Biotechnology and its Applications

Written and reviewed by Anjali Sharma, Biology faculty at UniDrill (M.Sc. Botany, Banaras Hindu University · 8 years teaching NCERT Biology). Based on NCERT Biology Class XII, Chapter 10.

Why does a cotton plant carry a bacterial gene?

A bacterium that lives in soil makes a protein that kills caterpillars. A cotton farmer's worst enemy is a caterpillar, the bollworm. Put the bacterial gene into the cotton plant and the plant makes its own protection. In the previous chapter you learned the tools of genetic engineering. This chapter shows what they build: crops, medicines, diagnostic tests and animals, and the rules that decide how far we should go.

Biotechnology, at this scale, is the industrial production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals. Its uses include therapeutics, diagnostics, GM crops, processed food, bioremediation, waste treatment and energy production. It rests on three research areas: (i) a better catalyst, an improved organism or a pure enzyme; (ii) engineering the best conditions for it to work; and (iii) downstream processing to purify the product.

Applications in agriculture

There are three ways to produce more food: agro-chemical based farming, organic farming, and genetically engineered crop-based farming. The Green Revolution tripled food supply, but it was not enough for the growing population. Most of its gain came from better management and agro-chemicals (fertilisers and pesticides), which are often too costly for farmers in developing countries.

Tissue culture and micropropagation

Plant tissue culture grows whole plants from tiny pieces. It relies on totipotency, the ability of a plant cell to give rise to a whole plant. Any piece of plant taken out and grown in a test tube under sterile conditions is an explant. It is grown on a nutrient medium containing a carbon source (sucrose), inorganic salts, vitamins, amino acids and growth regulators such as auxins and cytokinins.

Using tissue culture, thousands of plants can be raised from a single parent in a short time. This is micropropagation, and every plant is genetically identical to the parent, so the plants are called somaclones. Tomato, banana and apple have been produced commercially this way.

Virus-free plants. Even when a plant is infected with a virus, its meristem (apical and axillary) is free of the virus. If you culture the meristem, you get healthy plants. This has been done for banana, sugarcane and potato.

Beyond the textbook. For the exam: the meristem of an infected plant is virus-free, so meristem culture gives virus-free plants. In reality: the meristem is made of small, rapidly dividing cells with no developed vascular tissue, so viruses, which spread mostly through the vascular system, rarely reach it or keep pace with its growth. Why it matters: it explains why the tip of a shoot and not a leaf is chosen for this job.

Somatic hybridisation

Two plants that cannot be crossed by pollination can still be combined at the cell level. The walls of their cells are digested to get protoplasts (naked cells surrounded only by the plasma membrane). Protoplasts from two different varieties, each with a desirable character, are fused to make a somatic hybrid. The tomato and potato fusion called the pomato was made, but it did not have the right combination of characters for commercial use.

Pause and predict: a farmer wants a thousand identical banana plants that are free of a viral disease. Which technique should he use, and why does he not just plant cuttings of the diseased plant?

Check your answer: meristem culture followed by micropropagation. Cuttings of an infected plant would carry the virus into every new plant, but the meristem does not.

Genetically modified plants

Genetically modified organisms (GMOs) are plants, bacteria, fungi and animals whose genes have been altered by manipulation. GM crops are useful in several ways.

Beyond the textbook. For the exam: golden rice is vitamin A enriched rice. In reality: golden rice makes beta-carotene, a pigment that your body converts into vitamin A, and this is what gives the grain its golden colour. Why it matters: the target is a deficiency that causes blindness in many children in rice-eating regions, which is why the idea drew so much attention and debate.

Bt cotton: the protein that kills caterpillars

Some strains of the soil bacterium Bacillus thuringiensis make crystals of an insecticidal protein during a particular phase of their growth. The proteins kill certain insect groups: lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). So why does the toxin not kill the bacterium that makes it? Because it is made as an inactive protoxin. It becomes active only inside the insect, as the following chain shows.

How Bt toxin kills, in one line: insect eats the crystals → the alkaline pH of the gut dissolves them → the protoxin is activated → it binds to the midgut epithelial cells → it makes pores → the cells swell and burst (lyse) → the insect dies.

The toxin is coded by a gene named cry. Specific cry genes were isolated from Bacillus thuringiensis and put into crop plants such as cotton. Most Bt toxins are specific to an insect group, so the choice of gene depends on the crop and the pest.

Beyond the textbook. For the exam: Bt toxin is activated by the alkaline pH of the insect gut. In reality: this is also why the toxin spares us. A human stomach is acidic, not alkaline, and the toxin acts through receptors on gut cells that are specific to certain insect groups. Why it matters: it shows that "toxin" does not mean "toxic to everything" and answers the usual safety question about Bt crops.

Pause and predict: the Bt protein is produced inside the bacterium Bacillus thuringiensis itself. What prevents it from killing the bacterium?

Check your answer: it is stored as an inactive protoxin crystal. It needs the alkaline gut of an insect to be changed into its active form.

RNA interference: switching off a parasite's gene

A nematode, Meloidogyne incognita, infects tobacco roots and reduces the yield greatly. To protect the plant, scientists used a defence that every eukaryote has: RNA interference (RNAi). In RNAi, a double-stranded RNA (dsRNA) matching a specific messenger RNA (mRNA) binds to that mRNA and stops it from being translated. This is called silencing. In nature, the source of the matching RNA can be an infection by an RNA virus or mobile genetic elements (transposons) that replicate through an RNA intermediate.

