, genetically modified crops, Bt cotton, RNA interference, genetically engineered insulin, gene therapy, molecular diagnosis, transgenic animals, and the ethical questions of patents and biopiracy. It follows the current NCERT text section by section, and gives every name, year and number as NCERT states it (one misprinted species name is corrected and flagged), because these are asked directly in board and entrance examinations. Where a figure is dated, it is marked as the NCERT figure.

What does biotechnology deal with, and what are its three critical research areas?

Biotechnology essentially deals with the industrial-scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals. Its applications include therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.

NCERT lists three critical research areas of biotechnology. Learn them in this order.

1. Providing the best catalyst, in the form of an improved organism (usually a microbe) or a pure enzyme.
2. Creating optimal conditions, through engineering, for the catalyst to act.
3. Downstream processing technologies to purify the protein or organic compound.

Note: The chapter then looks at how biotechnology has improved human life in two fields: food production (Section 10.1) and health (Section 10.2). Transgenic animals (10.3) and ethical issues (10.4) complete it. A long-answer question can come from any one of the four.

Why was a new approach to food production needed?

NCERT names three options that can be thought of for increasing food production:

• agro-chemical based agriculture,
• organic agriculture, and
• genetically engineered crop-based agriculture.

The Green Revolution succeeded in tripling the food supply, yet this was not enough to feed the growing human population. The increased yields were partly due to improved crop varieties, but mainly due to better management practices and the use of agrochemicals (fertilisers and pesticides). Two problems remained. For farmers in the developing world, agrochemicals are often too expensive. And further increases in yield with existing varieties are not possible using conventional breeding.

Traditional breeding could not keep pace with demand or provide sufficiently fast and efficient systems for crop improvement. Two technologies answered this: tissue culture, and then genetic modification of crops.

What is tissue culture, and what can it do?

During the 1950s scientists learnt that whole plants could be regenerated from explants. An explant is any part of a plant taken out and grown in a test tube, under sterile conditions, in special nutrient media.

Definition: Totipotency is the capacity to generate a whole plant from any cell or explant.

The nutrient medium must provide:

• a carbon source such as sucrose,
• inorganic salts,
• vitamins,
• amino acids, and
• growth regulators such as auxins and cytokinins.

Micropropagation and somaclones

By tissue culture a large number of plants can be propagated in a very short time. This method of producing thousands of plants through tissue culture is called micro-propagation. Each of these plants is genetically identical to the original plant from which it was grown. Such plants are called somaclones. Many important food plants, such as tomato, banana and apple, have been produced on a commercial scale by this method.

Virus-free plants from the meristem

Tissue culture can also recover healthy plants from diseased plants. Even if a plant is infected with a virus, its meristem (apical and axillary) is free of the virus. The meristem can therefore be removed and grown in vitro to obtain virus-free plants. Scientists have succeeded in culturing meristems of banana, sugarcane and potato.

Somatic hybridisation

The steps of somatic hybridisation, as NCERT describes them, are:

1. Single cells are isolated from plants.
2. Their cell walls are digested, which leaves naked protoplasts surrounded only by plasma membranes.
3. Isolated protoplasts from two different varieties of plants, each with a desirable character, are fused.
4. The fusion gives hybrid protoplasts.
5. The hybrid protoplasts are grown further to form a new plant, called a somatic hybrid.

The standard example is the pomato: a protoplast of tomato was fused with a protoplast of potato, and the product was grown into hybrid plants combining tomato and potato characteristics. Unfortunately the pomato did not have all the desired combination of characteristics for commercial use.

Note: Somaclones and somatic hybrids are different things. Somaclones are genetically identical copies of one parent plant. A somatic hybrid combines the protoplasts of two different plants.

What are genetically modified organisms, and how have GM crops helped?

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

Genetically modified crops are a possible answer to two questions farmers face: how to get the maximum yield, and how to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced. NCERT lists five ways in which genetic modification of plants has been useful.

