Chapter 13: Biotechnology and its Applications

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Introduction

Overview

• Biotechnology involves large-scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants, and animals.


• Applications include therapeutics, diagnostics, GM crops, processed food, bioremediation, waste treatment, and energy production.


• Key research areas: improved catalysts (microbes/enzymes), engineering optimal conditions, and downstream purification technologies.

Memory Aids

• Biotech = Bio + Technology for large-scale biological products.


• Three research pillars: Catalyst, Conditions, Purification.

Quick Checkpoints

• What are the main applications of biotechnology?


• What are the three critical research areas in biotechnology?

Biotechnological Applications in Agriculture

Food Production Methods

• Three methods to increase food production: agrochemical-based, organic, and genetically engineered crops.


• Green Revolution tripled food supply but insufficient for growing population.


• Agrochemicals are costly for developing world farmers; conventional breeding limits yield improvement.


• Tissue culture developed for fast, efficient crop improvement.


• Plant tissue culture: growing plant cells/tissues/organs on sterile nutrient medium under controlled conditions.


• Explant: plant part grown in vitro; totipotency allows whole plant regeneration from any explant.


• Nutrient medium includes carbon source (sucrose), inorganic salts, vitamins, amino acids, auxins, cytokinins.


• Requirements: sterile environment, aeration, nutrient medium, optimum pH 5.7.


• Types based on growth:
  
  
  
  • Callus culture: explant on solid medium forms undifferentiated cell mass; needs auxin + cytokinin.
  
  
  • Suspension culture: single/small cell groups in liquid medium; auxin (2,4-D) only.
  
  
  
  


• Types based on explant:
  
  
  
  • Single cell culture: cloning via mitosis.
  
  
  • Shoot/root culture: regulated by cytokinin-auxin ratio; auxin excess promotes roots, cytokinin excess promotes shoots.
  
  
  • Somatic embryo culture: embryo from somatic cell.
  
  
  • Meristem culture: apical/axillary shoot meristems; used for virus-free plants, germplasm conservation, rapid multiplication.
  
  
  • Other types: anther, ovule, ovary cultures.
  
  
  
  

Memory Aids

• Totipotency = any cell can form whole plant.


• Callus = undifferentiated mass; needs auxin + cytokinin.


• Meristem culture = virus-free plants.

Quick Checkpoints

• What is totipotency?


• Difference between callus and suspension culture?


• Purpose of meristem culture?

Micropropagation

Technique and Uses

• Rapid vegetative multiplication using small explants under controlled conditions.


• Produces somaclones: genetically identical plantlets.


• Used commercially for tomato, banana, apple, etc.


• Recovers healthy plants from diseased ones by culturing virus-free meristems.


• Successful meristem culture in banana, sugarcane, potato.


• Advantages:
  
  
  
  • Easy, safe, economical.
  
  
  • Rapid multiplication irrespective of season.
  
  
  • Multiplication of sterile and ornamental plants with better growth and flowering.
  
  
  • Propagation of rare and endangered species.
  
  
  
  

Memory Aids

• Somaclones = clones from somatic cells.


• Meristem free of virus = healthy plants.

Quick Checkpoints

• What are somaclones?


• Why is micropropagation preferred for virus-free plants?

Protoplast production: enzymes cellulase and pectinase remove plant cell wall, leaving plasma membrane enclosed cell with nucleus and vacuole.

Somatic Hybridisation

Process and Examples

• Fusion of somatic cells from two varieties/species after cell wall removal (protoplast fusion).


• Also called protoplast culture.


• First somatic hybrid: tobacco; others include Brassica, Petunia, Solanum.


• Pomato: hybrid of potato and tomato.


• Bomato: hybrid of brinjal and tomato.


• Advantages:
  
  
  
  • Hybrid development in asexually reproducing plants.
  
  
  • New crop plants creation.
  
  
  • Production of useful allopolyploids.
  
  
  
  

Memory Aids

• Somatic hybrid = fusion of somatic protoplasts.


