# Biotechnology and its Applications (NEET Biology Class 12): Bt Cotton, RNAi, Insulin & Gene Therapy Source: https://tracknation.in/story/biotechnology-and-its-applications-neet-biology-class-12-bt-cotton-rnai-insulin-gene-therapy (TrackNation — free UPSC notes with PYQs) > NEET Class 12 Biotechnology and its Applications, mined from NCERT: tissue culture terms tabulated (explant, totipotency, micro-propagation, somaclones, somatic hybridisation), the five benefits of GM crops, Bt cotton with the full toxin-activation mechanism and a cry-gene-to-target-pest table, RNA interference, genetically engineered insulin (the 1983 Eli Lilly route, chains A and B, the C peptide), gene therapy for ADA deficiency, a molecular diagnosis table (PCR, radioactive probes, ELISA), a ## Tissue Culture & Genetically Modified Crops 📌 **NEET priority: Important.** *2–4 questions a year. The **cry gene → target pest table**, the **Bt toxin mechanism**, **RNAi**, the **1983 Eli Lilly insulin route** and the **five uses of transgenic animals** are where the marks reliably are.* ### Tissue culture — the foundation techniques | **Term** | **Definition** | | Explant | ANY part of a plant taken out and grown in a test tube under STERILE conditions in a special nutrient medium | | Totipotency | The capacity to generate a WHOLE PLANT from any cell or explant | | Micro-propagation | Producing THOUSANDS of plants through tissue culture in a very short duration. Used commercially for TOMATO, BANANA, APPLE | | Somaclones | The plants so produced — GENETICALLY IDENTICAL to the original plant | | Somatic hybridisation | Fusing naked PROTOPLASTS (cell walls digested away) from two different plant varieties, each with a desirable character, to get a SOMATIC HYBRID | - The nutrient medium must supply a **carbon source such as sucrose**, plus **inorganic salts, vitamins, amino acids and growth regulators (auxins, cytokinins)**. - **Recovering virus-free plants:** even in an infected plant, the **MERISTEM (apical and axillary) is FREE of virus**. Culturing the meristem in vitro therefore yields virus-free plants — done successfully for **banana, sugarcane and potato**. - **Pomato** — the tomato × potato somatic hybrid — was actually achieved, but **did NOT have the desired combination of characteristics** for commercial use. NEET likes this cautionary detail. ### Genetically Modified Organisms (GMO) — the five benefits - GM has (1) made crops **tolerant to abiotic stresses** (cold, drought, salt, heat); (2) **reduced reliance on chemical pesticides** via pest-resistant crops; (3) **reduced post-harvest losses**; (4) **increased efficiency of mineral usage**, preventing early exhaustion of soil fertility; and (5) **enhanced nutritional value** — e.g. **GOLDEN RICE, vitamin-A enriched rice**. ## Bt Cotton & RNA Interference ### Bt cotton — the mechanism NEET tests step by step - Some strains of ***Bacillus thuringiensis*** form **protein crystals** during a particular growth phase, containing a toxic insecticidal protein. These kill **lepidopterans** (tobacco budworm, armyworm), **coleopterans** (beetles) and **dipterans** (flies, mosquitoes). - **Why the toxin doesn't kill the Bacillus itself:** it exists as an **INACTIVE PROTOXIN**. Once an insect ingests it, the **ALKALINE pH of the insect gut** solubilises the crystals and converts it to the **ACTIVE toxin**. The activated toxin binds the **midgut epithelial cell surface**, creating **pores** that cause **cell swelling and lysis**, killing the insect. | **cry gene** | **Controls** | | cryIAc | Cotton BOLLWORMS | | cryIIAb | Cotton BOLLWORMS | | cryIAb | CORN BORER | The toxin is coded by genes named **cry**. **Most Bt toxins are insect-group SPECIFIC**, so the choice of gene depends on the crop and the targeted pest. Bt crops include **Bt cotton, Bt corn, rice, tomato, potato and soyabean**. ### RNA interference (RNAi) — pest-resistant plants - The nematode ***Meloidegyne incognitia*** infects **tobacco** roots and greatly reduces yield. - **RNAi occurs in ALL eukaryotic organisms as a method of CELLULAR DEFENCE.