Biotechnology and its Applications

2.1 Core concepts

• Biotechnology essentially deals with industrial-scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants and animals; its applications span therapeutics, diagnostics, GM crops for agriculture, processed food, bioremediation, waste treatment and energy production (NCERT §10 intro, p. 177).
• Three critical research areas of biotechnology are (i) providing the best catalyst in the form of an improved organism — usually a microbe or pure enzyme; (ii) creating optimal conditions through engineering for a catalyst to act; and (iii) downstream processing technologies to purify the protein/organic compound (NCERT §10 intro, p. 177).
• Three options exist for increasing food production — agro-chemical based agriculture, organic agriculture, and genetically engineered crop-based agriculture; the Green Revolution tripled food supply but was insufficient for growing demand. Increased yields were partly due to improved crop varieties but mainly due to better management practices and agro-chemicals (fertilisers and pesticides), which are often too expensive for farmers in the developing world (NCERT §10.1, p. 178).
• Tissue culture (developed in the 1950s) exploits totipotency — the capacity of any cell/explant to regenerate a whole plant. An explant is any part of a plant taken out and grown in a test tube under sterile conditions in special nutrient media containing a carbon source (sucrose), inorganic salts, vitamins, amino acids and growth regulators like auxins and cytokinins (NCERT §10.1, p. 178).
• Micro-propagation produces thousands of plants through tissue culture in very short durations; each plant is genetically identical to the original parent and is called a somaclone. Tomato, banana and apple have been produced on commercial scale by this method (NCERT §10.1, p. 178).
• Recovery of healthy plants from diseased plants is a second important application: even if a plant is infected with a virus, the meristem (apical and axillary) is free of virus; meristem culture yields virus-free banana, sugarcane and potato (NCERT §10.1, p. 178).
• Somatic hybridisation fuses naked protoplasts (cell walls digested, plasma membranes intact) of two different varieties — each having a desirable character — to make somatic hybrids. The tomato-potato fusion called pomato was achieved but lacked all the desired combination of characters for commercial use (NCERT §10.1, pp. 178-179).
• Genetically Modified Organisms (GMOs) are plants, bacteria, fungi or animals whose genes have been altered by manipulation. GM plants have been useful in many ways: (i) more tolerant to abiotic stresses (cold, drought, salt, heat); (ii) reduced reliance on chemical pesticides (pest-resistant crops); (iii) reduced post-harvest losses; (iv) increased efficiency of mineral usage (preventing early exhaustion of soil fertility); (v) enhanced nutritional value of food, e.g. golden rice — Vitamin 'A' enriched rice. GM has also created tailor-made plants supplying starches, fuels and pharmaceuticals (NCERT §10.1, p. 179).
• Bt toxin: Some strains of Bacillus thuringiensis produce proteins that kill insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). B. thuringiensis forms protein crystals during a particular phase of growth; these crystals contain a toxic insecticidal protein. The toxin does not kill the Bacillus because it exists as an inactive protoxin; once an insect ingests it, the alkaline pH of the gut solubilises the crystals, the activated toxin binds midgut epithelial cells, creates pores that cause cell swelling and lysis, and the insect dies (NCERT §10.1, p. 179).
• Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into crop plants such as cotton (Figure 10.1). The choice depends on the crop and the targeted pest, as most Bt toxins are insect-group specific. The toxin is coded by a gene named cry: proteins encoded by cryIAc and cryIIAb control cotton bollworms, while cryIAb controls corn borer (NCERT §10.1, p. 180).
• Pest-Resistant Plants by RNAi: Several nematodes parasitise plants and animals. The nematode Meloidogyne incognita infects roots of tobacco plants and causes a great reduction in yield. RNA interference (RNAi) — a method of cellular defence in all eukaryotes — silences a specific mRNA via a complementary dsRNA molecule that binds and prevents translation. The source of the complementary RNA can be infection by RNA viruses or mobile genetic elements (transposons) replicating via an RNA intermediate (NCERT §10.1, p. 180).
• Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant such that the DNA produced both sense and anti-sense RNA in host cells. The two complementary RNAs formed a double-stranded RNA (dsRNA) that initiated RNAi and silenced the specific mRNA of the nematode; the parasite could not survive in the transgenic host expressing specific interfering RNA (Figure 10.2) (NCERT §10.1, p. 180).
• The recombinant DNA technological processes have made immense impact in healthcare by mass-producing safe and more effective therapeutic drugs; recombinant therapeutics do not induce unwanted immunological responses common with products from non-human sources. At present, about 30 recombinant therapeutics are approved for human use worldwide; 12 are presently marketed in India (NCERT §10.2, p. 181).
