Biotechnology and Its ApplicationsNEET Botany · Class 12 · NCERT Chapter 8

Bt toxin mechanism and Bt crops

Bacillus thuringiensis is a soil bacterium that naturally produces proteins called Cry toxins (also called delta-endotoxins or Bt toxins). These are named after the cry (crystal) genes that encode them, because the proteins form crystalline inclusions in sporulating bacteria. Different cry genes produce proteins active against different insect orders.

Why Bt toxin is produced as a protoxin

Bt toxin is produced as an inactive protoxin (a harmless precursor). The protoxin is activated only under specific conditions: a highly alkaline environment (pH 9 to 10) and the presence of specific protease enzymes. This is exactly the environment in the midgut of certain insect larvae. In mammals, the stomach is acidic (pH 1 to 2), so the protoxin remains inactive and is safely digested.

Step-by-step mechanism in the insect gut

Bt crops and their target pests

GM crops: herbicide tolerance, virus resistance, golden rice

Herbicide-tolerant crops

The herbicide glyphosate (Roundup) inhibits EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase) — an enzyme in the shikimate pathway needed to synthesise aromatic amino acids. Without aromatic amino acids, plants die.

Roundup Ready soybean contains a mutant aroA gene from Agrobacterium tumefaciens, encoding an EPSP synthase that is insensitive to glyphosate. The transgenic crop makes aromatic amino acids normally and survives herbicide spraying; weeds (with wild-type EPSP synthase) are killed.

Virus-resistant transgenic plants

Transgenic tobacco was made resistant to Tobacco Mosaic Virus (TMV) by introducing the TMV coat protein gene into the tobacco plant. The plant produces viral coat protein, which interferes with viral replication (cross-protection). Similarly, papaya was made resistant to papaya ringspot virus using the viral coat protein gene approach.

Golden rice

Vitamin A deficiency causes preventable blindness (especially in children) in many developing countries. Rice endosperm lacks carotenoids (including beta-carotene, the precursor to Vitamin A). Golden rice was engineered by introducing two genes:

1. Phytoene synthase (Psy) — from daffodil (original) or maize (GR2E): converts geranylgeranyl pyrophosphate (GGPP) to phytoene.
2. Phytoene desaturase (CrtI) — from Erwinia uredovora: converts phytoene through lycopene to beta-carotene.

Beta-carotene (pro-vitamin A) accumulates in the endosperm, giving the rice a golden/yellow colour. When consumed, it is converted to Vitamin A in the body.

RNA interference (RNAi)

RNA interference is a natural cellular mechanism for silencing genes at the post-transcriptional level. It was discovered in 1998 by Andrew Fire and Craig Mello (Nobel Prize in Physiology or Medicine, 2006).

Mechanism of RNAi

1. Double-stranded RNA (dsRNA) is introduced into the cell (or produced by transgene expression).
2. The enzyme Dicer cleaves the dsRNA into short 21 to 23 nucleotide siRNA (short interfering RNA) duplexes.
3. The siRNA duplex is incorporated into the RISC (RNA-Induced Silencing Complex) along with Argonaute protein.
4. The sense strand of the siRNA is discarded; the antisense (guide) strand guides RISC to the complementary mRNA sequence.
5. RISC cleaves the target mRNA, leading to its degradation. The target gene is silenced at the post-transcriptional level.

Application: nematode control in tobacco

The nematode Meloidogyne incognita (root-knot nematode) is a major pest of tobacco. Transgenic tobacco was created that produces dsRNA specific to a gene essential for nematode survival (e.g., a gene expressed in the nematode pharynx). When nematodes feed on the transgenic root cells:

1. The dsRNA from the plant enters the nematode's cells through the gut.
2. RNAi is triggered inside the nematode.
3. The essential nematode gene is silenced.
4. The nematode cannot complete its life cycle in the plant root.
5. The plant is protected from nematode damage.

Recombinant insulin (humulin)

Before 1982, diabetic patients used insulin extracted from pig or cattle pancreas. Porcine insulin differs from human insulin by 1 amino acid; bovine insulin by 3 amino acids. These differences caused allergic reactions in some patients.

Production of recombinant human insulin (humulin)

Human insulin consists of two polypeptide chains: A chain (21 amino acids) and B chain (30 amino acids), linked by two disulfide bonds. It is produced by:

1. The human insulin A chain gene was synthesised chemically and cloned into an E. coli plasmid under the control of a lac promoter.
2. The human insulin B chain gene was similarly synthesised and cloned into a separate E. coli plasmid.
3. Each E. coli strain was grown separately; each produced the respective polypeptide chain as a fusion protein with beta-galactosidase.
4. The A and B chains were extracted, purified, and treated with cyanogen bromide to remove the beta-galactosidase fusion.
5. The purified A and B chains were mixed and incubated under conditions that allow the correct disulfide bonds (A6-A11, A7-B7, A20-B19) to form.
6. The resulting functional insulin was purified and formulated for injection.

Eli Lilly commercially launched humulin in 1982 — the first recombinant DNA pharmaceutical product approved for human use. Today, most recombinant insulin is produced in yeast (Saccharomyces cerevisiae) as a single proinsulin chain that is then processed to remove the connecting C-peptide, giving the A-B chain structure.