** It silences a specific mRNA using a **complementary dsRNA** that binds it and **prevents translation**. The natural source of such complementary RNA is infection by **RNA-genome viruses** or **transposons** (mobile genetic elements replicating via an RNA intermediate). - The strategy: using **Agrobacterium vectors**, nematode-specific genes were introduced into the host plant such that it produced **BOTH sense AND anti-sense RNA**. Being complementary, these formed **dsRNA**, initiating RNAi and silencing the nematode's mRNA. The parasite could not survive — the transgenic plant protected itself. ## Insulin, Gene Therapy & Molecular Diagnosis ### Genetically engineered insulin - Insulin has **two short polypeptide chains — A and B — linked by DISULPHIDE BRIDGES**. In mammals it is synthesised as a **PRO-HORMONE** containing an extra stretch called the **C PEPTIDE**, which is **removed during maturation** and is **NOT present in mature insulin**. - Insulin was earlier extracted from the **pancreas of slaughtered cattle and pigs**, which caused **allergy and immune reactions** in some patients. - **In 1983, Eli Lilly** (an American company) prepared two DNA sequences corresponding to **chains A and B**, introduced them into ***E. coli* plasmids**, produced the chains **SEPARATELY**, extracted them, and **combined them by creating disulfide bonds** to form human insulin. The main challenge was **getting insulin assembled into a mature form**. ### Gene therapy - **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**. - Existing options are **bone marrow transplantation** and **enzyme replacement therapy** (injecting functional ADA) — but **neither is completely curative**. - **The procedure:** lymphocytes from the patient's blood are grown in culture **outside the body**; a functional **ADA cDNA is introduced using a RETROVIRAL VECTOR**; the cells are returned to the patient. Because **these cells are not immortal**, the patient needs **PERIODIC INFUSION**. A **permanent cure** would require introducing the gene into cells at **early embryonic stages**. ### Molecular diagnosis | **Technique** | **Principle** | **Use** | | PCR | Amplification of the pathogen's nucleic acid | Detects VERY LOW concentrations of bacteria or virus BEFORE symptoms appear. Routinely used to detect HIV in suspected AIDS patients, and to detect gene mutations in suspected cancer patients | | Radioactive probe + autoradiography | A single-stranded DNA or RNA tagged with a radioactive molecule hybridises to its complementary DNA in a clone of cells | The clone with the MUTATED gene will NOT appear on the photographic film, because the probe has no complementarity with the mutated gene | | ELISA | Antigen-antibody interaction | Detects infection by the presence of ANTIGENS (proteins, glycoproteins) or by detecting ANTIBODIES synthesised against the pathogen | Conventional methods (serum and urine analysis) cannot achieve early detection — by the time symptoms appear, pathogen concentration is already very high. ## Transgenic Animals, Ethics & Why This Matters for NEET ### Transgenic animals — the five reasons they are made - Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, but **over 95 per cent of all existing transgenic animals are MICE**. | **Purpose** | **What it involves** | **Example** | | 1. Normal physiology and development | Study how genes are regulated and affect normal body function | Insulin-like growth factor — introducing genes from other species that alter its formation | | 2. Study of disease | Models for human diseases, enabling investigation of new treatments | Transgenic models for CANCER, CYSTIC FIBROSIS, RHEUMATOID ARTHRITIS and ALZHEIMER'S | | 3. Biological products | Introduce the DNA coding for a valuable human protein | α-1-antitrypsin to treat EMPHYSEMA; attempts for PKU and cystic fibrosis. In 1997 the first transgenic cow, ROSIE, produced human protein-enriched milk (2.4 g/litre) containing human ALPHA-LACTALBUMIN — nutritionally more balanced for human babies than natural cow milk | | 4. Vaccine safety | Transgenic mice test vaccine safety before human use | Testing the POLIO vaccine — could replace the use of MONKEYS for safety testing of vaccine batches | | 5. Chemical safety testing | Also called toxicity/safety testing. Animals carry genes making them MORE SENSITIVE to toxic