• Genetically Engineered Insulin: Adult-onset diabetes is managed by taking insulin at regular intervals. Insulin used earlier was extracted from the pancreas of slaughtered cattle and pigs, but animal-source insulin sometimes caused allergy or other reactions. Insulin consists of two short polypeptide chains — chain A and chain B — linked together by disulphide bridges (Figure 10.3) (NCERT §10.2.1, p. 181).
• In mammals, including humans, insulin is synthesised as a pro-hormone (like a pro-enzyme — it needs processing before becoming fully mature and functional) containing an extra stretch called the C peptide. The C peptide is not present in mature insulin and is removed during maturation. The main challenge for rDNA-based insulin was getting insulin assembled into a mature form. In 1983, Eli Lilly, an American company, prepared two DNA sequences corresponding to chains A and B of human insulin and introduced them into plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulphide bonds to form human insulin (NCERT §10.2.1, p. 182).
• Gene Therapy is a collection of methods that allows correction of a gene defect diagnosed in a child/embryo. Genes are inserted into a person's cells and tissues to treat a disease, especially hereditary; correction involves delivery of a normal gene to take over the function of the non-functional gene (NCERT §10.2.2, p. 182).
• The first clinical gene therapy was given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency. ADA is crucial for immune-system function; the disorder is caused by deletion of the gene for adenosine deaminase. In some children ADA deficiency is cured by bone marrow transplantation; in others it is treated by enzyme replacement therapy (functional ADA injected) — but neither approach is completely curative. As a first step toward gene therapy, lymphocytes from the patient's blood are grown in culture outside the body; a functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes and they are returned to the patient. Because these cells are not immortal, the patient requires periodic infusion of such engineered lymphocytes. Introducing the gene isolate into cells producing ADA at early embryonic stages could give a permanent cure (NCERT §10.2.2, p. 182).
• Molecular Diagnosis: Conventional methods (serum/urine analysis etc.) do not enable early detection. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno-Sorbent Assay (ELISA) serve early diagnosis. A pathogen is usually suspected only after symptoms appear, by which time concentration is already high; very low concentrations of bacteria/viruses can be detected by amplification of nucleic acid through PCR. PCR is now routinely used to detect HIV in suspected AIDS patients and mutations in suspected cancer patients, and is a powerful technique to identify many genetic disorders (NCERT §10.2.3, pp. 182-183).
• A single-stranded DNA or RNA tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells, followed by detection using autoradiography. A clone having the mutated gene will not appear on the photographic film because the probe will not have complementarity with the mutated gene. ELISA is based on the principle of antigen-antibody interaction; infection can be detected by presence of antigens (proteins, glycoproteins, etc.) or by detecting antibodies synthesised against the pathogen (NCERT §10.2.3, p. 183).
• Transgenic Animals are animals whose DNA has been manipulated to possess and express an extra (foreign) gene. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 per cent of all existing transgenic animals are mice. They serve five purposes (NCERT §10.3, pp. 183-184):
• (i) Normal physiology and development — e.g. study of complex factors like insulin-like growth factor by introducing genes that alter formation of the factor.
• (ii) Study of disease — models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.
• (iii) Biological products — human protein α-1-antitrypsin for emphysema; attempts for phenylketonuria (PKU) and cystic fibrosis. In 1997, the first transgenic cow Rosie produced human protein-enriched milk (2.4 grams per litre) containing human alpha-lactalbumin, nutritionally more balanced for human babies than natural cow milk.
• (iv) Vaccine safety — transgenic mice are being used to test the safety of the polio vaccine; if successful, they could replace monkeys.
• (v) Chemical safety testing (toxicity testing) — transgenic animals carrying genes that make them more sensitive to toxic substances yield results in less time.
• Ethical Issues: Manipulation of living organisms cannot go on without regulation; genetic modification can have unpredictable results when GMOs are introduced into the ecosystem. The Indian Government has set up the GEAC (Genetic Engineering Approval Committee) to take decisions regarding the validity of GM research and the safety of introducing GM organisms for public services (NCERT §10.4, p. 184).
• 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. Industrialised nations are financially rich but poor in biodiversity and traditional knowledge; the developing world is the opposite. Cases include the 1997 US patent on Basmati rice (the 'new' variety derived from Indian farmer varieties crossed with semi-dwarf varieties, with claims extending to functional equivalents), and patent attempts on turmeric and neem. The Indian Parliament has cleared the second amendment of the Indian Patents Bill, addressing emergency provisions, research and development (NCERT §10.4, pp. 184-185).

