Chapter 13: Biotechnology and its Applications

Introduction

Overview

• Biotechnology involves industrial 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.
• Three key research areas: improved catalysts (microbes/enzymes), optimal engineering conditions, and downstream purification technologies.

Memory Aids

• Biotech = Bio + Technology for large-scale biological products.
• Three research areas: Catalyst, Conditions, Purification.

Quick Checkpoints

• What are the main applications of biotechnology?
• What are the three critical research areas?

Biotechnological Applications in Agriculture

Food Production Methods

• Three ways to increase food production: agrochemical-based, organic, and genetically engineered crop-based agriculture.
• 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 = ability to regenerate whole plant from any cell.
• 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: solid medium, undifferentiated cell mass, needs auxin + cytokinin.
  • Suspension culture: liquid medium, single/small cell groups, auxin (2,4-D) only.
• Types based on explant:
  • Single cell culture: cloning via mitosis.
  • Shoot and root culture: regulated by cytokinin-auxin ratio.
  • Somatic embryo culture: embryo from somatic cell.
  • Meristem culture: shoot tips/nodal segments, virus-free plants, germplasm conservation, rapid multiplication.
  • Other types: anther, ovule, ovary culture.

Memory Aids

• Totipotency = any cell can form whole plant.
• Callus = undifferentiated mass; needs auxin + cytokinin.
• Meristem culture for 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 year-round.
  • Multiplication of sterile, ornamental, rare, endangered plants.

Memory Aids

• Somaclones = identical clones from tissue culture.
• Meristem free of virus = healthy plants.

Quick Checkpoints

• What are somaclones?
• Why is micropropagation preferred for virus-free plants?

This image shows production of a protoplast by enzymatic removal of plant cell wall using cellulase and pectinase. Protoplast is a plant cell without cell wall, enclosed by plasma membrane, retaining nucleus and vacuole.

Somatic Hybridisation

Process and Examples

• Somatic hybridisation: 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:
  • Develop hybrids of asexually reproducing plants.
  • Create new crop plants (e.g., pomato).
  • Produce useful allopolyploids.

Memory Aids

• Protoplast fusion = somatic hybridisation.
• Pomato = potato + tomato hybrid.

Quick Checkpoints

• What is somatic hybridisation?
• Examples of somatic hybrids?

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

Genetically Modified Organisms (GMO)

Definition and Benefits

• GMOs are organisms genetically modified by introducing foreign genes (transgenes).
• GM crops express useful foreign genes, called 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.
  • Enhanced nutritional value (e.g., golden rice with vitamin A).

Memory Aids

• Transgenes = foreign genes in GM plants.
• Flavr Savr tomato = longer shelf life.

Quick Checkpoints

• What are transgenic plants?
• List benefits of genetic modification in plants.

Production of Transgenic Plants

Method and Vector

• Vector: Ti plasmid from Agrobacterium tumefaciens, a natural genetic engineer.
• Ti plasmid transfers T-DNA into plant genome, causing crown gall tumors.
• Steps:
  • Isolate Ti plasmid and cut T-DNA with restriction enzyme.
  • Cut foreign DNA with same enzyme and insert into T-DNA (recombinant plasmid).
  • Introduce recombinant plasmid into bacterium.
  • Use bacterium to transfer T-DNA with foreign gene into plant cell.
  • Grow plant cells in culture to regenerate transgenic plant.
• Transgenic plants carry traits like disease resistance, insect resistance, herbicide resistance, improved food quality.

Memory Aids

• Ti plasmid = tumor inducing plasmid.
• Agrobacterium tumefaciens = natural genetic engineer.

Quick Checkpoints

• What is the role of Ti plasmid?
• Outline steps to produce transgenic plants.

Agrobacterium tumefaciens transfers T-DNA from Ti plasmid into plant cells causing crown gall tumors; used in genetic engineering.

Steps to produce transgenic plants using Ti plasmid: cut plasmid and foreign DNA, insert foreign gene, transform bacterium, infect plant cells, regenerate plants with new traits.