1. It has made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
2. It has reduced reliance on chemical pesticides (pest-resistant crops).
3. It has helped to reduce post-harvest losses.
4. It has increased the efficiency of mineral usage by plants, which prevents early exhaustion of the fertility of soil.
5. It has enhanced the nutritional value of food, for example golden rice, which is Vitamin A enriched rice.

In addition, genetic modification has been used to create tailor-made plants that supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.

How does Bt toxin kill insects without killing the bacterium that makes it?

Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short). The Bt toxin gene has been cloned from the bacterium and expressed in plants to give resistance to insects without the need for insecticides. In effect this creates a bio-pesticide. NCERT's examples are Bt cotton, Bt corn, rice, tomato, potato and soyabean.

Some strains of Bacillus thuringiensis produce proteins that kill certain insects:

The mechanism of action

1. B. thuringiensis forms protein crystals during a particular phase of its growth. These crystals contain a toxic insecticidal protein.
2. Inside the bacterium the Bt toxin protein exists as an inactive protoxin. This is why the toxin does not kill the Bacillus itself.
3. When an insect ingests the inactive toxin, the alkaline pH of its gut solubilises the crystals and the protoxin is converted into the active form of the toxin.
4. The activated toxin binds to the surface of the midgut epithelial cells and creates pores.
5. The pores cause cell swelling and lysis, and eventually the death of the insect.

The cry genes

Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into several crop plants such as cotton. The toxin is coded by a gene named cry, and there are a number of these genes. The choice of gene depends on the crop and the targeted pest, because most Bt toxins are insect-group specific.

A photograph of cotton bolls held in a hand. The boll labelled (a) has been destroyed by bollworms. The boll labelled (b) is a fully mature cotton boll, open and full of white fibre. The figure shows what Bt cotton is meant to prevent.

See Fig. 10.1 in your NCERT textbook

Note: Read the gene names letter by letter. The two genes for cotton bollworms are cryIAc and cryIIAb. The gene for corn borer is cryIAb. The names differ by a single character, and questions are set on exactly that difference. By convention the gene is written in italics with a small c (cry) and the protein it codes for is written Cry.

How does RNA interference protect tobacco plants from nematodes?

Several nematodes parasitise a wide variety of plants and animals, including human beings. The nematode Meloidogyne incognita infects the roots of tobacco plants and causes a great reduction in yield. (The NCERT text prints the name as Meloidegyne incognitia.) A novel strategy, based on RNA interference (RNAi), was adopted to prevent this infestation.

Definition: RNA interference is the silencing of a specific mRNA by a complementary double-stranded RNA (dsRNA) molecule that binds to the mRNA and prevents its translation. It takes place in all eukaryotic organisms as a method of cellular defence.

In nature, the complementary RNA could come from an infection by viruses that have RNA genomes, or from mobile genetic elements (transposons) that replicate via an RNA intermediate.

The strategy, step by step

1. Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant.
2. The DNA was introduced in such a way that it produced both sense and anti-sense RNA in the host cells.
3. These two RNAs, being complementary to each other, formed a double-stranded RNA (dsRNA).
4. The dsRNA initiated RNAi and so silenced the specific mRNA of the nematode.
5. The parasite could not survive in a transgenic host expressing the specific interfering RNA, so the transgenic plant was protected from the parasite.

Two photographs of tobacco roots side by side. Panel (a) shows the roots of a typical control plant, with swellings on the roots marked by arrows. Panel (b) shows the roots of a transgenic plant 5 days after deliberate infection with the nematode. These roots are protected by the RNAi mechanism and look thin and clean.

See Fig. 10.2 in your NCERT textbook

Note: In this strategy the plant is the transgenic organism, but the gene that is silenced belongs to the nematode. RNAi stops translation of the mRNA. It does not remove or cut the gene.

How is human insulin made by recombinant DNA technology?

Recombinant DNA technological processes have made an immense impact on healthcare by enabling the mass production of safe and more effective therapeutic drugs. Recombinant therapeutics also do not induce unwanted immunological responses, which are common with similar products isolated from non-human sources. According to NCERT, about 30 recombinant therapeutics have been approved for human use the world over, and 12 of these are being marketed in India.

