Biotechnology and Its Applications

Introduction to Biotechnology

Biotechnology primarily focuses on the industrial-scale production of biopharmaceuticals and biologicals using genetically modified microbes, fungi, plants, and animals.

Key applications of biotechnology include:

• Therapeutics
• Diagnostics
• Genetically modified crops for agriculture
• Processed food
• Bioremediation
• Waste treatment
• Energy production

Three critical research areas in biotechnology are:

• Providing the best catalyst (e.g., improved microbe or pure enzyme).
• Creating optimal conditions through engineering for the catalyst to act.
• Downstream processing technologies to purify the protein/organic compound.

Biotechnological Applications in Agriculture

For increasing food production, three main options are considered:

• Agro-chemical based agriculture
• Organic agriculture
• Genetically engineered crop-based agriculture

The Green Revolution significantly increased food supply, mainly due to better management practices and agrochemicals, but it wasn’t enough to feed the growing population, and agrochemicals were expensive for developing world farmers. Traditional breeding techniques also struggled to keep pace.

Tissue Culture

Tissue culture is a technology developed in the 1950s where whole plants can be regenerated from explants (any part of a plant grown in a test tube under sterile conditions in special nutrient media).

• Totipotency: The capacity of any plant cell/explant to generate a whole plant.
• Nutrient medium: Must provide a carbon source (e.g., sucrose), inorganic salts, vitamins, amino acids, and growth regulators (e.g., auxins, cytokinins).
• Micro-propagation: The method of producing thousands of plants in very short durations through tissue culture.
• Somaclones: Plants produced through micro-propagation that are genetically identical to the original plant.
• Commercial application: Used for plants like tomato, banana, apple.
• Recovery of healthy plants: Even if a plant is virus-infected, its meristem (apical and axillary) is often virus-free, allowing for virus-free plant regeneration from meristem culture (e.g., banana, sugarcane, potato).

Somatic Hybridisation

Scientists can isolate naked protoplasts (cells without cell walls, surrounded by plasma membranes) from plants.

• Process: Protoplasts from two different plant varieties, each with a desirable character, can be fused to create hybrid protoplasts.
• Somatic hybrids: The new plants grown from these hybrid protoplasts.
• Example: Fusion of tomato and potato protoplasts resulted in pomato, though it lacked the desired commercial characteristics.

Genetically Modified Organisms (GMO)

GMOs are plants, bacteria, fungi, and animals whose genes have been altered by manipulation.

Benefits of GM plants:

• Increased tolerance to abiotic stresses (cold, drought, salt, heat).
• Reduced reliance on chemical pesticides (pest-resistant crops).
• Reduced post-harvest losses.
• Increased efficiency of mineral usage by plants, preventing early soil exhaustion.
• Enhanced nutritional value of food (e.g., golden rice, enriched with Vitamin ‘A’).
• Creation of tailor-made plants to supply alternative resources for industries (starches, fuels, pharmaceuticals).

Pest Resistant Plants

Biotechnology helps produce pest-resistant plants, decreasing pesticide use.

Bt Toxin

• Source: Bacillus thuringiensis (Bt), a bacterium, produces Bt toxin.
• Mechanism:
  • Bt toxin gene is cloned from the bacteria and expressed in plants, providing insect resistance.
  • Bt toxin protein exists as inactive protoxins in bacteria, thus not harming the Bacillus itself.
  • When an insect ingests the inactive toxin, the alkaline pH of its gut converts it into an active form.
  • The activated toxin binds to midgut epithelial cells, creating pores, causing cell swelling, lysis, and ultimately insect death.
• Specificity: Most Bt toxins are insect-group specific.
• Examples: Bt cotton, Bt corn, rice, tomato, potato, soyabean.
• Genes: Specific Bt toxin genes (named cry) are isolated and incorporated into crops. For example, cryIAc and cryIIAb control cotton bollworms, while cryIAb controls corn borer.

RNA Interference (RNAi)

This novel strategy prevents infestation by nematodes, such as Meloidegyne incognitia which infects tobacco plant roots.

• Mechanism:
  • RNAi is a cellular defense method in all eukaryotic organisms involving the silencing of specific mRNA due to a complementary dsRNA (double-stranded RNA) molecule. This dsRNA binds to and prevents translation of the mRNA.
  • Using Agrobacterium vectors, nematode-specific genes are introduced into the host plant.
  • This introduction causes the host cells to produce both sense and anti-sense RNA specific to the nematode.
  • These complementary RNAs form a dsRNA, which initiates RNAi, silencing the nematode’s specific mRNA.
  • Result: The parasite cannot survive in the transgenic host, protecting the plant.

Biotechnological Applications in Medicine

Recombinant DNA technology has had a huge impact on healthcare, enabling mass production of safe and effective therapeutic drugs. These recombinant therapeutics are less likely to cause unwanted immunological responses compared to similar products from non-human sources.

Genetically Engineered Insulin

• Challenge: Traditional insulin was extracted from slaughtered cattle and pigs, which could cause allergies or immune reactions in patients. The challenge was producing human insulin without such issues.
• Insulin structure: Consists of two short polypeptide chains, Chain A and Chain B, linked by disulphide bridges.
• Pro-hormone: In humans, insulin is synthesized as a pro-hormone (pro-insulin) containing an extra stretch called the C peptide, which is removed during maturation to active insulin.
• Eli Lilly’s approach (1983): This American company prepared two DNA sequences corresponding to the A and B chains of human insulin.
  • These sequences were introduced into plasmids of E. coli to produce chains A and B separately.
  • The chains were extracted and then combined by creating disulfide bonds to form mature human insulin. This produced human insulin with a structure identical to the natural molecule.