Insect Resistance in Transgenic Plants

Bt Toxin and Application

• Bacillus thuringiensis produces proteins toxic to specific insects (lepidopterans, coleopterans, dipterans).
• Bt forms protein crystals containing inactive protoxins activated in insect gut alkaline pH.
• Activated toxin binds midgut cells, creates pores causing cell lysis and insect death.
• Bt toxin genes (cry genes) inserted into crops like cotton and corn for pest resistance.
• First transgenic crop in India: Bt cotton.
• First transgenic plant: tobacco with glyphosate resistance.
• GM plants also produce biopharmaceuticals like hirudin (blood clotting inhibitor) in Brassica napus.

Memory Aids

• Bt toxin = insect-specific protein from B. thuringiensis.
• Cry genes code for Bt toxin.

Quick Checkpoints

• How does Bt toxin kill insects?
• Which crops have Bt genes?

Pest Resistant Plants

RNA Interference (RNAi) Mechanism

• Nematode Meloidogyne incognita infects tobacco roots reducing yield.
• RNAi inhibits gene activity by producing anti-sense RNA complementary to mRNA.
• Anti-sense RNA binds mRNA forming double-stranded RNA, blocking translation.
• Double-stranded RNA often degraded, silencing gene expression.
• Agrobacterium vectors introduce nematode-specific anti-sense genes into plants.
• Transgenic plants express anti-sense RNA, preventing nematode infestation.

Memory Aids

• RNAi = gene silencing via anti-sense RNA.
• Anti-sense RNA blocks mRNA translation.

Quick Checkpoints

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

RNAi process: dsRNA cut by Dicer into siRNA, siRNA forms RISC complex, guides RISC to target mRNA, which is cleaved to prevent protein synthesis.

Example: Flavr Savr tomato blocks polygalacturonase gene to delay fruit softening and extend shelf life.

Advantages and Disadvantages of Transgenic Plants

Pros and Cons

• Advantages:
  • Increased tolerance to abiotic stresses.
  • Reduced chemical pesticide use.
  • Lower post-harvest losses.
  • Improved mineral use efficiency.
  • Enhanced nutritional content (e.g., vitamin A rice).
• Disadvantages:
  • Potential production of toxic/allergic metabolites.
  • Unexpected susceptibility to pathogens.
  • Gene transfer to related weed species.
  • Ecological disturbances due to altered interactions.

Memory Aids

• GM plants = benefits + ecological risks.

Quick Checkpoints

• List advantages of transgenic plants.
• What are possible ecological risks?

Biotechnological Applications in Medicine

Overview

• Recombinant DNA technology enables mass production of safe, effective therapeutic drugs.
• Recombinant therapeutics avoid unwanted immune responses common with non-human products.
• About 30 recombinant therapeutics approved worldwide; 12 marketed in India.
• Escherichia coli initially used as host for cloning; yeast increasingly preferred for protein production.

Memory Aids

• Recombinant = Safe + Effective + Human-compatible drugs
• Yeast > E. coli for protein production

Quick Checkpoints

• What advantages do recombinant therapeutics have over animal-derived drugs?
• Why is yeast preferred over E. coli for recombinant protein production?

Genetically Engineered Insulin

Production and Structure

• 1921: Insulin extracted from dog pancreas; later from pig and cattle pancreas.
• Animal insulin effective but may cause allergic reactions.
• Insulin: 51 amino acids, two chains (A: 21, B: 30) linked by disulphide bridges.
• Human insulin synthesized as proinsulin with extra C-peptide (33 amino acids) removed during maturation.
• Main challenge: assembling mature insulin using recombinant DNA techniques.
• 1983: Eli Lilly produced separate DNA sequences for A and B chains, inserted into E. coli plasmids.
• Chains produced separately, combined by disulphide bonds to form human insulin (Humulin).

