Biotechnological Applications In Agriculture

Biotechnology involves industrial-scale production of biopharmaceuticals and biologicals using genetically modified organisms (GMOs). Its applications span various fields, including therapeutics, diagnostics, agriculture (GM crops), food processing, bioremediation, waste treatment, and energy production.

Three crucial research areas in biotechnology that drive innovation are:

1. Developing the best catalyst: Often an improved microbe or a pure enzyme.
2. Creating optimal conditions: Engineering environments for the catalyst to function efficiently (e.g., bioreactors).
3. Downstream processing: Technologies for purifying the desired protein or organic compound after production.

Biotechnology has been instrumental in improving human life quality, particularly in food production and healthcare.

Increasing food production is a major challenge. Three potential approaches are considered:

1. Agro-chemical based agriculture (relying heavily on fertilizers and pesticides).
2. Organic agriculture (avoiding synthetic chemicals).
3. Genetically engineered crop-based agriculture.

While the Green Revolution significantly increased food supply, primarily through improved varieties, better management, and agrochemicals, these approaches face limitations, especially for farmers in developing countries (high cost of chemicals) and limitations in yield potential with conventional breeding.

Genetically modified crops offer a potential solution to increase yields further and reduce reliance on chemical inputs.

Genetically Modified Organisms (GMOs) are plants, bacteria, fungi, or animals whose genes have been altered through genetic manipulation.

Genetically Modified (GM) plants offer several benefits:

• Increased tolerance to abiotic stresses like cold, drought, salt, and heat.
• Reduced need for chemical pesticides (development of pest-resistant crops).
• Minimised post-harvest losses.
• Enhanced efficiency of nutrient uptake, preserving soil fertility.
• Improved nutritional value of food (e.g., Golden rice, enriched with Vitamin A).

GM technology also allows for the creation of plants tailored to produce specific industrial products like starches, fuels, and pharmaceuticals.

A significant application in agriculture is the development of pest-resistant plants, aimed at reducing pesticide use.

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Bt Cotton

Certain strains of the bacterium Bacillus thuringiensis (Bt) produce proteins that are toxic to specific groups of insects, such as lepidopterans (caterpillars), coleopterans (beetles), and dipterans (flies, mosquitoes).

• B. thuringiensis produces these insecticidal proteins as inactive protoxin crystals during a phase of their growth.
• These protoxins are harmless to the bacteria themselves.
• When an insect ingests the inactive toxin crystals, the alkaline pH of the insect's gut solubilises and activates the protoxin into its active form.
• The activated toxin binds to the epithelial cells of the insect's midgut, creating pores.
• These pores cause the cells to swell and lyse, ultimately leading to the death of the insect.

Scientists have isolated the genes (called cry genes) that encode these specific Bt toxins from Bacillus thuringiensis and introduced them into various crop plants, including cotton, corn, rice, tomato, potato, and soybean.

The choice of the specific cry gene introduced depends on the target pest. For example, the genes cryIAc and cryIIAb control cotton bollworms, while cryIAb controls corn borer. Plants expressing these genes produce the Bt toxin protein, making them resistant to these insects without the need for external insecticides, effectively acting as a bio-pesticide.

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Pest Resistant Plants

Many pests, including nematodes, cause significant damage to crops. For example, the nematode Meloidegyne incognitia infects the roots of tobacco plants, severely reducing yield.

A novel strategy to protect plants from such nematode infestations is based on RNA interference (RNAi).

• RNA interference is a natural process of cellular defense found in all eukaryotic organisms.
• It involves the silencing of a specific messenger RNA (mRNA) molecule by a complementary double-stranded RNA (dsRNA) molecule. The dsRNA binds to the target mRNA, preventing its translation into protein.
• The source of this dsRNA can be viral RNA genomes or mobile genetic elements.

Using Agrobacterium vectors, nematode-specific genes were introduced into tobacco plants. The introduced DNA was engineered to produce both a 'sense' and an 'anti-sense' RNA molecule within the host plant cells.

These sense and anti-sense RNA molecules, being complementary, hybridised to form a dsRNA molecule. This dsRNA initiated RNAi, specifically silencing the mRNA of the nematode that is essential for its survival or function.

As a result, the nematode parasite could not survive in the transgenic tobacco plant, which had effectively protected itself through this novel RNAi mechanism, reducing yield loss.

Biotechnological Applications In Medicine

Recombinant DNA technology has had a profound impact on healthcare, enabling the large-scale production of safe and more effective therapeutic drugs and diagnostics.

• Safety: Recombinant therapeutics are identical to human proteins, reducing the risk of unwanted immunological responses compared to products isolated from non-human sources (e.g., animal proteins).
• Effectiveness: They are often purer and more consistently active.
• Availability: Mass production in microbes or cell cultures makes them more readily available.

