Chapter Notes

Biotechnology and its Applications

Biotechnology is a field that uses genetically modified microbes, fungi, plants, and animals for the industrial-scale production of biopharmaceuticals and other biological products. Its wide range of applications includes:

• Therapeutics (medicines)
• Diagnostics (disease detection)
• Genetically modified crops for agriculture
• Processed food
• Bioremediation (cleaning up pollution)
• Waste treatment
• Energy production

There are three key research areas that drive biotechnology forward:

1. Finding the best catalyst: This involves improving an organism (like a microbe) or a pure enzyme to make biological processes more efficient.
2. Creating optimal conditions: This uses engineering principles to create the perfect environment for the catalyst to work effectively.
3. Downstream processing: This involves developing technologies to purify the final product, whether it's a protein or another organic compound.

Let's explore how biotechnology has revolutionized food production and healthcare.

Biotechnological Applications in Agriculture

To meet the food demands of a growing population, we have three main options:

1. Agro-chemical based agriculture: Using fertilizers and pesticides.
2. Organic agriculture: Farming without synthetic chemicals.
3. Genetically engineered crop-based agriculture: Using genetically modified crops.

The Green Revolution greatly increased the food supply, but it relied heavily on agrochemicals, which are expensive for many farmers and can harm the environment. Conventional breeding methods are often too slow to keep up with demand. This led to the development of new biotechnological approaches.

Tissue Culture

During the 1950s, scientists discovered that whole plants could be regenerated from small pieces of a plant, called explants. This process, done in a test tube under sterile conditions with a special nutrient medium, is known as tissue culture.

• Totipotency: This is the amazing ability of a single plant cell or explant to grow into a complete plant.
• Nutrient Medium: For tissue culture to succeed, the medium must contain a carbon source (like sucrose), inorganic salts, vitamins, amino acids, and growth regulators (like auxins and cytokinins).

A major application of tissue culture is micro-propagation, which is a method for producing thousands of plants in a very short time. All the plants produced this way are genetically identical to the original parent plant and are called somaclones. This technique is used commercially for plants like tomato, banana, and apple.

Another key use is recovering healthy plants from diseased ones. Even if a plant is infected with a virus, its meristem (the actively growing tissues at the apical and axillary buds) is usually virus-free. By culturing the meristem, scientists can grow virus-free plants of species like banana, sugarcane, and potato.

Somatic Hybridisation

Scientists can go a step further by isolating individual plant cells and digesting their cell walls to get naked protoplasts (cells surrounded only by a plasma membrane).

• Somatic Hybridisation: This is the process of fusing protoplasts from two different plant varieties to create a hybrid protoplast.
• Somatic Hybrids: These are the new plants grown from the hybrid protoplasts, combining desirable characters from both parent plants.

Genetically Modified Organisms (GMOs)

A more direct way to improve crops is by using genetic modification.

Genetically Modified Organisms (GMOs) are plants, bacteria, fungi, or animals whose genes have been altered through manipulation. GM plants, in particular, have been beneficial in many ways:

• Increased tolerance to abiotic stresses like cold, drought, salt, and heat.
• Reduced reliance on chemical pesticides by creating pest-resistant crops.
• Helped to reduce post-harvest losses.
• Increased efficiency of mineral usage, which prevents soil from becoming infertile quickly.
• Enhanced nutritional value of food, a famous example being golden rice, which is enriched with Vitamin A.

GM technology is also used to create "tailor-made" plants that can supply industries with resources like starches, fuels, and pharmaceuticals.

Pest Resistant Plants

Bt Toxin

One of the most significant applications in agriculture is the creation of pest-resistant plants, which reduces the need for chemical pesticides. This is often achieved using the Bt toxin.