Memory Aids

• Insulin = A + B chains + disulphide bridges
• Proinsulin = Insulin + C-peptide (removed)
• Humulin = Recombinant human insulin

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 genetic defects by compensating for non-functional genes.
• First clinical gene therapy in 1990 for 4-year-old girl with ADA deficiency causing SCID.
• ADA enzyme vital for immune function; deficiency treated by bone marrow transplant or enzyme replacement but not fully cured.
• Introducing ADA gene into early embryonic cells can provide permanent cure.

Memory Aids

• Gene therapy = Gene insertion for disease correction
• ADA deficiency = SCID immune disorder

Quick Checkpoints

• What is the goal of gene therapy?
• How does gene therapy treat ADA deficiency?

Monoclonal Antibody Production

Hybridoma Technology

• Monoclonal antibodies produced by immortalizing antigen-activated B-cells fused with myeloma cells.
• Antigen injected into mouse; B-lymphocytes isolated from spleen.
• B-cells fused with myeloma cells to form hybridoma cells producing specific antibodies.
• Technique developed by Kohler and Milstein in 1975.
• Hybridomas selected on HAT medium; only fused cells survive and multiply.
• Hybridomas produce pure monoclonal antibodies for research and therapy.

Memory Aids

• Hybridoma = B-cell + Myeloma fusion
• HAT medium selects hybrid cells
• Monoclonal = Single antibody type

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 diagnosis crucial for effective treatment.
• Conventional methods (serum, urine analysis) not effective for early detection.
• Recombinant DNA technology, PCR, and ELISA enable early detection.
• PCR amplifies nucleic acids to detect low pathogen levels before symptoms appear.
• PCR used to detect HIV, gene mutations in cancer, and other genetic disorders.
• Radioactive DNA/RNA probes hybridize to complementary DNA for mutation detection via autoradiography.
• ELISA detects infection by identifying antigens or antibodies based on antigen-antibody interaction.

Memory Aids

• PCR = Amplify DNA for early pathogen detection
• ELISA = Detect antigens or antibodies
• Autoradiography = Detect gene mutations with probes

Quick Checkpoints

• Why is PCR preferred for early pathogen detection?
• What principle does ELISA use for diagnosis?

Transgenic Animals

Overview

• A transgenic animal has genes (transgenes) introduced by transfection.
• Transfection: introduction of DNA (naked or vector-integrated) into animal cells.
• In other organisms, this process is called transformation.

Reasons for Production

• Normal physiology and development: Study gene regulation and effects on body functions, e.g., insulin-like growth factor.
• Study of disease: Model human diseases like cancer, cystic fibrosis, rheumatoid arthritis, Alzheimer's for treatment research.
• Biological products: Produce medicines like human proteins (e.g., a-1-antitrypsin) cheaply; example: transgenic cow "Rosie" producing human protein-enriched milk.
• Vaccine safety: Use transgenic mice to test vaccine safety, potentially replacing monkeys in polio vaccine testing.
• Chemical safety testing: Create animals sensitive to toxins to study toxic effects.

Memory Aids

• Transgene = introduced gene in animal genome.
• Transfection = DNA insertion in animal cells.
• Rosie the cow = human protein milk producer.
• Transgenic mice = vaccine and disease models.

Quick Checkpoints

• What is a transgene?
• Difference between transfection and transformation?
• Why produce transgenic animals?
• Examples of diseases modeled by transgenic animals?

Examples of Transgenic Animals

Transgenic Cattle

• Produced by microinjection of fertilised ova (surgically recovered or in vitro cultured).
• Objectives: increase milk/meat, molecular farming, improve milk casein, regulate lactose and butter fat, enhance disease resistance.

Transgenic Mice

• Preferred model due to short estrous cycle, gestation, large litters, in vitro fertilisation, embryo culture, embryonic stem cell lines.
• Used extensively for gene transfer and transgenic production studies.

Transgenic Rabbits

• Used for gene/molecular farming to produce pharmaceutically important proteins.
• Human genes transferred include interleukin 2, growth hormone, tissue plasminogen activator, a-1-antitrypsin.