Globally, around 30 recombinant therapeutic products have been approved for human use, with about 12 being marketed in India.

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Genetically Engineered Insulin

Insulin is a hormone required for managing diabetes. Historically, insulin for diabetic patients was extracted from the pancreas of slaughtered cattle and pigs. However, animal insulin could sometimes cause allergic reactions or other immune responses in humans.

Insulin consists of two short polypeptide chains, chain A and chain B, linked by disulphide bridges.

In mammals (including humans), insulin is synthesised as a pro-hormone called pro-insulin. Pro-insulin contains an extra sequence called the C peptide, which is removed during maturation to form the active, mature insulin (consisting only of A and B chains).

Producing mature human insulin using recombinant DNA technology faced the challenge of assembling the A and B chains correctly, as the pro-insulin requires processing.

In 1983, the American company Eli Lilly successfully produced human insulin using rDNA techniques:

• They prepared two separate DNA sequences corresponding to the A and B chains of human insulin.
• These DNA sequences were introduced into separate plasmids of *E. coli*.
• The bacteria produced the A and B chains separately.
• The A and B chains were then extracted, purified, and combined *in vitro* by forming disulphide bonds to create mature, functional human insulin.

This genetically engineered insulin, identical in structure to natural human insulin, can be mass-produced, overcoming the limitations and issues associated with animal-sourced insulin.

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Gene Therapy

Gene therapy is a collection of methods that aim to correct a genetic defect diagnosed in a child or embryo. It involves introducing normal genes into a person's cells or tissues to treat a disease caused by a non-functional or defective gene. The goal is to replace or compensate for the function of the faulty gene.

The first successful clinical gene therapy was performed in 1990 on a 4-year-old girl with adenosine deaminase (ADA) deficiency. ADA is an enzyme essential for the proper functioning of the immune system. The deficiency is caused by a genetic defect (deletion) in the gene encoding ADA.

Traditional treatments for ADA deficiency included:

• Bone marrow transplantation: Can be curative but requires a suitable donor.
• Enzyme replacement therapy: Functional ADA enzyme is injected into the patient. This provides temporary relief but is not a permanent cure and requires repeated injections.

As a step towards gene therapy, lymphocytes from the patient's blood were grown in culture. A functional ADA cDNA (complementary DNA, derived from the normal ADA gene) was introduced into these lymphocytes using a retroviral vector. These genetically modified lymphocytes, now capable of producing functional ADA, were then returned to the patient.

However, since lymphocytes have a limited lifespan, the patient required periodic infusions of these genetically engineered cells. A potential permanent cure for ADA deficiency would involve introducing the gene isolated from bone marrow cells (producing ADA) into cells at early embryonic stages. This would ensure the gene is present in all cells and inherited.

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Molecular Diagnosis

Early and accurate diagnosis is vital for effective disease treatment. Conventional diagnostic methods (like biochemical tests of serum or urine) can often detect diseases only when symptoms have appeared and the pathogen concentration is already high.

Molecular diagnostic techniques allow for earlier detection even when pathogen levels are very low and symptoms are not yet visible. Important molecular diagnostic tools include:

• Recombinant DNA technology: Used in various ways, including probe-based detection.
• Polymerase Chain Reaction (PCR): Can detect very low concentrations of pathogens or genetic material. PCR can amplify a specific DNA or RNA sequence. If a pathogen's nucleic acid is present in a sample, even in tiny amounts, PCR can amplify it sufficiently for detection. PCR is routinely used for:
  • Detecting HIV in suspected AIDS patients even before symptoms appear.
  • Identifying mutations in genes that predispose individuals to diseases like cancer.
  • Diagnosing many other genetic disorders by amplifying specific gene regions.
• ELISA (Enzyme-Linked Immunosorbent Assay): Based on the principle of antigen-antibody interaction. Can detect the presence of specific antigens (from pathogens) or antibodies produced by the host in response to infection.

Another molecular diagnostic technique involves using a single-stranded DNA or RNA probe, tagged with a radioactive molecule. This probe is complementary to a specific DNA sequence (e.g., a gene). When the probe is allowed to hybridise with the DNA of cells or cloned DNA fragments, it will bind only to the complementary sequence. Detection using autoradiography reveals the presence of the target sequence.

This technique can be used to detect mutations: if a gene is mutated, the probe complementary to the normal gene sequence will not hybridise to the mutated gene in the sample. Clones with the mutated gene will therefore not show up on the autoradiograph, indicating the presence of the mutation.