The problem with animal insulin

Adult-onset diabetes can be managed by taking insulin at regular time intervals. Insulin used for diabetes was earlier extracted from the pancreas of slaughtered cattle and pigs. Insulin from an animal source caused some patients to develop allergy or other types of reactions to the foreign protein.

The structure of insulin

Insulin consists of two short polypeptide chains, chain A and chain B, linked together by disulphide bridges. In mammals, including humans, insulin is synthesised as a pro-hormone. Like a pro-enzyme, the pro-hormone needs to be processed before it becomes a fully mature and functional hormone. The pro-hormone contains an extra stretch called the C peptide. The C peptide is not present in mature insulin. It is removed during maturation into insulin.

At the top is proinsulin: one continuous chain in which the A peptide and the B peptide are joined by a looping C peptide, with S-S (disulphide) bridges between the A and B parts. An arrow points down to the products: the A peptide and the B peptide held together by the S-S bridges, which is insulin, and a separate free C peptide. To draw it, show three segments in one chain, mark the S-S bridges, then show the C peptide cut away.

See Fig. 10.3 in your NCERT textbook

How Eli Lilly made it

The main challenge in producing insulin by rDNA techniques was getting the insulin assembled into a mature form. In 1983, Eli Lilly, an American company, solved it as follows.

1. Two DNA sequences were prepared, corresponding to the A chain and the B chain of human insulin.
2. The sequences were introduced into plasmids of E. coli to produce the insulin chains.
3. Chains A and B were produced separately.
4. The chains were extracted.
5. The chains were combined by creating disulphide bonds, to form human insulin.

Human insulin made in bacteria has a structure absolutely identical to that of the natural molecule. This is why, as the NCERT summary says, recombinant therapeutics do not induce unwanted immunological responses and are free from the risk of infection that was seen with products isolated from non-human sources.

Note: NCERT asks whether insulin can be given orally. It cannot. Insulin is a protein, so the protein-digesting enzymes of the alimentary canal would digest it before it could act. It is therefore injected.

What is gene therapy, and how was ADA deficiency treated?

Definition: Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child or embryo. Genes are inserted into a person's cells and tissues to treat a disease.

Correction of a genetic defect involves delivering a normal gene into the individual or embryo, to take over the function of the non-functional gene and compensate for it.

The first clinical gene therapy was given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency. The enzyme adenosine deaminase is crucial for the immune system to function. The disorder is caused by the deletion of the gene for adenosine deaminase.

The steps of ADA gene therapy

1. Lymphocytes from the blood of the patient are grown in a culture outside the body.
2. A functional ADA cDNA is introduced into these lymphocytes, using a retroviral vector.
3. The genetically engineered lymphocytes are returned to the patient.
4. Because these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes.

The NCERT summary adds that gene therapy works by replacing a defective mutant allele with a functional one, or by gene targeting, which involves gene amplification. Viruses that attack their hosts and introduce their genetic material into the host cell as part of their replication cycle are used as vectors to transfer healthy genes or, more recently, portions of genes.

How does molecular diagnosis detect a disease early?

Early diagnosis and an understanding of pathophysiology are very important for the effective treatment of a disease. With conventional methods of diagnosis, such as serum and urine analysis, early detection is not possible. Normally the presence of a pathogen (bacteria, viruses and so on) is suspected only when it has produced a disease symptom. By that time the concentration of the pathogen in the body is already very high.

Some of the techniques that serve the purpose of early diagnosis are recombinant DNA technology, the Polymerase Chain Reaction (PCR) and the Enzyme Linked Immuno-sorbent Assay (ELISA).

Note: NCERT asks how PCR can detect very low amounts of DNA. PCR makes copies of the target DNA again and again, and each cycle roughly doubles the number of copies. A few molecules of the pathogen's nucleic acid are multiplied into enough DNA to be detected.

What are transgenic animals, and why are they produced?

Definition: Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals.

Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although NCERT states that over 95 per cent of all existing transgenic animals are mice. NCERT gives five common reasons for producing them.

Rosie was the first transgenic cow. In 1997 she produced human protein-enriched milk, with 2.4 grams of the human protein per litre. The milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies than natural cow milk.