2.2 Definitions to memorise

2.3 Diagrams / processes to remember

• Figure 10.1 (p. 180): Cotton boll — (a) destroyed by bollworms vs. (b) a fully mature cotton boll. Visual demonstration of crop loss prevented by introducing cryIAc/cryIIAb into cotton to make Bt cotton.
• Figure 10.2 (p. 181): RNAi-protected tobacco roots — (a) roots of typical control plants vs. (b) transgenic plant roots 5 days after deliberate infection of nematode but protected through the novel RNAi mechanism. Shows clean, healthy transgenic roots despite challenge.
• Figure 10.3 (p. 182): Maturation of pro-insulin into insulin (simplified) — single proinsulin ring shows chain A + chain B + C peptide held together by disulphide bridges; cleavage releases the free C peptide plus mature insulin where chains A and B remain joined by S-S bonds. The B peptide and A peptide labels with disulphide cross-links are diagnostic for figure-based MCQs.
• Bt toxin activation pathway (p. 179): inactive crystalline protoxin in bacterium -> ingestion by insect -> alkaline gut pH solubilises crystals -> active toxin -> binds midgut epithelial cells -> creates pores -> cell swelling and lysis -> insect death.
• RNAi flowchart (p. 180): Agrobacterium vector delivers nematode-specific DNA into host -> host transcribes both sense and anti-sense RNA -> the two pair to form dsRNA -> dsRNA initiates RNAi -> nematode mRNA silenced -> parasite cannot survive -> transgenic plant protected.
• Recombinant insulin assembly (p. 182): two DNA sequences for chains A and B prepared -> inserted separately into plasmids of E. coli -> chains A and B produced separately -> extracted -> combined via disulphide bonds -> mature human insulin (identical to natural molecule).
• ADA gene therapy steps (p. 182): lymphocytes drawn from patient -> cultured outside the body -> functional ADA cDNA introduced using a retroviral vector -> engineered lymphocytes returned to the patient -> periodic infusion required (lymphocytes are not immortal) -> permanent cure would require gene introduction at early embryonic stage.

2.4 Common confusions / NTA trap points

• ***cryIAc* vs. cryIIAb vs. cryIAb — the first two control cotton bollworms**; cryIAb (single I, single A, lowercase b) controls corn borer. NTA loves to swap these in matching items.
• Bt toxin is not lethal to Bacillus itself because it exists as an inactive protoxin in crystalline form — not because the bacterium is "resistant" (Exercise option a) or "encloses it in a special sac" (Exercise option d), and not because the toxin is "immature" (option b).
• The first ADA gene therapy patient (1990) was a 4-year-old girl; her treatment is not curative because lymphocytes are not immortal — periodic infusion is needed. Embryonic-stage introduction is the suggested permanent cure.
• Eli Lilly produced insulin chains A and B separately in E. coli plasmids and joined them via disulphide bonds — NCERT does not say the proinsulin/C-peptide gene was cloned as one unit, nor that yeast was used.
• Rosie the cow (1997) produced milk with human alpha-lactalbumin at 2.4 g/L, not human insulin and not ADA. α-1-antitrypsin treats emphysema, not PKU; PKU and cystic fibrosis are listed as targets being attempted.
• GEAC stands for Genetic Engineering Approval Committee — not "Appraisal", not "Authority", not "Agency".
• Pomato is the tomato-potato somatic hybrid; it failed commercially because it lacked the desired combination of characters — NCERT does not say it was infertile or banned.
• Meristem (apical and axillary) is virus-free, hence used for virus-free banana/sugarcane/potato — not for somatic hybridisation. NTA may swap "meristem culture" with "protoplast fusion".
• Probe-autoradiography logic is inverted: the clone with the mutated gene does not appear on the film because the probe is complementary to the normal gene. Many students wrongly mark "the mutated clone glows".
• 30 recombinant therapeutics worldwide; 12 in India — exact numbers are frequently asked. Do not confuse with "12 worldwide".
• Golden rice = Vitamin A enriched rice, not iron or protein enriched.
• Basmati patent was granted by the US Patent and Trademark Office in 1997; the variety was derived by crossing Indian Basmati with semi-dwarf varieties — not "American long-grain".

2.5 Key processes / classifications