• Source: The Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt).
• Mechanism: The bacterium produces protein crystals containing a toxic insecticidal protein. This toxin exists as an inactive protoxin inside the bacterium, so it doesn't harm the bacterium itself. When an insect eats the plant containing this protoxin, the alkaline pH of the insect's gut activates the toxin. The active toxin then binds to the surface of the midgut cells, creating pores. This causes the cells to swell and burst (lysis), eventually killing the insect.
• Application: The gene responsible for producing the Bt toxin (the cry gene) has been cloned from the bacteria and inserted into crop plants like cotton, corn, rice, and tomato. This creates a "bio-pesticide" within the plant itself.
• Specificity: Different Bt toxins are specific to different groups of insects. For example, the proteins encoded by the genes cryIAc and cryIIAb control cotton bollworms, while the protein from the gene cryIAb controls the corn borer.

RNA Interference (RNAi)

Another strategy to create pest-resistant plants involves a natural process called RNA interference (RNAi). This is a method of cellular defense found in all eukaryotic organisms.

• Mechanism: RNAi involves silencing a specific messenger RNA (mRNA) molecule. This happens when a complementary double-stranded RNA (dsRNA) molecule binds to the mRNA, preventing it from being translated into a protein.
• Application in Tobacco: The nematode Meloidegyne incognitia infects the roots of tobacco plants, severely reducing their yield. To combat this, scientists used Agrobacterium vectors to introduce nematode-specific genes into the tobacco plant.
• Process: The introduced DNA was designed to produce both sense and anti-sense RNA in the host plant's cells. These two RNA strands are complementary and bind together to form a dsRNA. This dsRNA triggers the RNAi process, silencing the specific mRNA of the nematode. As a result, the parasite cannot survive in the transgenic host, and the plant is protected from infestation.

Biotechnological Applications in Medicine

Recombinant DNA technology has had a huge impact on healthcare, allowing for the mass production of safer and more effective therapeutic drugs. Unlike medicines derived from non-human sources, these recombinant therapeutics do not cause unwanted immune responses. Currently, about 30 recombinant therapeutics are approved for human use worldwide, with 12 available in India.

Genetically Engineered Insulin

For people with diabetes, managing the disease often requires taking insulin. Before biotechnology, the insulin used was extracted from the pancreas of slaughtered cattle and pigs. This animal-source insulin sometimes caused allergies or other immune reactions in patients.

• Structure of Human Insulin: Insulin is made of two short polypeptide chains, chain A and chain B, linked by disulfide bridges.
• Pro-hormone: In humans, insulin is first synthesized as a pro-hormone (pro-insulin), which contains an extra stretch called the C peptide. This C peptide is removed during maturation to create the functional hormone.
• The Challenge: The main difficulty in producing insulin with rDNA technology was getting the A and B chains assembled correctly into a mature form.
• The Solution: In 1983, an American company named Eli Lilly developed a solution. They prepared two separate DNA sequences corresponding to chains A and B of human insulin. They introduced these sequences into plasmids of E. coli bacteria to produce the two chains separately. The chains were then extracted and combined by creating disulfide bonds to form functional human insulin.

Gene Therapy

Gene therapy is a collection of methods that aims to correct a gene defect diagnosed in a child or embryo. It involves inserting genes into a person's cells and tissues to treat a disease. The goal is to deliver a normal, functional gene to compensate for a non-functional or mutated one.

• First Clinical Case: The first clinical gene therapy was performed in 1990 on a 4-year-old girl with adenosine deaminase (ADA) deficiency. This enzyme is vital for the immune system to function. The disorder is caused by the deletion of the gene for ADA.
• Treatment Methods:
  1. Enzyme Replacement Therapy: The patient is given injections of functional ADA. This is not a permanent cure.
  2. Bone Marrow Transplantation: This can be a cure, but a suitable donor is needed.
  3. Gene Therapy Approach: Lymphocytes (a type of white blood cell) are taken from the patient's blood and grown in a culture. A functional ADA cDNA (complementary DNA) is introduced into these lymphocytes using a retroviral vector. These genetically engineered cells are then returned to the patient.
• Limitation and Permanent Cure: The limitation of this method is that lymphocytes are not immortal. Therefore, the patient needs periodic infusions of these modified cells. A permanent cure could be possible if the gene for ADA is introduced into cells from the bone marrow at an early embryonic stage.

