Unit 10: Biotechnology and its Application

1. Biotechnological Applications in Agriculture

Biotechnology in agriculture involves the manipulation of crops and organisms to increase yield, resistance, and nutritional value. Three major options are available for food production: agro-chemical based agriculture, organic agriculture, and genetically engineered crop-based agriculture.

Genetically Modified Organisms (GMOs)

Plants, bacteria, fungi, and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMOs). Genetic modification has:

• Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).
• Reduced reliance on chemical pesticides (pest-resistant crops).
• Helped to reduce post-harvest losses.
• Increased efficiency of mineral usage by plants (preventing early exhaustion of fertility of soil).
• Enhanced nutritional value of food (e.g., Vitamin A enriched rice).

Pest-Resistant Crops (Bt Cotton)

Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt). Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles), and dipterans (flies, mosquitoes).

Definition: Bt toxin gene has been cloned from the bacteria and been expressed in plants to provide resistance to insects without the need for insecticides; thus creating a bio-pesticide.

How Bt Cotton Works:

1. Bacillus thuringiensis forms protein crystals during a particular phase of their growth.
2. These protein crystals contain a toxic insecticidal protein.
3. The toxin is present in the bacterium as an inactive protoxin.
4. When an insect ingests the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut, which solubilizes the crystals.
5. The activated toxin binds to the surface of midgut epithelial cells and creates pores that cause cell swelling and lysis, eventually leading to the death of the insect.

Pest-Resistant Plants (RNA Interference / RNAi)

RNA interference (RNAi) is a method of cellular defense used in all eukaryotic organisms. This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing).

• Example: A nematode Meloidogyne incognita infects the roots of tobacco plants and causes a great reduction in yield.
• Strategy: Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA produced both sense and anti-sense RNA in the host cells.
• Since these two RNA are complementary to each other, they formed a double stranded RNA (dsRNA) that initiated RNAi and silenced the specific mRNA of the nematode.
• The transgenic plant therefore got itself protected from the parasite as the parasite could not survive in a transgenic host expressing specific interfering RNA.

2. Biotechnological Applications in Medicine

Recombinant DNA technology processes have made a massive impact in the healthcare domain by enabling the mass production of safe and more effective therapeutic drugs. Unlike products isolated from non-human sources, recombinant therapeutics do not induce unwanted immunological responses.

Genetically Engineered Insulin

Management of adult-onset diabetes requires taking regular insulin. Earlier, insulin was extracted from pancreas of slaughtered cattle and pigs, which caused allergies or other reactions in some patients.

• Insulin consists of two short polypeptide chains: Chain A and Chain B, linked together by disulphide bridges.
• In mammals, insulin is synthesized as a pro-hormone containing an extra stretch called the C-peptide, which is removed during maturation into insulin.
• The Recombinant Challenge: The main challenge was assembling insulin into mature, functional chains. In 1983, an American company Eli Lilly prepared two DNA sequences corresponding to A and B-chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains.
• Chains A and B were produced separately, extracted, and combined by creating disulfide bonds to form human insulin.

Gene Therapy

Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child or embryo. Here, genes are inserted into a person's cells and tissues to treat a disease.

• First Clinical Gene Therapy: Given in 1990 to a 4-year-old girl with adenosine deaminase (ADA) deficiency.
• Cause of ADA Deficiency: Deletion of the gene for adenosine deaminase, which is crucial for the immune system to function.
• Treatment Steps: Lymphocytes from the patient's blood are grown in culture outside the body. A functional human ADA cDNA is introduced using a retroviral vector into these lymphocytes, which are then infused back into the patient.
• Limitation: Since these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. If the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

Molecular Diagnosis

Early diagnosis is crucial for effective treatment. Traditional methods of serum and urine analysis do not allow early diagnosis. Recombinant DNA technology, Polymerase Chain Reaction (PCR), and Enzyme Linked Immunosorbent Assay (ELISA) are some techniques that serve the purpose of early diagnosis.

3. Transgenic Animals

Definition: Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals.

Rats, rabbits, pigs, sheep, cows, and fish have been produced, although over 95 percent of all existing transgenic animals are mice.

Why are Transgenic Animals Produced?

• 1. Normal Physiology and Development: Transgenic animals can be specifically designed to allow us to study how genes are regulated and how they affect the normal functions of the body and its development (e.g., studying complex factors involved in growth such as insulin-like growth factor).
• 2. Study of Disease: Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. These serve as models for human diseases such as cancer, cystic fibrosis, rheumatoid arthritis, and Alzheimer's.
• 3. Biological Products: Transgenic animals can be made that produce useful biological products by introducing the portion of DNA (or genes) which codes for a particular product such as human protein alpha-1-antitrypsin used to treat emphysema. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 grams per litre). The milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies than natural cow-milk.
• 4. Vaccine Safety: Transgenic animals are being used to test the safety of vaccines before they are used on humans. Polysafety testing of vaccines was traditionally done on monkeys; transgenic mice are now being used to replace them.
• 5. Chemical Safety Testing: This is known as toxicity/safety testing. Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. Toxicity testing in such animals will allow us to obtain results in less time.

4. Ethical Issues

The manipulation of organisms by biotechnology cannot go unregulated. Unpredictable problems can arise when genetically modified organisms are introduced into the ecosystem.

Genetic Modification and Patenting

• Modification of living organisms can create public concerns because people consider it going against natural laws.
• There is also the problem of patents granted for biological entities and products derived from them, sometimes without proper authorization or compensation to the indigenous people/countries (e.g., attempting to patent Indian basmati rice strains).
• Biopiracy: The use of bio-resources by multinational companies and other organizations without proper authorization from the countries and people concerned without compensatory payment.

Indian Government and Regulation

• The Indian Government has set up organizations such as GEAC (Genetic Engineering Appraisal Committee), which makes decisions regarding the validity of GM research and the safety of introducing GM-organisms for public services.
• The colonial exploitation of traditional knowledge, bio-resources, and patenting without permissions needs strict legal frameworks.

Common Mistakes to Avoid: Do not confuse RNA interference (RNAi) with PCR; RNAi silences gene expression via double-stranded RNA, whereas PCR amplifies DNA fragments. Always distinguish between somatic gene therapy and germ-line gene therapy in terms of permanence and ethics.