substances | Allows results to be obtained in LESS TIME | ### Ethical issues, GEAC and biopiracy - The Indian Government set up the **GEAC — Genetic Engineering Approval Committee** — to decide on the validity of GM research and the safety of introducing GM organisms for public services. - **Biopiracy** is the use of bio-resources by multinational companies and other organisations **without proper authorisation** from the countries and people concerned. - **The Basmati case:** India has an estimated **200,000 varieties of rice**, including **27 documented varieties of Basmati**. In **1997 an American company got patent rights on Basmati rice** through the US Patent and Trademark Office — a 'new' variety actually derived from Indian farmers' varieties, crossed with semi-dwarf varieties and claimed as an invention. Similar patent attempts have been made on **turmeric and neem**. ### Why this matters for NEET - High-value one-liners: **cryIAc and cryIIAb → bollworm**, **cryIAb → corn borer**; Bt protoxin activated by **ALKALINE gut pH**; RNAi uses **dsRNA**; insulin = **Eli Lilly, 1983**, chains **A and B** joined by **disulphide bridges**, **C peptide removed**; first gene therapy **1990, ADA deficiency**; **Rosie, 1997**, **2.4 g/L** alpha-lactalbumin; **95% of transgenic animals are mice**; **GEAC**; Basmati patent **1997**. - **Trap:** the **Bt toxin does NOT kill the Bacillus** because it is an inactive **protoxin** — it only activates in the insect's alkaline gut. This "why doesn't it kill its own producer" question is a NEET favourite. Also, the **C peptide is absent from MATURE insulin** — it exists only in the pro-hormone. - **Second trap:** in gene therapy for ADA deficiency, the treatment is **NOT a permanent cure** because the engineered lymphocytes are **not immortal** and need periodic reinfusion — a permanent cure would need the gene introduced at the **early embryonic stage**. And **somaclones are genetically IDENTICAL** to the parent, unlike somatic hybrids which combine two different varieties. ## Test Yourself: MCQs, PYQs & Active Recall Answer these, then close the article and do an Active Recall. Reveal each answer only after you commit to one. ## Practice Questions **Q1.** The capacity to generate a whole plant from any cell or explant is called: - (a) Totipotency - (b) Transformation - (c) Somatic hybridisation - (d) Micro-propagation **Answer: (a)** — Totipotency is the capacity of any plant cell or explant to regenerate an entire plant. **Q2.** Plants produced through tissue culture that are genetically identical to the parent are called: - (a) Somaclones - (b) Somatic hybrids - (c) Protoplasts - (d) Transgenics **Answer: (a)** — Micro-propagation produces somaclones — plants genetically identical to the original plant. **Q3.** Virus-free plants can be obtained by culturing which plant part? - (a) Leaf lamina - (b) Mature stem - (c) Meristem (apical and axillary) - (d) Root tip **Answer: (c)** — Even in an infected plant the meristem is free of virus, so culturing it in vitro gives virus-free plants. **Q4.** Pomato, produced by somatic hybridisation of tomato and potato: - (a) Is a transgenic plant - (b) Did not have the desired combination of characteristics for commercial use - (c) Is widely cultivated commercially - (d) Was never actually produced **Answer: (b)** — The hybrid was achieved, but it did not have all the desired combination of characteristics for commercial utilisation. **Q5.** Golden rice is an example of genetic modification that: - (a) Increases drought tolerance - (b) Enhances nutritional value with vitamin A - (c) Increases pest resistance - (d) Reduces post-harvest losses **Answer: (b)** — Golden rice is vitamin A-enriched rice — an example of GM enhancing nutritional value. **Q6.** Bt toxin does not kill the Bacillus that produces it because: - (a) It only affects plants - (b) It exists as an inactive protoxin, activated only by the alkaline pH of the insect gut - (c) It is stored outside the cell - (d) Bacillus has an antidote **Answer: (b)** — The toxin exists as an inactive protoxin; the alkaline pH of the insect gut solubilises the crystals and activates it. **Q7.