Gene therapy and ADA deficiency

Gene therapy is the introduction of a normal, functional allele into cells that carry a defective allele, to treat a genetic disease. It is applicable only when the disease is due to a loss-of-function mutation in a single gene.

ADA (adenosine deaminase) deficiency

ADA deficiency is caused by deletion or mutation in the ADA gene. ADA converts adenosine to inosine in the purine salvage pathway. Without ADA, toxic deoxyadenosine accumulates in T lymphocytes, causing their death and resulting in Severe Combined Immunodeficiency (SCID) — the patient has essentially no immune system.

ADA gene therapy protocol (1990)

1. Blood is drawn from the patient. T lymphocytes are isolated.
2. A functional ADA gene is inserted into a retroviral vector (replication-defective retrovirus).
3. The patient's lymphocytes are infected with the retroviral vector in cell culture.
4. The retrovirus integrates the ADA gene stably into the lymphocyte genome.
5. The genetically corrected lymphocytes are expanded in culture and then reinfused into the patient.
6. The corrected lymphocytes can now produce ADA enzyme and survive. Immune function is partially restored.

Other biotechnology-derived medicines

Molecular diagnostics: PCR and ELISA

Traditional diagnostic methods (culturing bacteria, microscopy) often require large amounts of pathogen material. Molecular diagnostic tools can detect pathogens at much lower concentrations, often before symptoms appear.

PCR-based diagnosis

PCR (Polymerase Chain Reaction) can amplify a single copy of pathogen DNA or RNA to detectable levels. Applications:

• HIV diagnosis: PCR detects HIV proviral DNA in lymphocytes or viral RNA (after reverse transcription) during the window period (2 to 6 weeks post-infection), before antibodies reach detectable levels.
• Cancer diagnosis: PCR can detect mutated oncogene sequences (e.g., KRAS, TP53, BCR-ABL fusion gene in CML) even when very few cancer cells are present.
• Bacterial infections: PCR detects pathogen DNA (e.g., Mycobacterium tuberculosis, Chlamydia trachomatis) in clinical samples faster than culture.
• Prenatal diagnosis: PCR on fetal DNA from chorionic villus sampling (CVS) or amniocentesis can detect genetic disorders.

ELISA (Enzyme Linked Immunosorbent Assay)

ELISA detects antigen-antibody interactions and converts them to a quantifiable colour signal. It is highly sensitive and specific. The basic principle:

1. A capture antibody (or antigen) is coated onto the surface of a microwell plate.
2. The patient's sample is added; the target antigen (or antibody) binds to the coated antibody.
3. An enzyme-conjugated secondary antibody (which binds to the primary antibody) is added.
4. After washing away unbound antibody, the enzyme substrate is added.
5. The enzyme converts the colourless substrate to a coloured product. Colour intensity is proportional to the amount of antigen/antibody in the sample.
6. Absorbance is measured by a spectrophotometer. Results compared to a standard curve.

ELISA applications: HIV antibody detection, hepatitis B surface antigen (HBsAg) detection, food allergen testing, pregnancy tests (HCG hormone), hormone assays (TSH, LH, FSH), drug monitoring.

Bioethics and biopiracy

The power of modern biotechnology to alter living organisms raises profound ethical questions. Two major issues frequently tested in NEET are biopiracy and the regulation of GM organisms.

Biopiracy

Biopiracy is the unauthorised appropriation of biological resources (plants, animals, microorganisms) or traditional knowledge about them (typically held by indigenous communities in developing countries) by companies or researchers, without prior informed consent of the originating community and without equitable sharing of benefits.

• Turmeric (Curcuma longa): The US Patent Office granted a patent for using turmeric to heal wounds. Indian scientists showed this was traditional Indian knowledge documented in Sanskrit texts. The patent was revoked in 1997.
• Basmati rice: A US company obtained a patent for a hybrid rice variety resembling basmati. India successfully challenged this, arguing basmati is a traditional Indian product with centuries of documented history.
• Neem (Azadirachta indica): European and US patents on neem-based pesticides and antifungals were challenged by India and other countries; several were revoked because traditional uses were already documented.

Convention on Biological Diversity (CBD) and the Nagoya Protocol

The CBD (1992, Rio Earth Summit) established three principles: (1) conservation of biodiversity, (2) sustainable use of its components, and (3) fair and equitable sharing of benefits arising from genetic resources. The Nagoya Protocol (2010, in force 2014) added detailed Access and Benefit-Sharing (ABS) requirements — countries providing genetic resources must give prior informed consent (PIC) and negotiate benefit-sharing agreements before those resources are used commercially.

Regulation of GM organisms in India

Other bioethics concerns in biotechnology

• Allergenicity of GM foods: A gene from one organism may encode a protein that is allergenic in humans, even if the original organism was safe (e.g., Brazil nut 2S albumin in soybean).
• Gene flow: Pollen from GM crops can fertilise wild relatives, introducing transgenes into natural populations (gene escape).
• Biodiversity loss: Large-scale cultivation of one GM variety can displace traditional diverse cultivars.
• Gene therapy ethics: Germ-line gene therapy (modifying embryo or gamete DNA) would affect future generations who cannot consent. Somatic gene therapy affects only the individual patient.
• Human cloning and designer babies: Use of reproductive technologies to select traits raises concerns about eugenics.

Worked problems

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