NEET BiologyNCERT Class 12Chapter 10

Biotechnology and its Applications: NEET notes

This chapter takes the tools of recombinant DNA technology out of the lab and into farms, hospitals and law courts. It covers tissue culture and somatic hybrids, GM crops such as Bt cotton and RNAi-protected tobacco, recombinant insulin, gene therapy for ADA deficiency, molecular diagnosis, transgenic animals, and the ethics of GM organisms and biopiracy.

What NEET asks

NEET asks why Bt toxin spares the bacterium (inactive protoxin, activated by alkaline gut pH), which cry gene controls which pest, how RNAi silences nematode mRNA, how Eli Lilly made insulin (A and B chains separately in E. coli, C peptide absent), the ADA gene-therapy steps, PCR, probes and ELISA in diagnosis, the uses of transgenic animals with Rosie's 2.4 grams per litre, and GEAC, biopiracy and the Basmati patent. Marks are lost by saying the toxin is activated by acid, by mixing up cryIAb and cryIAc, and by forgetting that ADA gene therapy with lymphocytes is not permanent.

1. Three research areas

NCERT §10 (chapter introduction)

Biotechnology, in the sense of the previous chapter, is mainly about making biopharmaceuticals and biologicals on an industrial scale, using genetically modified microbes, fungi, plants and animals.
Its applications span therapeutics, diagnostics, genetically modified crops, processed food, bioremediation, waste treatment and energy production.
Research in biotechnology has three critical areas. The first is supplying a superior catalyst: an improved organism (usually a microbe) or a pure enzyme.
The second is engineering optimal conditions in which that catalyst can act.
The third is downstream processing: the technologies that purify the protein or organic compound once it has been made.
This chapter follows those tools into two fields that touch daily life most: food production (agriculture) and health (medicine).

2. Food production and tissue culture

NCERT §10.1

Three routes can raise food production: agriculture based on agro-chemicals, organic agriculture, and agriculture based on genetically engineered crops.
The Green Revolution tripled the food supply, yet it still fell short of feeding a growing population. Its gains came partly from improved varieties but mainly from better management and agrochemicals (fertilisers and pesticides).
Agrochemicals are often too costly for farmers in developing countries, and conventional breeding cannot push the yield of existing varieties much higher.
Because traditional breeding could not keep pace with demand, tissue culture was developed. In the 1950s scientists learnt that a whole plant can be regenerated from an explant: any part of a plant removed and cultured in a test tube on a special nutrient medium, with everything kept sterile.
The capacity of any cell or explant to give rise to a whole plant is totipotency.
The medium must supply a carbon source such as sucrose, plus inorganic salts, vitamins, amino acids and growth regulators such as auxins and cytokinins.
Producing thousands of plants this way in a very short time is micro-propagation.
Every plant so produced is genetically identical to the parent it came from; such plants are somaclones.
Tomato, banana and apple are among the important food plants produced commercially by micro-propagation.

3. Meristem culture and somatic hybrids

NCERT §10.1

Tissue culture can also recover healthy plants from diseased ones. Even in a virus-infected plant the meristem, apical and axillary, stays free of virus.
So the meristem is removed and grown in vitro to give virus-free plants. Meristems of banana, sugarcane and potato have been cultured this way.
Single plant cells can be isolated and their cell walls digested away, leaving naked protoplasts bounded only by the plasma membrane.
Protoplasts from two varieties, each with a desirable character, can be fused into hybrid protoplasts that grow into a new plant.
Such plants are somatic hybrids, and the process is somatic hybridisation.
Fusing a tomato protoplast with a potato protoplast gave the pomato. It did not combine all the desired characters, so it was not used commercially.

4. Genetically modified crops

NCERT §10.1

Genetic modification offers a way to raise yields while cutting the use of fertilisers and chemicals, and so their harm to the environment.
Any plant, bacterium, fungus or animal whose genes have been deliberately manipulated is a Genetically Modified Organism (GMO).
GM crops have been made more tolerant of abiotic stresses: cold, drought, salt and heat.
Pest-resistant GM crops reduce reliance on chemical pesticides.
Genetic modification has also helped cut post-harvest losses and made plants use minerals more efficiently, which stops the soil's fertility from being exhausted early.
It has enhanced the nutritional value of food. Golden rice is rice enriched in vitamin A.
GM has also produced tailor-made plants that supply industries with alternative resources: starches, fuels and pharmaceuticals.