Memory Aids

• Supermouse = first transgenic mouse with growth hormone gene.
• "Superbug" = genetically engineered Pseudomonas putida for oil spill cleanup.

Quick Checkpoints

• What technique is used for transgenic cattle?
• Why are mice preferred for gene transfer studies?
• Which proteins are produced using transgenic rabbits?
• What is the significance of the supermouse?

Ethical Issues

Bioethical Concerns

• Introduction of transgenes across species violates species integrity.
• Biotechnology may cause unforeseen environmental risks, including biodiversity loss.
• Human gene transfer to animals dilutes the concept of humanness.
• Animals used for pharmaceutical protein production suffer and are treated as factories.
• Exploitation of living beings for human benefit is disrespectful.
• Potential for unknown biological damage exists.
• Risk of creating new infectious agents accidentally.

Memory Aids

• Transgene transfer = Species integrity violation.
• Biotech risks = Environment + biodiversity.
• Human-animal gene mix = Humanness dilution.
• Animals as factories = Ethical concern.
• Respect life, avoid exploitation.
• Unknown risks always possible.
• New pathogens risk present.

Quick Checkpoints

• What is the role of GEAC?
• How does transgene introduction affect species?
• What are the environmental risks of biotechnology?
• Why is gene transfer between humans and animals controversial?
• What ethical issues arise from using animals in biotech?
• What biological risks cannot be ruled out?
• How can new infectious agents be created accidentally?

Biopatent

Definition and Examples

• Patent: Government right to prevent commercial use of an invention.
• Biopatents: Patents on biological entities and derived 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

• Patent = exclusive commercial rights.
• Biopatent = biological product patent.
• Common plants patented: pepper, rice, mustard, pomegranate, turmeric, neem.

Quick Checkpoints

• What is a biopatent?
• Which plants have biopatents?
• Why are biopatents important?

Biopiracy

Concept and Examples

• Biopiracy: Unauthorized exploitation or patenting of biological resources of other nations.
• Example: 1997 US patent on basmati rice germplasm indigenous to India.
• India has ~200,000 rice varieties.
• Indian Patents Bill amended to prevent unauthorized use of bio-resources and traditional knowledge.

Memory Aids

• Biopiracy = illegal bio-resource exploitation.
• Basmati rice patent = key example.
• Indian law protects bio-resources.

Quick Checkpoints

• What is biopiracy?
• Give an example of biopiracy.
• How does India protect its bio-resources?

Biowar

Definition and Details

• Biowar: Use of infectious pathogens, spores, toxins as biological weapons against humans, crops, animals.
• Agents: Viruses, bacteria, fungi kept active in special containers for delivery by missile or aircraft.
• Delivery forms: Spray and powder.
• Examples of pathogens: Bacillus anthracis, Vibrio cholerae, smallpox virus, Pasteurella/Yersinia pestis, botulinum toxin.
• Bioweapons are invisible, low cost, cause more casualties than conventional/chemical weapons.
• Defenses: Gas masks, protective shelters, vaccination, antibiotics, decontamination, sensitive detection.

Memory Aids

• Biowar = biological weapons using pathogens.
• Delivery: spray/powder via missiles/aircraft.
• Key pathogens: anthrax, cholera, smallpox, plague, botulinum.
• Defenses include masks, vaccines, antibiotics.

Quick Checkpoints

• What is biowar?
• Which agents are used in bioweapons?
• How are bioweapons delivered?
• What are defenses against bioweapons?

Hybridoma Technology

Overview

• Hybridoma technology is used to produce monoclonal antibodies.
• It involves fusion of antibody-producing B-cells with myeloma cells.
• Resulting hybrid cells (hybridomas) can grow indefinitely and produce specific antibodies.

Memory Aids

• Hybridoma = B-cell + myeloma fusion.
• Produces monoclonal antibodies.
• Hybridomas grow indefinitely.

Quick Checkpoints

• What cells are fused in hybridoma technology?
• What is the product of hybridoma cells?
• Why are hybridomas important?