** The activated Bt toxin kills insects by: - (a) Blocking digestion of protein - (b) Binding midgut epithelial cells and creating pores causing swelling and lysis - (c) Blocking respiration - (d) Preventing moulting **Answer: (b)** — The activated toxin binds the surface of midgut epithelial cells and creates pores causing cell swelling and lysis. **Q8.** The cry gene that controls the corn borer is: - (a) cryIAb - (b) cryIAc - (c) cryIIAb - (d) cryIIIA **Answer: (a)** — cryIAb controls the corn borer, while cryIAc and cryIIAb control cotton bollworms. **Q9.** The nematode that infects tobacco roots, controlled using RNAi, is: - (a) Wuchereria bancrofti - (b) Caenorhabditis elegans - (c) Ascaris lumbricoides - (d) Meloidegyne incognitia **Answer: (d)** — Meloidegyne incognitia infects tobacco roots and causes a great reduction in yield. **Q10.** RNA interference works by: - (a) A complementary dsRNA binding to and preventing translation of a specific mRNA - (b) Removing introns - (c) Amplifying the gene - (d) Cutting the DNA **Answer: (a)** — RNAi silences a specific mRNA because a complementary double-stranded RNA binds it and prevents translation. **Q11.** RNA interference occurs naturally in eukaryotes as a method of: - (a) Gene amplification - (b) Cellular defence - (c) Protein folding - (d) DNA replication **Answer: (b)** — RNAi takes place in all eukaryotic organisms as a method of cellular defence. **Q12.** Insulin consists of: - (a) Four chains as H2L2 - (b) Two chains A and B linked by disulphide bridges - (c) A single polypeptide chain - (d) Three chains including C peptide **Answer: (b)** — Insulin has two short polypeptide chains, A and B, linked together by disulphide bridges. **Q13.** The C peptide of pro-insulin is: - (a) Present in mature insulin - (b) A disulphide bridge - (c) Removed during maturation and absent from mature insulin - (d) The active site **Answer: (c)** — The C peptide is an extra stretch in the pro-hormone that is removed during maturation into insulin. **Q14.** Human insulin was produced by recombinant DNA technology in 1983 by: - (a) Genentech - (b) Pfizer - (c) Eli Lilly - (d) Monsanto **Answer: (c)** — In 1983 Eli Lilly prepared DNA sequences for chains A and B, introduced them into E. coli plasmids and combined the chains by disulfide bonds. **Q15.** The first clinical gene therapy in 1990 was given for: - (a) Adenosine deaminase (ADA) deficiency - (b) Phenylketonuria - (c) Sickle cell anaemia - (d) Cystic fibrosis **Answer: (a)** — The first clinical gene therapy was given in 1990 to a 4-year-old girl with ADA deficiency. **Q16.** ADA deficiency is caused by: - (a) A point mutation - (b) Deletion of the gene for adenosine deaminase - (c) Trisomy - (d) A viral infection **Answer: (b)** — The disorder is caused due to the deletion of the gene for adenosine deaminase, an enzyme crucial for immune function. **Q17.** Gene therapy for ADA deficiency requires periodic infusion because: - (a) The retrovirus is unstable - (b) The patient develops immunity - (c) The gene degrades - (d) The genetically engineered lymphocytes are not immortal **Answer: (d)** — Because the treated lymphocytes are not immortal, the patient requires periodic infusion; a permanent cure needs early embryonic introduction. **Q18.** What percentage of all existing transgenic animals are mice? - (a) 50 per cent - (b) 75 per cent - (c) Over 95 per cent - (d) About 20 per cent **Answer: (c)** — Although many species have been made transgenic, over 95 per cent of all existing transgenic animals are mice. **Q19.** Rosie, the first transgenic cow (1997), produced milk containing: - (a) Growth hormone - (b) Insulin - (c) Human alpha-lactalbumin at 2.4 grams per litre - (d) Alpha-1-antitrypsin **Answer: (c)** — Rosie produced human protein-enriched milk containing human alpha-lactalbumin at 2.4 grams per litre. **Q20.** The committee set up by the Indian Government to decide on the validity of GM research is: - (a) ICMR - (b) NACO - (c) CSIR - (d) GEAC — Genetic Engineering Approval Committee **Answer: (d)** — GEAC makes decisions regarding the validity of GM research and the safety of introducing GM organisms for public services. ### NEET Previous Year Questions (PYQs) *Real NEET previous-year questions on this chapter, with explanations in our own words.