5. Bt cotton

NCERT §10.1

Bt toxin is made by the bacterium Bacillus thuringiensis (Bt). Its gene has been cloned and expressed in plants, giving insect resistance without insecticides: in effect, a bio-pesticide.
Crops given Bt genes include cotton and corn, and also rice, tomato, potato and soyabean.
Some strains of B. thuringiensis make proteins lethal to particular insects: lepidopterans such as the tobacco budworm and armyworm, coleopterans (beetles), and dipterans (flies and mosquitoes).
During one phase of growth the bacterium forms protein crystals that contain a toxic insecticidal protein.
The toxin does not kill the Bacillus because it is stored as an inactive protoxin.
In an insect that eats it, the alkaline pH of the gut solubilises the crystals and converts the protoxin into the active toxin.
The active toxin binds to the surface of the midgut epithelial cells and creates pores, so the cells swell and lyse and the insect dies.
Most Bt toxins act on one insect group, so the gene chosen depends on the crop and its pest. The toxin genes are named cry.
Proteins coded by cryIAc and cryIIAb control cotton bollworms; the protein of cryIAb controls the corn borer.

6. RNA interference

NCERT §10.1

Several nematodes parasitise a wide range of plants and animals, humans included. The nematode Meloidogyne incognita infects tobacco roots and greatly reduces the yield.
A new strategy against it uses RNA interference (RNAi), a cellular defence that operates in all eukaryotic organisms.
In RNAi a specific mRNA is silenced: a complementary double-stranded RNA (dsRNA) binds to it and prevents its translation.
In nature this complementary RNA may come from an infection by viruses with RNA genomes, or from transposons (mobile genetic elements) that replicate through an RNA intermediate.
Nematode-specific genes were carried into the tobacco plant by Agrobacterium vectors, inserted so that the plant's cells made both sense and anti-sense RNA.
The two RNAs, being complementary, paired into dsRNA that set off RNAi and silenced the specific mRNA of the nematode.
The parasite could not survive in the transgenic host expressing the interfering RNA, so the plant was protected.

7. Genetically engineered insulin

NCERT §10.2, §10.2.1

Recombinant DNA technology allows mass production of safe and more effective therapeutic drugs. Recombinant therapeutics do not provoke the unwanted immune responses common with similar products taken from non-human sources.
Worldwide, about 30 recombinant therapeutics have approval for use in humans; 12 of these are currently sold in India.
Adult-onset diabetes can be managed by taking insulin at regular intervals.
Insulin was earlier extracted from the pancreas of slaughtered cattle and pigs. Being a foreign protein, animal insulin caused allergy or other reactions in some patients.
Insulin is made of two short polypeptide chains, A and B, linked by disulphide bridges.
In mammals, humans included, insulin is made as a pro-hormone that must be processed before it works. The pro-hormone carries an extra stretch, the C peptide, which is removed during maturation and is absent from mature insulin.
The main difficulty in making insulin by rDNA techniques was getting it assembled into the mature form.
In 1983 the American company Eli Lilly made two DNA sequences, one for the A chain and one for the B chain of human insulin, and placed them in E. coli plasmids.
The A and B chains were produced separately, extracted, and joined by creating disulphide bonds, giving human insulin.

8. Gene therapy

NCERT §10.2.2

Gene therapy is a set of methods for correcting a gene defect diagnosed in a child or an embryo. It treats disease by putting genes into the person's cells and tissues.
A normal gene is delivered into the person or embryo to take over the function of the non-functional gene and compensate for it.
The first clinical gene therapy was in 1990: the patient, a girl aged 4, had adenosine deaminase (ADA) deficiency.
ADA is an enzyme crucial for the immune system to work. The disorder arises from deletion of the gene for ADA.
Some children with ADA deficiency can be cured by bone marrow transplantation; others receive enzyme replacement therapy, injections of working ADA. Neither approach is completely curative.
In gene therapy, lymphocytes from the patient's blood are grown in culture outside the body, a functional ADA cDNA is introduced into them using a retroviral vector, and the cells are returned to the patient.
These lymphocytes are not immortal, so the patient needs periodic infusions of the engineered cells.
Putting the ADA gene, isolated from marrow cells, into cells of an early embryo could cure the disorder permanently.