In the study of growth, genes from other species that alter the formation of insulin-like growth factor are introduced, and the biological effects that result are studied. This gives information about the biological role of the factor in the body.

What ethical issues does biotechnology raise?

The manipulation of living organisms by the human race cannot go on any further without regulation. Ethical standards are required to evaluate the morality of all human activities that might help or harm living organisms. Beyond morality, the biological significance matters too: genetic modification of organisms can have unpredictable results when such organisms are introduced into the ecosystem.

GEAC

The Indian Government has set up organisations such as GEAC (Genetic Engineering Approval Committee), which make decisions regarding the validity of GM research and the safety of introducing GM organisms for public services.

Note: Write the name as NCERT gives it, Genetic Engineering Approval Committee. The committee has since been renamed the Genetic Engineering Appraisal Committee, so you may see either name outside the textbook.

Patents and the Basmati case

The modification and use of living organisms for public services, as food and medicine sources for example, has created problems with the patents granted for them. There is growing public anger that certain companies are being granted patents for products and technologies that make use of genetic materials, plants and other biological resources that have long been identified, developed and used by farmers and indigenous people of a specific region or country.

• Rice has been present in Asia's agricultural history for thousands of years. NCERT gives an estimate of 200,000 varieties of rice in India alone.
• Basmati rice is distinct for its unique aroma and flavour. NCERT records 27 documented varieties of Basmati grown in India, and Basmati is referred to in ancient texts, folklore and poetry.
• In 1997 an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed the company to sell a "new" variety of Basmati in the US and abroad.
• The "new" variety had actually been derived from Indian farmers' varieties. Indian Basmati was crossed with semi-dwarf varieties and claimed as an invention or a novelty.
• The patent extends to functional equivalents, which implies that other people selling Basmati rice could be restricted by the patent.

Several attempts have also been made to patent uses, products and processes based on Indian traditional herbal medicines, for example turmeric and neem. NCERT warns that if we are not vigilant and do not immediately counter such patent applications, other countries or individuals may encash on our rich legacy.

Biopiracy

Definition: Biopiracy is the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned, and without compensatory payment.

Most industrialised nations are rich financially but poor in biodiversity and traditional knowledge. The developing and underdeveloped world, in contrast, is rich in biodiversity and in traditional knowledge related to bio-resources. That traditional knowledge can be exploited to develop modern applications, and can save time, effort and expenditure during their commercialisation.

There is a growing realisation of the injustice, inadequate compensation and benefit sharing between developed and developing countries. Some nations are therefore developing laws to prevent such unauthorised exploitation of their bio-resources and traditional knowledge. NCERT records that the Indian Parliament has cleared the second amendment of the Indian Patents Bill, which takes such issues into consideration, including patent terms, emergency provisions, and research and development initiatives.

How do you answer the NCERT exercise questions?

These are short pointers for the exercise questions that the chapter text itself answers.

Which part of the plant is best suited for making virus-free plants, and why?

The meristem, apical or axillary. Even when the plant is infected with a virus, the meristem is free of the virus, so a meristem grown in vitro gives virus-free plants.

What is the major advantage of producing plants by micropropagation?

A large number of plants can be produced in a very short time, and every plant is genetically identical to the original plant (a somaclone).

What are the components of the medium used for propagating an explant in vitro?

A carbon source such as sucrose, inorganic salts, vitamins, amino acids, and growth regulators such as auxins and cytokinins.

Why do crystals of Bt toxin not kill the bacteria that produce them?

The correct option is (c), the toxin is inactive. It exists in the bacterium as an inactive protoxin and becomes active only in the alkaline gut of the insect.

What are transgenic bacteria? Give one example.

Bacteria whose genes have been altered by manipulation so that they carry and express a foreign gene. The chapter's example is E. coli carrying plasmids with the DNA sequences for the A and B chains of human insulin.

What are the advantages and disadvantages of genetically modified crops?