* **Q21.** Bt toxin crystals are toxic to which insect groups? (NEET PYQ) - (a) All arthropods equally - (b) Lepidopterans, coleopterans and dipterans - (c) Only beetles - (d) Only lepidopterans **Answer: (b)** — Bacillus thuringiensis proteins kill lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). **Q22.** Alpha-1-antitrypsin produced by transgenic animals is used to treat: (NEET PYQ) - (a) Emphysema - (b) Cystic fibrosis - (c) Phenylketonuria - (d) Diabetes **Answer: (a)** — Human alpha-1-antitrypsin is used to treat emphysema; similar attempts are being made for PKU and cystic fibrosis. **Q23.** Transgenic mice are being used to test the safety of which vaccine? (NEET PYQ) - (a) Polio vaccine - (b) Rabies vaccine - (c) Hepatitis-B vaccine - (d) BCG vaccine **Answer: (a)** — Transgenic mice are used to test polio vaccine safety and could replace the use of monkeys for testing vaccine batches. **Q24.** Biopiracy refers to: (NEET PYQ) - (a) Use of bio-resources by organisations without proper authorisation from the countries and people concerned - (b) Illegal cloning of animals - (c) Illegal sale of GM seeds - (d) Theft of laboratory equipment **Answer: (a)** — Biopiracy is the use of bio-resources by multinational companies and other organisations without proper authorisation. **Q25.** An American company obtained patent rights on Basmati rice in the year: (NEET PYQ) - (a) 1990 - (b) 1983 - (c) 1997 - (d) 2003 **Answer: (c)** — In 1997 an American company got patent rights on Basmati rice through the US Patent and Trademark Office. **Q26.** The source of complementary RNA that naturally triggers RNAi could be: (NEET PYQ) - (a) Ribosomal RNA - (b) tRNA - (c) mRNA capping - (d) Infection by RNA viruses or transposons replicating via an RNA intermediate **Answer: (d)** — The source could be infection by viruses having RNA genomes or mobile genetic elements (transposons) replicating via an RNA intermediate. **Q27.** PCR is routinely used in molecular diagnosis to detect: (NEET PYQ) - (a) HIV in suspected AIDS patients and mutations in suspected cancer patients - (b) Only bacterial infections - (c) Blood groups - (d) Only genetic disorders **Answer: (a)** — PCR detects very low concentrations of pathogens before symptoms appear, and is used for HIV detection and cancer gene mutations. **Q28.** In the radioactive probe method of molecular diagnosis, the clone with the mutated gene: (NEET PYQ) - (a) Appears brightest on the film - (b) Cannot be cultured - (c) Will NOT appear on the photographic film - (d) Turns blue **Answer: (c)** — The probe has no complementarity with the mutated gene, so that clone does not hybridise and does not appear on the film. **Q29.** The nutrient medium for plant tissue culture must contain a carbon source such as: (NEET PYQ) - (a) Glucose only - (b) Starch - (c) Cellulose - (d) Sucrose **Answer: (d)** — The medium must provide a carbon source such as sucrose, plus inorganic salts, vitamins, amino acids and growth regulators. **Q30.** Naked plant cells obtained after digesting away the cell wall are called: (NEET PYQ) - (a) Protoplasts - (b) Explants - (c) Somaclones - (d) Callus **Answer: (a)** — Isolated protoplasts, surrounded only by plasma membranes, can be fused in somatic hybridisation. ### Active Recall Prompt Write everything you can recall about **Biotechnology and its Applications**, naming each part first: **tissue culture** (explant, totipotency, micro-propagation, somaclones, somatic hybridisation, virus-free plants, pomato); the **five benefits of GM crops**; **Bt cotton** (which insect groups, why the toxin doesn't kill the Bacillus, the full activation mechanism, and each cry gene with its target); **RNAi** (the nematode, the mechanism, the natural source of dsRNA); **genetically engineered insulin** (its structure, the C peptide, and the 1983 Eli Lilly route); **gene therapy** (the 1990 case, the full procedure, why it isn't permanent); **molecular diagnosis** (PCR, probes, ELISA); the **five uses of transgenic animals** with their examples; and **ethical issues** (GEAC, biopiracy, the Basmati case). Begin each fact with its topic and end it with a full stop.