Transgenic Animals

Transgenic animals are animals whose genetic material (DNA) has been altered by introducing and expressing an extra (foreign) gene from another species.

Many types of transgenic animals have been created, including rats, rabbits, pigs, sheep, cows, fish, and predominantly mice (over 95% of existing transgenic animals are mice).

Transgenic animals are produced for various reasons, providing significant benefits to humans:

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Applications Of Transgenic Animals

1. Study of Normal Physiology and Development: Transgenic animals are designed to study how genes are regulated, how they interact, and how they affect the normal functions and development of the body. By introducing genes and observing the resulting effects, scientists can gain insights into the biological roles of specific factors (e.g., genes involved in growth).
2. Study of Disease: Many transgenic animals serve as models for human diseases. They are created to carry specific mutations or express genes that mimic human disease conditions (e.g., models for cancer, cystic fibrosis, rheumatoid arthritis, Alzheimer's disease). These models allow researchers to study disease progression, mechanisms, and test potential new treatments more effectively.
3. Production of Biological Products: Transgenic animals can be engineered to produce valuable biological products (proteins, pharmaceuticals) that are difficult or expensive to obtain otherwise. The introduced foreign gene codes for the desired product, which is often secreted in accessible body fluids like milk.
   • Example: Production of alpha-1-antitrypsin, a human protein used to treat emphysema, in transgenic animals.
   • Attempts are being made to produce treatments for phenylketonuria (PKU) and cystic fibrosis using transgenic animals.
   • The first transgenic cow, Rosie (in 1997), produced milk containing the human protein alpha-lactalbumin. This human protein-enriched milk (2.4 grams per litre) was nutritionally more balanced for human babies than regular cow's milk.
4. Vaccine Safety Testing: Transgenic mice are used to test the safety of vaccines before they are used on humans. For instance, transgenic mice are used to test the safety of the polio vaccine. If these models prove reliable, they can replace the use of monkeys for vaccine safety testing, addressing ethical concerns about animal use and potentially being more standardised.
5. Chemical Safety Testing (Toxicity Testing): Transgenic animals can be engineered to be more sensitive to toxic substances than non-transgenic animals. When exposed to toxic chemicals, these animals show effects more readily, allowing for faster and more efficient toxicity testing. This process is similar to testing the toxicity of drugs.

Ethical Issues

The ability to manipulate living organisms through biotechnology raises significant ethical questions. As humans gain more power to alter life forms, there is a growing need for ethical guidelines to assess the morality and potential consequences of these actions on living organisms and the environment.

Genetic modification can have unpredictable effects when the modified organisms are introduced into the ecosystem. There are concerns about potential harm to non-target organisms, disruption of ecological balance, and the spread of engineered genes into wild populations.

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Regulatory Aspects

Governments worldwide are establishing regulatory bodies to oversee GM research and the release of GM organisms. In India, the Genetic Engineering Approval Committee (GEAC) is responsible for evaluating the validity of GM research and assessing the safety of introducing GM organisms for public use.

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Patents And Biopiracy

The commercial use of genetically modified organisms and bio-resources has also led to complex issues regarding patents. Concerns have been raised when companies are granted patents for products, technologies, or genetic materials that are derived from traditional knowledge or biological resources developed and used by indigenous people or farmers over centuries, without proper compensation or authorisation.

A prominent example involves Basmati rice. India has cultivated Basmati rice for thousands of years, with an estimated 27 documented varieties known for their unique aroma and flavour. Despite this long history and rich diversity in India, an American company was granted patent rights on Basmati rice by the US Patent and Trademark Office in 1997. This patent allowed the company to market a 'new' variety of Basmati globally. This 'new' variety was allegedly derived from Indian farmer's varieties, specifically by crossing Indian Basmati with semi-dwarf varieties and claiming it as an invention.

The patent was broad, potentially restricting others selling Basmati rice. Such incidents, along with attempts to patent traditional Indian medicinal knowledge and bio-resources (like turmeric and neem), highlight the issue of biopiracy.

Biopiracy is the term used for the unauthorised use of bio-resources and traditional knowledge by multinational companies and other organisations, without proper permission from the countries and people concerned and without compensatory payment. Industrialised nations are often rich financially but lack the biodiversity and traditional knowledge found in developing countries. This traditional knowledge is valuable for modern applications and commercialisation, saving significant time and cost.

There is growing recognition of the injustice in inadequate compensation and unfair benefit sharing between developed countries exploiting traditional knowledge/bio-resources and the developing countries where these resources originate. This has led some nations to develop laws to prevent such exploitation.

In India, amendments to the Indian Patents Bill have been enacted to address these issues, including provisions related to patent terms, emergency use, and research and development initiatives involving traditional knowledge and bio-resources.

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