• Pomato = potato + tomato hybrid.

Quick Checkpoints

• What is somatic hybridisation?


• Why is cell wall removal necessary?

Protoplast fusion: cell walls removed, protoplasts fused using PEG, nuclei fuse forming hybrid cell with combined genetic material.

Genetically Modified Organisms (GMO)

Definition and Benefits

• GMOs are organisms genetically modified by introducing foreign genes; includes plants, animals, microbes.


• GM crops express useful foreign genes (transgenes); plants expressing these are transgenic plants.


• Genetic modification benefits:
  
  
  
  • Abiotic stress tolerance (cold, drought, salt, heat).
  
  
  • Reduced chemical pesticide use (pest resistance).
  
  
  • Reduced post-harvest losses (e.g., Flavr Savr tomato).
  
  
  • Improved mineral use efficiency, preserving soil fertility.
  
  
  • Enhanced nutritional value (e.g., vitamin A enriched golden rice).
  
  
  
  

Memory Aids

• Transgene = foreign gene inserted.


• Transgenic plant = expresses transgene.


• Golden rice = vitamin A enriched.

Quick Checkpoints

• What defines a transgenic plant?


• List benefits of GM crops.

Production of Transgenic Plants

Method and Vector

• Agrobacterium tumefaciens plasmid (Ti plasmid) used as vector to transfer foreign genes into plant genome.


• Ti plasmid naturally transfers DNA causing crown gall tumors in plants.


• Process:
  
  
  
  • Isolate Ti plasmid and cut T-DNA region with restriction enzyme.
  
  
  • Cut foreign DNA with same enzyme and insert into T-DNA, forming recombinant plasmid.
  
  
  • Introduce recombinant plasmid into Agrobacterium.
  
  
  • Infect plant cells; T-DNA with foreign gene integrates into plant genome.
  
  
  • Grow transformed cells in culture to regenerate whole transgenic plant.
  
  
  
  


• Transgenic plants carry traits like disease resistance, insect resistance, herbicide resistance, improved quality.

Memory Aids

• Ti plasmid = natural genetic engineer.


• Recombinant plasmid = foreign gene inserted into T-DNA.

Quick Checkpoints

• What is the role of Ti plasmid?


• Outline steps to produce transgenic plants using Agrobacterium.

Agrobacterium tumefaciens transfers T-DNA from Ti plasmid into plant genome causing crown gall tumor; used as genetic engineering tool.

Steps: cut Ti plasmid and foreign DNA, insert foreign DNA into T-DNA, transform bacterium, infect plant cells, regenerate transgenic plant.

Insect Resistance in Transgenic Plants

Bt Toxin and Application

• Bacillus thuringiensis produces insecticidal proteins toxic to specific insect groups (lepidopterans, coleopterans, dipterans).


• Bt toxin forms inactive protoxins as crystals; activated in insect gut alkaline pH.


• Activated toxin binds midgut cells, forms pores causing cell lysis and insect death.


• Bt toxin genes (cry genes) inserted into crops like cotton and corn for pest resistance.


• Examples: cry1Ac and cry2Ab in cotton control bollworms; cry1Ab in Bt corn controls corn borer.


• First transgenic crop in India: Bt cotton.


• First transgenic plant: tobacco with glyphosate resistance gene.


• GM plants also produce biopharmaceuticals like hirudin in Brassica napus.

Memory Aids

• Bt toxin = insect-specific protein from B. thuringiensis.


• cry genes = code for Bt toxin.


• Bt cotton = first transgenic crop in India.

Quick Checkpoints

• How does Bt toxin kill insects?


• Which crops have Bt toxin genes?

Pest Resistant Plants

RNA Interference (RNAi) and Application

• Nematode Meloidogyne incognita infects tobacco roots, reducing yield.


• RNA interference (RNAi) inhibits gene activity by producing RNA complementary to mRNA (anti-sense RNA).