Advantages: higher crop yield, tolerance to abiotic stresses, less reliance on chemical pesticides, lower post-harvest losses, more efficient mineral usage, and enhanced nutritional value. Disadvantages: genetic modification can have unpredictable results when the organisms are introduced into the ecosystem, which is why GEAC must decide on their safety; and the patents granted for modified living organisms and biological resources, as in the Basmati case, have raised ethical problems.

What are Cry proteins, and how has man exploited them?

Cry proteins are the insecticidal proteins coded by the cry genes of Bacillus thuringiensis. The genes have been isolated and incorporated into crop plants such as cotton, so that the plant makes its own bio-pesticide: cryIAc and cryIIAb against cotton bollworms, and cryIAb against corn borer.

What is gene therapy? Illustrate with ADA deficiency.

Define gene therapy, state the 1990 case, and give the four steps listed above: lymphocytes cultured outside the body, functional ADA cDNA introduced with a retroviral vector, cells returned to the patient, and periodic infusion because the cells are not immortal. End with the possible permanent cure at the early embryonic stage.

Glossary

• Explant — Any part of a plant taken out and grown in a test tube under sterile conditions in special nutrient media.
• Totipotency — The capacity to generate a whole plant from any single cell or explant of that plant.
• Micro-propagation — The method of producing thousands of plants through tissue culture in a very short duration.
• Somaclones — Plants produced by tissue culture that are genetically identical to the original plant from which they were grown.
• Protoplast — A plant cell whose cell wall has been digested, leaving it surrounded only by its plasma membrane.
• Somatic hybrid — A plant grown from a hybrid protoplast, formed by fusing protoplasts of two different varieties of plants.
• Genetically Modified Organism (GMO) — A plant, bacterium, fungus or animal whose genes have been altered by manipulation.
• Bt toxin — An insecticidal protein produced by the bacterium Bacillus thuringiensis, coded by the cry genes and used as a bio-pesticide.
• Protoxin — The inactive form in which Bt toxin exists in the bacterium, before the alkaline pH of the insect gut activates it.
• RNA interference (RNAi) — Silencing of a specific mRNA by a complementary double-stranded RNA molecule that binds to it and prevents translation.
• Transposons — Mobile genetic elements; those that replicate via an RNA intermediate can be a source of complementary RNA for RNAi.
• Pro-hormone — The precursor form of a hormone, which must be processed before it becomes a fully mature and functional hormone.
• C peptide — The extra stretch present in proinsulin that is removed during maturation and is absent from mature insulin.
• Gene therapy — A collection of methods that allows correction of a gene defect diagnosed in a child or embryo, by inserting genes into cells.
• Probe — A single-stranded DNA or RNA tagged with a radioactive molecule, used to find its complementary DNA by hybridisation.
• ELISA — Enzyme Linked Immuno-sorbent Assay, a diagnostic technique based on the principle of antigen-antibody interaction.
• Transgenic animal — An animal whose DNA has been manipulated so that it possesses and expresses an extra (foreign) gene.
• Biopiracy — The use of bio-resources by multinational companies and other organisations without proper authorisation and without compensatory payment.

Common errors and misconceptions

• Misconception: Bt toxin does not harm the bacterium because the bacterium is resistant to it. Correct: The toxin exists in the bacterium as an inactive protoxin. It becomes active only after an insect ingests it.
• Misconception: The protoxin is activated by the acid in the insect's gut. Correct: It is the alkaline pH of the insect gut that solubilises the crystals and activates the toxin.
• Misconception: cryIAb controls cotton bollworms. Correct: cryIAc and cryIIAb control cotton bollworms. cryIAb controls corn borer.
• Misconception: RNAi destroys the nematode's gene. Correct: RNAi silences a specific mRNA. The dsRNA binds to the mRNA and prevents its translation.
• Misconception: Eli Lilly made proinsulin in bacteria and then removed the C peptide. Correct: Chains A and B were produced separately in E. coli, extracted, and joined by creating disulphide bonds.
• Misconception: Mature insulin has three chains: A, B and C. Correct: Mature insulin has only chains A and B. The C peptide is removed during maturation.
• Misconception: The ADA gene therapy given with lymphocytes is a permanent cure. Correct: The lymphocytes are not immortal, so periodic infusion is required. A permanent cure could come from introducing the gene at early embryonic stages.
• Misconception: Rosie was a transgenic sheep. Correct: Rosie was the first transgenic cow (1997). Her milk contained human alpha-lactalbumin, at 2.4 grams of the human protein per litre.
• Misconception: In the probe method, the clone with the mutated gene shows up on the photographic film. Correct: It does not appear, because the probe has no complementarity with the mutated gene.
• Misconception: Somaclones and somatic hybrids mean the same thing. Correct: Somaclones are genetically identical to one parent plant. Somatic hybrids come from fused protoplasts of two different plants.

