Biotechnology

The Ti plasmid (Tumour-inducing plasmid) is found in Agrobacterium tumefaciens, a soil bacterium that naturally transfers its T-DNA into plant cells, causing crown gall disease. Genetic engineers disarm the Ti plasmid (remove tumour-causing genes) and replace them with the gene of interest. A. tumefaciens then acts as a natural 'vector' to deliver foreign DNA into plant cells. It works best on dicots (tobacco, tomato, soybean). For monocots (rice, maize), biolistics (gene gun) is preferred.

Bt cotton in India (Bollgard I) contains the Cry1Ac gene from Bacillus thuringiensis, producing a crystal protein toxic to Helicoverpa armigera (American bollworm) and other lepidopteran pests. Bollgard II contains both Cry1Ac and Cry2Ab for broader and more durable resistance. The Cry proteins are pore-forming toxins — they bind to receptors in insect midgut cells, causing osmotic lysis. They are non-toxic to mammals and humans. Bt cotton approved by GEAC in 2002 — first transgenic crop approved in India.

Golden Rice is a transgenic rice variety engineered to produce beta-carotene (provitamin A) in the endosperm — the part people eat. Normal rice produces no beta-carotene in endosperm. Golden Rice 2 (GR2) uses the psy gene from maize and the crt1 gene from Erwinia, producing up to 37 µg beta-carotene/gram dry weight — enough to address Vitamin A Deficiency (VAD), which causes blindness in ~500,000 children/year globally. Developed by Potrykus and Beyer.

Molecular markers detect DNA sequence differences between individuals: RFLP (Restriction Fragment Length Polymorphism) — oldest, uses restriction enzymes + Southern blot, codominant; RAPD (Random Amplified Polymorphic DNA) — PCR-based, arbitrary primers, dominant markers, cheap; SSR/Microsatellites — PCR-based, short tandem repeats, highly polymorphic, codominant, most used in plant breeding; SNP (Single Nucleotide Polymorphism) — single base changes, most abundant in genomes, used in GWAS; AFLP — combines restriction digest + PCR, high multiplex. SSR and SNP markers are now standard in marker-assisted selection (MAS).

The Cartagena Protocol on Biosafety (2000, entered force 2003) is an international treaty under the Convention on Biological Diversity that governs transboundary movement of Living Modified Organisms (LMOs/GMOs). India ratified it in 2003. In India, GMO approvals go through: RCGM (Review Committee on Genetic Manipulation) under DBT for contained research, and GEAC (Genetic Engineering Appraisal Committee) under MoEF for environmental release and commercial cultivation. GEAC approved Bt cotton (2002) and recommended Bt Brinjal (2010, but placed under moratorium by the government).

PCR amplifies a specific DNA segment through 3 thermocyclic steps: (1) Denaturation — heat to 94–96°C; double-stranded DNA separates into single strands; (2) Annealing — cool to 50–65°C; primers bind (anneal) to complementary sequences flanking the target; (3) Extension/Elongation — heat to 72°C; Taq DNA polymerase (from Thermus aquaticus) extends the primers, synthesising new DNA. Each cycle doubles the target: n cycles → 2ⁿ copies. Typical PCR = 30–35 cycles → ~1 billion copies from a single DNA molecule in 2–3 hours.