• Anti-sense RNA binds mRNA forming double-stranded RNA, blocking translation.


• Double-stranded RNA often degraded, silencing gene expression.


• Anti-sense genes specific to nematodes introduced into plants via Agrobacterium vectors.


• Transgenic plants express anti-sense RNA, preventing nematode infestation.


• Example: Flavr Savr tomato blocks polygalacturonase gene to delay fruit softening.

Memory Aids

• RNAi = gene silencing via anti-sense RNA.


• Anti-sense RNA + mRNA = translation block.


• Flavr Savr = delayed fruit softening.

Quick Checkpoints

• What is RNA interference?


• How does anti-sense RNA protect plants from nematodes?

RNAi process: dsRNA cut by Dicer into siRNA; siRNA guides RISC to degrade target mRNA, silencing gene expression.

Advantages and Disadvantages of Transgenic Plants

Pros and Cons

• Advantages:
  
  
  
  • Increased tolerance to abiotic stresses (cold, drought, salt, heat).
  
  
  • Reduced chemical pesticide use via pest-resistant crops.
  
  
  • Lower post-harvest losses.
  
  
  • Improved mineral use efficiency, preserving soil fertility.
  
  
  • Enhanced nutritional content (e.g., vitamin A enriched rice).
  
  
  
  


• Disadvantages:
  
  
  
  • Potential production of toxic or allergenic metabolites.
  
  
  • Unexpected susceptibility to pathogens.
  
  
  • Gene flow to related weed species.
  
  
  • Ecological disturbances due to altered interactions with parasites, symbionts, competitors.
  
  
  
  

Memory Aids

• GM crops = benefits + ecological risks.


• Gene flow = risk to wild relatives.

Quick Checkpoints

• List advantages of transgenic plants.


• What are ecological risks of GM plants?

Biotechnological Applications in Medicine

Overview

• Recombinant DNA technology enables mass production of safe, effective therapeutic drugs.


• Recombinant therapeutics reduce unwanted immune responses compared to non-human sources.


• About 30 recombinant therapeutics approved worldwide; 12 marketed in India.


• Escherichia coli was initially used as host for cloning due to ease; yeast is now preferred for recombinant protein production.

Memory Aids

• Recombinant drugs = safer + effective + less immune reaction.


• Yeast > E. coli for protein production now.

Quick Checkpoints

• Why are recombinant therapeutics preferred over animal-derived drugs?


• Which host organisms are used for recombinant protein production?

Genetically Engineered Insulin

Production and Structure

• Insulin discovered in 1921 from dog pancreas; later extracted from pig and cattle pancreas.


• Animal insulin effective but may cause allergic reactions in some patients.


• Insulin consists of 51 amino acids: Chain A (21 aa) and Chain B (30 aa) linked by disulphide bridges.


• Human insulin synthesized as proinsulin with an extra C-peptide (33 aa) removed during maturation.


• Main challenge: assembling mature insulin from recombinant chains.


• In 1983, Eli Lilly produced separate DNA sequences for A and B chains, inserted into E. coli plasmids.


• Chains produced separately, combined via disulphide bonds to form human insulin (Humulin).

Memory Aids

• Insulin = 2 chains + disulphide bridges + C-peptide removed.


• Humulin = recombinant human insulin from separate A & B chains.

Quick Checkpoints

• What is the role of C-peptide in insulin synthesis?


• How is recombinant insulin produced using E. coli?

Gene Therapy

Concept and Application

• Gene therapy inserts normal genes into cells to treat genetic diseases.


• Corrects defective genes by delivering functional copies to patient cells or embryos.


• First clinical gene therapy in 1990 treated a 4-year-old girl with adenosine deaminase (ADA) deficiency causing SCID.


• Other treatments (bone marrow transplant, enzyme replacement) are not permanent cures.


• Introducing ADA gene into early embryonic cells can provide permanent cure.

Memory Aids

• Gene therapy = fix gene defect by adding normal gene.