Exam-style questions with model answers

Q1. Name the nematode that infects the roots of tobacco plants, and the process used to protect the plants from it. [1 mark]

1. The nematode is Meloidogyne incognita (printed in NCERT as Meloidegyne incognitia). The plants are protected by RNA interference (RNAi).

Q2. Why does the Bt toxin not kill the bacterium that produces it, although it kills the insect? [2 marks]

1. In the bacterium the Bt toxin exists as an inactive protoxin, inside protein crystals, so it does not harm the Bacillus.
2. When an insect ingests it, the alkaline pH of the insect gut solubilises the crystals and converts the protoxin into the active toxin, which kills the insect.

Q3. How can virus-free plants be obtained from a virus-infected plant? Name two plants in which this has been done. [2 marks]

1. Even in a virus-infected plant the meristem (apical and axillary) is free of the virus. The meristem is removed and grown in vitro to obtain virus-free plants.
2. Meristems of banana, sugarcane and potato have been cultured in this way (any two).

Q4. Describe the structure of proinsulin. How did Eli Lilly produce human insulin in 1983? [3 marks]

1. Insulin is synthesised as a pro-hormone, a single chain in which chain A and chain B are joined by an extra stretch called the C peptide (Figure 10.3). The C peptide is removed during maturation, leaving chains A and B linked by disulphide bridges.
2. Eli Lilly prepared two DNA sequences corresponding to the A and B chains of human insulin and introduced them into plasmids of E. coli.
3. Chains A and B were produced separately, extracted and combined by creating disulphide bonds to form human insulin.

Q5. Explain how RNA interference was used to make tobacco plants resistant to a nematode. [3 marks]

1. Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant, in such a way that both sense and anti-sense RNA were produced in the host cells.
2. The two RNAs were complementary and formed a double-stranded RNA, which initiated RNAi and silenced the specific mRNA of the nematode.
3. The parasite could not survive in the transgenic host expressing the specific interfering RNA, so the plant was protected.

Q6. What is biopiracy? Explain with the example of Basmati rice. [3 marks]

1. Biopiracy is the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned, and without compensatory payment.
2. In 1997 an American company got patent rights on Basmati rice through the US Patent and Trademark Office, which allowed it to sell a "new" variety in the US and abroad.
3. The variety had actually been derived from Indian farmers' varieties, by crossing Indian Basmati with semi-dwarf varieties. NCERT records 27 documented varieties of Basmati grown in India, and the patent could restrict other people selling Basmati rice.

Q7. Give any three reasons for which transgenic animals are produced, with one example of each. [3 marks]

1. Study of disease: transgenic animals serve as models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.
2. Biological products: transgenic animals produce useful products such as human α-1-antitrypsin, used to treat emphysema; Rosie, the first transgenic cow, produced milk containing human alpha-lactalbumin.
3. Vaccine safety: transgenic mice are being used to test the safety of the polio vaccine and could replace monkeys. (Normal physiology and development, and chemical safety testing, are also acceptable.)