Gene Therapy

Gene therapy is a collection of methods allowing for the correction of a gene defect diagnosed in a child/embryo. Genes are inserted into a person’s cells and tissues to treat a disease, delivering a normal gene to compensate for a non-functional one.

• First clinical gene therapy: Given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency.
• ADA deficiency: Caused by the deletion of the gene for adenosine deaminase, an enzyme crucial for the immune system.
• Treatment options for ADA deficiency:
  • Bone marrow transplantation: Can cure some children.
  • Enzyme replacement therapy: Functional ADA is given by injection.
  • Limitations: Both above methods are not completely curative.
  • Gene therapy procedure:
    1. Lymphocytes from the patient’s blood are grown in culture outside the body.
    2. A functional ADA cDNA (using a retroviral vector) is introduced into these lymphocytes.
    3. The engineered lymphocytes are returned to the patient.
    4. Limitation: As these cells are not immortal, periodic infusions are required.
  • Potential permanent cure: If the ADA-producing gene from marrow cells is introduced into cells at early embryonic stages, it could be a permanent cure.

Molecular Diagnosis

Early diagnosis and understanding disease pathophysiology are crucial for effective treatment. Conventional methods (serum and urine analysis) often cannot detect diseases early.

Techniques for early diagnosis:

• Recombinant DNA technology
• Polymerase Chain Reaction (PCR)
• Enzyme-Linked Immunosorbent Assay (ELISA)

Polymerase Chain Reaction (PCR)

• Application: Detects very low concentrations of bacteria or viruses (even before symptoms appear) by amplifying their nucleic acid.
• Routine uses: Detecting HIV in suspected AIDS patients, detecting mutations in genes in suspected cancer patients, identifying other genetic disorders.
• Detection of mutated genes: A single-stranded DNA or RNA probe (tagged with a radioactive molecule) is allowed to hybridize to its complementary DNA in a cell clone. Detection uses autoradiography. The clone with a mutated gene will not appear on the photographic film because the probe won’t have complementarity.

Enzyme-Linked Immunosorbent Assay (ELISA)

• Principle: Based on the antigen-antibody interaction.
• Detection: Infection by a pathogen can be detected by identifying either the presence of antigens (proteins, glycoproteins) or the antibodies synthesized against the pathogen.

Transgenic Animals

Transgenic animals are animals whose DNA has been manipulated to possess and express an extra (foreign) gene. Over 95% of existing transgenic animals are mice, though rats, rabbits, pigs, sheep, cows, and fish have also been produced.

Common reasons for producing transgenic animals:

• Normal physiology and development:

  • Designed to study how genes are regulated and how they affect body functions and development.
  • Example: Studying complex factors like insulin-like growth factor by introducing genes that alter its formation and observing the biological effects.

• Study of disease:

  • Created as models for human diseases to increase understanding of how genes contribute to disease development and to investigate new treatments.
  • Models exist for diseases like cancer, cystic fibrosis, rheumatoid arthritis, and Alzheimer’s.

• Biological products:

  • Used to produce expensive biological products needed to treat certain human diseases.
  • Introduction of genes coding for specific human proteins.
  • Example: Production of human protein α-1-antitrypsin to treat emphysema. Attempts are also made for phenylketonuria (PKU) and cystic fibrosis.
  • Rosie (1997): The first transgenic cow, produced human protein-enriched milk (2.4 grams/litre) containing human alpha-lactalbumin, which was nutritionally more balanced for human babies than natural cow-milk.

• Vaccine safety testing:

  • Transgenic mice are developed to test the safety of vaccines before human use.
  • Example: Testing the safety of the polio vaccine in transgenic mice, potentially replacing the use of monkeys.

• Chemical safety testing (Toxicity/Safety Testing):

  • Transgenic animals are made more sensitive to toxic substances by carrying specific genes.
  • They are exposed to toxic substances, and the effects are studied, allowing for faster results.

Ethical Issues

The manipulation of living organisms requires ethical standards and regulation due to potential harm to organisms and unpredictable results when genetically modified organisms are introduced into the ecosystem.

• GEAC (Genetic Engineering Approval Committee): Set up by the Indian Government to decide on the validity of GM research and the safety of introducing GM-organisms for public services.

Biopiracy

Biopiracy is the term for the use of bio-resources by multinational companies and other organizations without proper authorization from the concerned countries and people, and without compensatory payment.

• Problem: Companies are granted patents for products and technologies using genetic materials, plants, and other biological resources that have been long identified, developed, and used by farmers and indigenous people of a specific region/country.
• Context: Industrialized nations are financially rich but poor in biodiversity and traditional knowledge, while developing/underdeveloped countries are rich in both. Traditional knowledge can be exploited for modern applications, saving time and expenditure.
• Examples:
  • Basmati Rice: India has an estimated 200,000 varieties of rice, with Basmati being distinct for its aroma and flavor, having 27 documented varieties and a history of centuries. In 1997, an American company obtained patent rights on Basmati rice through the US Patent and Trademark Office, allowing them to sell a ‘new’ variety derived from Indian farmer’s varieties (Indian Basmati crossed with semi-dwarf varieties). This patent extended to functional equivalents, potentially restricting others from selling Basmati.
  • Traditional Herbal Medicines: Attempts have been made to patent uses, products, and processes based on Indian traditional herbal medicines like turmeric and neem.
• Response: There is growing realization of injustice and inadequate compensation. Some nations are developing laws to prevent unauthorized exploitation of bio-resources and traditional knowledge. The Indian Parliament recently cleared the second amendment of the Indian Patents Bill to address these issues, including patent terms of emergency provisions, and research and development initiatives.