• ADA gene therapy treats SCID permanently.

Quick Checkpoints

• What is the principle behind gene therapy?


• How does gene therapy differ from enzyme replacement?

Monoclonal Antibody Production

Hybridoma Technology

• Monoclonal antibodies are identical antibodies from a single B-cell clone.


• Produced by fusing antigen-activated B-lymphocytes with myeloma (cancer) cells to form hybridomas.


• Antigen injected into mouse to stimulate B-cell production.


• B-cells from spleen fused with myeloma cells using polyethylene glycol (PEG).


• Hybridomas selected on HAT medium; only fused cells survive.


• Hybridomas proliferate and produce specific monoclonal antibodies continuously.


• Monoclonal antibodies are purified for research and therapy.

Memory Aids

• Hybridoma = B-cell + myeloma fusion.


• HAT medium selects hybrid cells only.

Quick Checkpoints

• What is the purpose of fusing B-cells with myeloma cells?


• How are hybridomas selected and maintained?

Molecular Diagnosis

Techniques and Importance

• Early disease diagnosis is crucial for effective treatment.


• Conventional methods (serum, urine tests) often detect disease late.


• Recombinant DNA technology, PCR, and ELISA enable early detection.


• PCR amplifies nucleic acids to detect low pathogen levels before symptoms appear.


• PCR routinely detects HIV and gene mutations in cancer and genetic disorders.


• Radioactive DNA/RNA probes hybridize to complementary sequences to detect mutations via autoradiography.


• ELISA detects pathogens by antigen-antibody interactions, identifying antigens or antibodies in samples.

Memory Aids

• PCR = amplify DNA/RNA for early pathogen detection.


• ELISA = antigen-antibody based detection.

Quick Checkpoints

• Why is PCR preferred for early diagnosis?


• What is the principle behind ELISA?

Transgenic Animals

Definition and Purpose

• Transgenic animals have genes (transgenes) introduced into their genome via transfection.


• Transfection: introduction of DNA (naked or vector-integrated) into animal cells; called transformation in other organisms.


• Used to study gene regulation, disease models, produce biological products, test vaccine and chemical safety.

Applications

• Normal physiology: Study gene roles in growth and development (e.g., insulin-like growth factor).


• Disease study: Models for human diseases like cancer, cystic fibrosis, rheumatoid arthritis, Alzheimer's.


• Biological products: Produce human proteins (e.g., a-1-antitrypsin) for treatment; example: transgenic cow \"Rosie\" producing human protein-enriched milk.


• Vaccine safety: Transgenic mice test vaccine safety, potentially replacing monkeys (e.g., polio vaccine).


• Chemical safety: Transgenic animals with sensitivity genes used for toxicity testing.

Memory Aids

• Transgenic = Transferred gene in animal genome.


• Applications: Physiology, Disease, Products, Vaccine, Chemicals (P-D-P-V-C).


• \"Rosie\" cow = human protein milk producer.

Quick Checkpoints

• What is a transgene?


• Difference between transfection and transformation?


• List main reasons for producing transgenic animals.


• Example of a transgenic animal used for vaccine safety.

Transgenic Cattle

Techniques and Objectives

• Microinjection of fertilised ova is the only successful transfection method.


• Ova obtained surgically or from slaughtered cow ovaries cultured in vitro.


• Objectives: increase milk/meat production, molecular farming, improve milk casein, regulate lactose and butter fat, enhance disease resistance.

Memory Aids

• Microinjection = main method in cattle.


• Focus on milk quality and disease resistance.

Quick Checkpoints

• What is the main transfection technique in cattle?


• List objectives of transgenic cattle production.

Transgenic Mice

Advantages and Uses

• Preferred model due to short estrous cycle, gestation, and generation time.


• Produce multiple offspring per pregnancy (litters).


• Embryos can be fertilised and cultured in vitro.


• Embryonic stem (ES) cell lines available and maintained.


• Diverse genetic stocks facilitate gene transfer studies.