Q8. What is gene therapy? Describe how it was used to treat adenosine deaminase (ADA) deficiency, and state why it was not a permanent cure. [5 marks]

1. Gene therapy is a collection of methods that allows correction of a gene defect diagnosed in a child or embryo. A normal gene is delivered into the individual or embryo to take over the function of the non-functional gene.
2. The first clinical gene therapy was given in 1990 to a 4-year-old girl with ADA deficiency. The enzyme is crucial for the immune system, and the disorder is caused by deletion of the gene for adenosine deaminase.
3. Lymphocytes from the patient's blood are grown in a culture outside the body.
4. A functional ADA cDNA is introduced into these lymphocytes using a retroviral vector, and the lymphocytes are returned to the patient.
5. These cells are not immortal, so the patient requires periodic infusion of the genetically engineered lymphocytes. If the gene isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Q9. What are Cry proteins? Explain how Bt cotton resists insect attack, naming the genes involved. [5 marks]

1. Cry proteins are insecticidal proteins produced by some strains of Bacillus thuringiensis and coded by genes named cry. They kill certain lepidopterans, coleopterans and dipterans.
2. The bacterium forms protein crystals containing the toxin as an inactive protoxin, which is why the bacterium itself is not killed.
3. When an insect ingests the protoxin, the alkaline pH of its gut solubilises the crystals and converts it into the active toxin.
4. The active 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.
5. Specific Bt toxin genes have been incorporated into cotton. The proteins encoded by cryIAc and cryIIAb control cotton bollworms (cryIAb controls corn borer), so the crop needs less insecticide.

Key takeaways

• Tissue culture rests on totipotency: a whole plant can be regenerated from an explant grown in sterile nutrient medium, and micro-propagation gives thousands of genetically identical somaclones.
• The meristem of a virus-infected plant is free of virus, so meristem culture gives virus-free banana, sugarcane and potato plants.
• Bt toxin is an inactive protoxin in the bacterium and is activated by the alkaline pH of the insect gut, where it creates pores in midgut epithelial cells.
• The genes cryIAc and cryIIAb control cotton bollworms, while cryIAb controls corn borer; most Bt toxins are insect-group specific.
• RNA interference silences a specific mRNA with a complementary dsRNA; it protected tobacco roots from the nematode Meloidogyne incognita.
• Eli Lilly made human insulin in 1983 by producing chains A and B separately in E. coli and joining them with disulphide bonds.
• The first clinical gene therapy was given in 1990 to a 4-year-old girl with ADA deficiency, using her own lymphocytes and a retroviral vector.
• Recombinant DNA technology, PCR and ELISA serve early diagnosis; PCR can detect a very low concentration of a pathogen before any symptom is visible, and a labelled probe with autoradiography can detect a mutated gene.
• NCERT states that over 95 per cent of transgenic animals are mice; Rosie, the first transgenic cow, produced milk with human alpha-lactalbumin in 1997.
• GEAC decides on the validity of GM research and the safety of GM organisms; the Basmati patent of 1997 is the standard example of biopiracy.

Test yourself

What is totipotency?

Totipotency is the capacity to generate a whole plant from any cell or explant of that plant.

What are somaclones?

Somaclones are plants produced through tissue culture that are genetically identical to the original plant from which they were grown.

Which bacterium produces Bt toxin, and what activates the toxin in the insect?

Bt toxin is produced by Bacillus thuringiensis. The alkaline pH of the insect gut solubilises the crystals and converts the inactive protoxin into the active toxin.

Which cry genes control cotton bollworms, and which controls corn borer?

The proteins encoded by cryIAc and cryIIAb control cotton bollworms, and the protein encoded by cryIAb controls corn borer.

What does dsRNA do in RNA interference?

The complementary dsRNA binds to a specific mRNA and prevents its translation, which silences that mRNA.

Which vector was used to introduce nematode-specific genes into the tobacco plant?

Agrobacterium vectors were used to introduce the nematode-specific genes into the host tobacco plant.

Which part of proinsulin is absent from mature insulin?

The C peptide is present in proinsulin but is removed during maturation, so mature insulin has only chains A and B.

When and to whom was the first clinical gene therapy given?

The first clinical gene therapy was given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency.

On what principle is ELISA based?

ELISA is based on the principle of antigen-antibody interaction, detecting either the antigens of the pathogen or the antibodies made against it.

What did the milk of Rosie, the first transgenic cow, contain?

Rosie's milk contained the human protein alpha-lactalbumin, at 2.4 grams of the human protein per litre, which made it more balanced for human babies.