9. Molecular diagnosis

NCERT §10.2.3

Effective treatment needs early diagnosis and an understanding of the disease's pathophysiology. Conventional methods such as serum and urine analysis cannot detect disease early.
Early diagnosis is possible with three techniques: recombinant DNA technology, PCR (Polymerase Chain Reaction) and ELISA (Enzyme Linked Immuno-sorbent Assay).
A pathogen is usually suspected only after it causes symptoms, by which time its concentration in the body is already very high.
PCR amplifies the nucleic acid of a bacterium or virus, so it can be detected at a very low concentration, before symptoms appear.
PCR is now a routine test for HIV when AIDS is suspected, and it finds gene mutations when cancer is suspected. It also identifies many other genetic disorders.
A probe is single-stranded DNA or RNA carrying a radioactive tag. The probe is left to pair (hybridise) with complementary DNA in a clone of cells and is then located by autoradiography.
A clone carrying a mutated gene does not show on the photographic film, because the probe is not complementary to the mutated gene.
ELISA works on antigen-antibody interaction. Infection is detected either from the pathogen's antigens (proteins, glycoproteins and so on) or from the antibodies made against the pathogen.

10. Transgenic animals

NCERT §10.3

Transgenic animals have had their DNA manipulated so that they carry and express an extra, foreign gene.
Rats, rabbits, pigs, sheep, cows and fish have all been made transgenic, yet mice make up over 95 per cent of the transgenic animals that exist.
Normal physiology and development: they reveal how genes are regulated and how they affect body function and development, for example the role of insulin-like growth factor, studied by adding genes from other species that alter its formation.
Study of disease: they serve as models of human diseases, so new treatments can be investigated. Transgenic models exist for cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's disease.
Biological products: animals given the gene for a product can make costly medicines, such as human α-1-antitrypsin, a treatment for emphysema. Work along the same lines targets phenylketonuria (PKU) and cystic fibrosis.
In 1997 Rosie, the first transgenic cow, produced milk enriched with human protein (2.4 grams per litre). It contained human alpha-lactalbumin and was nutritionally more balanced for human babies than natural cow milk.
Vaccine safety: transgenic mice now check that the polio vaccine is safe before it reaches humans. If reliable, they could replace monkeys in testing vaccine batches.
Chemical safety (toxicity) testing: animals carrying genes that make them more sensitive to toxic substances than normal animals are exposed to the substances, giving results in less time.

11. Ethical issues and biopiracy

NCERT §10.4

Manipulating living organisms cannot continue without regulation; ethical standards are needed to judge the morality of activities that may help or harm organisms.
Beyond morality there is biological risk: a GM organism released into an ecosystem may have unpredictable effects.
The Indian Government has set up bodies such as GEAC (Genetic Engineering Approval Committee), which rule on whether GM research is valid and whether releasing GM organisms for public services is safe.
Using organisms for public services such as food and medicine has also raised problems over patents, including public anger at patents granted on genes, plants and bio-resources that farmers and indigenous people of a region found, developed and used long ago.
India has an estimated 200,000 varieties of rice, and 27 documented varieties of Basmati, which is distinct for its aroma and flavour and is mentioned in ancient texts, folklore and poetry.
In 1997 an American company was granted a US patent on Basmati rice. Its 'new' variety came from Indian farmers' varieties crossed with semi-dwarf varieties, and the patent covered functional equivalents, which could restrict others selling Basmati.
There have also been attempts to patent the uses, products and processes of Indian traditional herbal medicines, such as turmeric and neem.
Biopiracy: multinational companies or other organisations using a country's bio-resources without proper authorisation from that country and its people, and without paying compensation.
Most industrialised nations have money but little biodiversity or traditional knowledge; developing and underdeveloped nations have plenty of both. Traditional knowledge can save time, effort and expense in commercialisation.
Some nations are framing laws against such unauthorised use. The Indian Parliament has cleared the second amendment of the Indian Patents Bill, which takes these issues into account, including patent terms, emergency provisions and research and development initiatives.