• Techniques for gene transfer and transgenic production developed using mice.

Memory Aids

• Mouse = model mammal for gene transfer.


• Short cycles and many offspring aid research.

Quick Checkpoints

• Why are mice preferred for transgenic studies?


• What features make mice suitable for gene transfer?

Transgenic Rabbits

Applications and Examples

• Used in gene/molecular farming to produce pharmaceutically important proteins.


• Human genes transferred include interleukin 2, growth hormone, tissue plasminogen activator, a-1-antitrypsin.


• First transgenic animal was a mouse with growth hormone gene, called \"supermouse\".


• Genetically engineered Pseudomonas putida (\"superbug\") cleans oil spills.

Memory Aids

• Rabbits for protein production.


• Supermouse = first transgenic animal.


• Superbug = engineered bacterium for oil cleanup.

Quick Checkpoints

• What proteins are produced using transgenic rabbits?


• What was the first transgenic animal?


• What is the \"superbug\" and its function?

Ethical Issues

Bioethical Concerns in Biotechnology

• Transferring genes between species breaches 'integrity of species'.


• Biotechnology may cause unforeseen environmental risks, threatening biodiversity.


• Human-animal gene transfer blurs the concept of 'humanness'.


• Using animals as protein factories causes them suffering and reduces their status.


• Exploitation of living beings for human benefit is disrespectful.


• Potential unknown biological damages cannot be ruled out.


• Risk of accidentally creating new infectious agents exists.

Memory Aids

• \"Species integrity\" means no gene mixing across species.


• Biotech risks = environment + ethics + unknown harms.


• Animals in biotech = factories causing suffering.

Quick Checkpoints

• What is the role of GEAC?


• Why is gene transfer between species ethically controversial?


• What are the environmental risks of biotechnology?


• How does biotechnology affect animal welfare?

Biopatent

Definition and Examples

• Patent: Government-granted exclusive rights to an inventor to prevent commercial use by others.


• Biopatents: Patents on biological entities or their products.


• Examples: Black pepper (Piper nigrum), basmati rice (Oryza sativa), Indian mustard (Brassica campestris), pomegranate (Punica granatum), turmeric (Curcuma domestica), neem (Azadirachta indica).

Memory Aids

• Biopatent = patent + biological product.


• Common patented plants: spices and crops of India.

Quick Checkpoints

• What is a biopatent?


• Which plants have been patented?


• Why are biopatents important?

Biopiracy

Concept and Examples

• Biopiracy: Unauthorized exploitation or patenting of biological resources of a country by others.


• Example: 1997 US patent on basmati rice germplasm indigenous to India.


• India has ~200,000 rice varieties, highlighting the richness of its bioresources.


• Indian Patents Bill amended to prevent unauthorized use of bioresources and traditional knowledge.

Memory Aids

• Biopiracy = bio + piracy (stealing biological resources).


• India protects its bioresources legally.

Quick Checkpoints

• What is biopiracy?


• Give an example of biopiracy involving basmati rice.


• How does India prevent biopiracy?

Biowar

Definition and Details

• Biowar: Use of infectious pathogens, spores, or toxins as biological weapons against humans, crops, or animals.


• Agents include viruses, bacteria, fungi kept virulent in special containers for delivery via missiles or aircraft.


• Delivery forms: spray and powder.


• Potential pathogens: Bacillus anthracis, Vibrio cholerae, smallpox virus, Pasteurella/Yersinia pestis, botulinum toxin.


• Bioweapons are low-cost, nearly invisible, and can cause more casualties than conventional weapons.


• Defenses: gas masks, protective shelters, vaccination, antibiotics, decontamination, sensitive detection systems.

Memory Aids

• Biowar = biological agents as weapons.


• Common agents: anthrax, cholera, smallpox, plague, botulinum toxin.


• Protection includes masks and vaccines.

Quick Checkpoints

• What is biowar?


• Name common bioweapon agents.


• How can bioweapons be defended against?

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