Molecular Diagnosis

For effective treatment, early diagnosis of a disease is crucial. Conventional methods like serum and urine analysis often detect a disease only after symptoms appear, by which time the concentration of the pathogen (like bacteria or viruses) is already high. Modern molecular techniques allow for much earlier detection.

• Polymerase Chain Reaction (PCR): This technique can amplify nucleic acids (DNA or RNA). It can detect very low concentrations of a pathogen's DNA or RNA long before symptoms are visible. PCR is routinely used to detect HIV in suspected AIDS patients and to identify gene mutations in suspected cancer patients.
• Autoradiography with Probes: A single-stranded DNA or RNA molecule, tagged with a radioactive molecule, is called a probe. This probe is allowed to hybridize (bind) to its complementary DNA in a sample of cells. The sample is then viewed using autoradiography. A cell clone with a mutated gene will not show up on the photographic film because the probe will not bind to the altered DNA sequence.
• Enzyme Linked Immuno-sorbent Assay (ELISA): This technique works on the principle of antigen-antibody interaction. An infection can be detected either by the presence of antigens (like proteins from the pathogen) or by detecting the antibodies that the body has produced against the pathogen.

Transgenic Animals

Transgenic animals are animals that have had their DNA manipulated to carry and express an extra, foreign gene. Transgenic rats, rabbits, pigs, sheep, cows, and fish have been created, though over 95% of all existing transgenic animals are mice.

These animals are created for several common reasons:

1. Normal physiology and development: To study how genes are regulated and how they affect the body's normal functions. For example, by introducing a gene that alters insulin-like growth factor, scientists can learn about its biological role in growth.
2. Study of disease: Many transgenic animals are designed as models for human diseases like cancer, cystic fibrosis, rheumatoid arthritis, and Alzheimer's. This allows for the investigation of new treatments.
3. Biological products: Transgenic animals can be created to produce useful biological products. For example, human protein ($\alpha-1$-antitrypsin), used to treat emphysema, can be produced this way. In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 grams per litre). This milk contained human alpha-lactalbumin, making it a more nutritionally balanced product for human babies than natural cow's milk.
4. Vaccine safety: Transgenic mice are being developed to test the safety of vaccines, like the polio vaccine, before they are used on humans. This could replace the use of monkeys for vaccine testing.
5. Chemical safety testing (toxicity testing): Transgenic animals can be made more sensitive to toxic substances than non-transgenic animals. When these animals are exposed to a substance, its toxic effects can be studied much faster.

Ethical Issues

The power to manipulate living organisms raises significant ethical questions. Regulations are needed to evaluate the morality of human activities that might help or harm living organisms.

• Ecological Risks: The genetic modification of organisms can have unpredictable and potentially harmful results when these organisms are introduced into an ecosystem.
• Regulation in India: The Indian Government has established organizations like the GEAC (Genetic Engineering Approval Committee). The GEAC makes decisions regarding the validity of GM research and the safety of introducing GM organisms for public use.
• Patents and Biopiracy: The modification and use of living organisms have led to problems with patents. There is growing public anger over companies being granted patents for products and technologies that use genetic materials, plants, and traditional knowledge that have been developed and used by indigenous people and farmers for centuries.

• Biopiracy: This term refers to the use of bio-resources by multinational companies and other organizations without proper authorization from the countries and people concerned and without compensatory payment.
• Global Imbalance: Industrialized nations are often financially rich but poor in biodiversity and traditional knowledge. In contrast, developing and underdeveloped nations are rich in biodiversity and traditional knowledge. This knowledge can be exploited to save time and money in developing modern applications.
• Legal Protections: In response to this injustice, some nations are developing laws to prevent the unauthorized exploitation of their bio-resources. The Indian Parliament has amended the Indian Patents Bill to address these issues, including patent terms and research initiatives.