Must-know facts

Three research areas: superior catalyst (organism or pure enzyme), optimal conditions for it, downstream processing.
Three routes to more food: agro-chemical, organic, and GM crop-based agriculture; the Green Revolution tripled food supply.
Totipotency: any cell or explant can regenerate a whole plant; micro-propagation gives genetically identical somaclones.
Tissue culture medium: sucrose as carbon source, inorganic salts, vitamins, amino acids, auxins and cytokinins.
Virus-free plants come from the meristem (apical and axillary), which stays free of virus; banana, sugarcane, potato.
Protoplast fusion gives somatic hybrids; tomato + potato gave the pomato, which was not commercially useful.
Golden rice is vitamin A enriched rice.
Bt toxin is an inactive protoxin; the insect's alkaline gut pH solubilises the crystal and activates it; it makes pores in midgut epithelial cells.
Bt proteins act on lepidopterans, coleopterans and dipterans; cryIAc and cryIIAb control cotton bollworms, cryIAb controls corn borer.
RNAi against Meloidogyne incognita in tobacco: Agrobacterium vectors, sense + anti-sense RNA form dsRNA that silences nematode mRNA.
About 30 recombinant therapeutics approved worldwide; 12 marketed in India.
Insulin: chains A and B joined by disulphide bridges; pro-insulin carries a C peptide removed at maturation.
1983, Eli Lilly: A and B chain DNA put in E. coli plasmids, chains made separately and joined by disulphide bonds.
First clinical gene therapy: 1990, a 4-year-old girl with ADA deficiency; lymphocytes + ADA cDNA via retroviral vector; periodic infusion needed.
PCR detects pathogens at very low concentration and is routine for HIV; radioactive probes + autoradiography; ELISA uses antigen-antibody interaction.
Over 95 per cent of transgenic animals are mice.
Rosie (1997), the first transgenic cow: milk with human alpha-lactalbumin, 2.4 grams per litre.
Human α-1-antitrypsin from transgenic animals treats emphysema; transgenic mice test polio vaccine safety.
GEAC = Genetic Engineering Approval Committee; decides on GM research and release.
Basmati patent 1997 by an American company; India has about 200,000 rice varieties and 27 documented Basmati varieties.

Common traps

Saying Bt toxin does not kill the bacterium because the bacterium is resistant to it.

The toxin is stored as an inactive protoxin; only the alkaline gut of the insect activates it.

Saying the protoxin is activated by the acidic pH of the insect gut.

The insect gut is alkaline; the alkaline pH solubilises the crystal and activates the toxin.

Pairing cryIAb with cotton bollworm.

cryIAc and cryIIAb control cotton bollworms; cryIAb controls the corn borer.

Thinking RNAi in the tobacco plant kills the nematode with a toxin protein.

The plant makes dsRNA that silences a specific nematode mRNA, so the parasite cannot survive; no toxin protein is involved.

Writing that Eli Lilly's E. coli made pro-insulin with the C peptide.

Separate DNA for chains A and B was used; the chains were made separately and joined by disulphide bonds, so no C peptide was involved.

Calling ADA gene therapy with lymphocytes a permanent cure.

Lymphocytes are not immortal, so infusions are repeated; only a gene put into early embryonic cells could be permanent.

Saying somaclones are genetically varied.

Micro-propagated plants are genetically identical to the parent; that is what somaclone means here.

Taking the whole infected plant for virus-free culture.

Only the apical or axillary meristem is virus-free, so the meristem is cultured.

Expanding GEAC as Genetic Engineering Advisory Committee.

It is the Genetic Engineering Approval Committee.

Key terms

Explant: Any plant part taken out and grown in a test tube on a sterile nutrient medium.
Totipotency: The ability of a single cell or explant to regenerate a whole plant.
Micro-propagation: Producing thousands of plants quickly by tissue culture.
Somaclone: A tissue-culture plant genetically identical to the parent it came from.
Protoplast: A plant cell whose wall has been digested away, bounded only by the plasma membrane.
Somatic hybridisation: Fusing protoplasts of two varieties and growing the hybrid cell into a plant.
GMO: An organism whose genes have been altered by manipulation.
Bt toxin: Insecticidal protein of Bacillus thuringiensis, stored as an inactive protoxin crystal.
cry gene: A gene coding for a Bt crystal toxin protein, such as cryIAc or cryIAb.
RNA interference: Silencing of a specific mRNA by a complementary double-stranded RNA that blocks its translation.
C peptide: The extra stretch in pro-insulin, removed when it matures into insulin.
Gene therapy: Correcting a gene defect by delivering a normal gene into a person's cells or an embryo.
ADA deficiency: Immune disorder caused by deletion of the gene for adenosine deaminase.
Probe: A radioactively tagged single-stranded DNA or RNA that finds its complementary sequence.
ELISA: Enzyme Linked Immuno-sorbent Assay: detects infection by antigen-antibody interaction.
Transgenic animal: An animal whose DNA has been manipulated to carry and express a foreign gene.
GEAC: Genetic Engineering Approval Committee, which rules on GM research and release in India.
Biopiracy: Using a country's bio-resources without authorisation and without compensatory payment.

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