Here is how it was used. Using Agrobacterium as a vector, a nematode-specific gene was put into the tobacco plant in such a way that the plant made sense and anti-sense RNA. The two strands are complementary, so they paired to form dsRNA. This silenced the nematode's own mRNA when the nematode fed on the plant, and the parasite could not survive in the transgenic plant.

Pause and predict: why must the plant make both sense and antisense RNA, not just one of them?

Check your answer: one strand alone would not form a double-stranded molecule. RNAi is triggered by dsRNA, and a sense strand and its complementary antisense strand are what pair up to make it.

Applications in medicine

Recombinant DNA technology has produced safe and effective drugs in large amounts. Recombinant therapeutics do not cause the unwanted immune responses often seen with products from non-human sources. About 30 recombinant therapeutics are approved for human use worldwide, and 12 are marketed in India.

Genetically engineered insulin

People with diabetes need insulin. Earlier, it was taken from the pancreas of slaughtered cattle and pigs, and this animal insulin sometimes caused allergy or other reactions. Human insulin made by bacteria avoids that.

Insulin has two short polypeptide chains, chain A and chain B, joined by disulphide bridges. In the body it is first made as a pro-hormone, proinsulin, which has an extra stretch called the C peptide. The C peptide is removed as the hormone matures, so mature insulin has no C peptide. The problem for rDNA scientists was to get a correctly assembled mature insulin.

In 1983, Eli Lilly, an American company, solved it. It prepared two DNA sequences, one for chain A and one for chain B, and put them into plasmids of E. coli. The chains were made separately, extracted, and joined by creating disulphide bonds to form human insulin.

Beyond the textbook. For the exam: chains A and B were produced separately in E. coli and joined by disulphide bonds. In reality: later methods express the whole proinsulin gene in bacteria or yeast and then remove the C peptide with enzymes, which skips the joining step. Why it matters: the exam asks about the 1983 method, so do not mix the two together.

Gene therapy

Gene therapy is a set of methods that corrects a gene defect found in a child or embryo. A normal gene is delivered into the person's cells to take over the job of the non-functional gene.

The first clinical gene therapy was given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency. ADA is crucial for the working of the immune system, and the disorder is caused by deletion of the gene for the enzyme. Some children can be treated by bone marrow transplantation and others by enzyme replacement therapy, in which functional ADA is injected, but neither is completely curative.

The gene therapy steps were: lymphocytes from the patient's blood are grown in culture → a functional ADA cDNA is introduced into them using a retroviral vector → the engineered lymphocytes are returned to the patient. The cells are not immortal, so the patient needs periodic infusions. A permanent cure needs the gene to be introduced into cells at an early embryonic stage that produce ADA.

Beyond the textbook. For the exam: ADA gene therapy uses lymphocytes and a retroviral vector, and needs repeated infusions. In reality: ADA deficiency is a form of severe combined immunodeficiency, and later treatments have targeted bone marrow stem cells instead, because stem cells keep renewing themselves and give a longer-lasting effect. Why it matters: it explains why lymphocytes, which are not immortal, give only a temporary effect.

Molecular diagnosis

Conventional tests of serum or urine detect a disease only when symptoms appear and the pathogen is already numerous. Molecular tools help earlier.

The probe logic, which students get wrong. The probe is complementary to the normal gene. A clone that carries the mutated gene will not bind the probe, so it does not appear on the photographic film. You look for what is missing.

Transgenic animals

Transgenic animals are animals whose DNA has been manipulated to carry and express an extra, foreign gene. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, but over 95 per cent of all transgenic animals are mice. They are made for five reasons.

Attempts are also being made to make products for phenylketonuria (PKU) and cystic fibrosis.

Ethical issues

Genetic modification can have unpredictable effects when GMOs are released into the ecosystem, so the process cannot go on without rules. The Government of India set up the GEAC (Genetic Engineering Approval Committee) to decide on the validity of GM research and on the safety of introducing GM organisms for public use.

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. It matters because industrialised nations are rich in money but poor in biodiversity and traditional knowledge, while developing nations are the opposite. Many companies have tried to patent products and technologies based on these resources.

The most famous case is Basmati rice. In 1997 an American company got a patent from the US Patent and Trademark Office on a "new" Basmati variety, derived from Indian farmer varieties crossed with semi-dwarf varieties, with claims extending to functional equivalents. Patents were also attempted on turmeric and neem. The Indian Parliament has since cleared the second amendment of the Indian Patents Bill, which deals with such issues, including patent terms, emergency provisions and research and development initiatives.

Beyond the textbook. For the exam: Basmati, turmeric and neem are the biopiracy examples, and the 1997 Basmati patent was granted in the USA. In reality: India challenged these. The turmeric and neem patents were later revoked, and the Basmati patent lost its most important claims. Why it matters: the cases show that traditional knowledge can be defended once it is documented, which is why India has since built databases of such knowledge.

Exam traps

60-second revision

Where this chapter connects

• Previous: Biotechnology: Principles and Processes gives the tools (restriction enzymes, vectors, PCR, bioreactors) used in every application here.
• Microbes link: Microbes in Human Welfare introduces Bacillus thuringiensis as a spray-on biocontrol agent before this chapter turns it into Bt cotton.
• Immunity link: Human Health and Disease uses the same ELISA test for HIV and the recombinant hepatitis B vaccine.
• Genetics link: Molecular Basis of Inheritance covers the RNA, mRNA and translation steps that RNAi interrupts.