Answer:
Breeding is the controlled reproduction of organisms to develop new varieties or strains with desired characteristics. It can be traditional (conventional breeding) or modern (using biotechnology).
Plant Breeding:
Objectives of Plant Breeding:
- Increase crop yield
- Improve nutritional quality
- Develop resistance to pests, diseases, drought, and salinity
- Earlier maturation (shorter growing season)
- Improve mechanical harvesting characteristics
Methods of Plant Breeding:
Animal Breeding:
Objectives: Increase milk production, meat yield, egg production; improve disease resistance; faster growth; better fibre quality.
Methods of Animal Breeding:
Answer:
Recombinant DNA technology (genetic engineering) is the set of techniques used to isolate, manipulate, combine, and express genes from different organisms. It was developed in the early 1970s (Cohen and Boyer, 1973).
Tools Required in Recombinant DNA Technology:
Steps of Recombinant DNA Technology:
Step 1: Isolation of the Gene of Interest
- The gene to be cloned is identified and isolated from the donor organism's genome
- Method A: Cut the donor organism's DNA using restriction enzymes
- Method B (preferred for eukaryotic genes): Extract mRNA for the protein of interest → synthesise complementary DNA (cDNA) using reverse transcriptase → this avoids introns (non-coding sequences that bacteria cannot process)
Step 2: Preparation of the Vector
- The vector (usually a plasmid) is cut open with the same restriction enzyme used to cut the gene of interest
- This creates compatible sticky ends on both the gene and the vector
Step 3: Ligation — Making Recombinant DNA
- The gene of interest is mixed with the cut vector
- DNA ligase joins the gene into the vector through the complementary sticky ends
Step 4: Transformation — Introduction into Host Cell
- The recombinant plasmid is introduced into the host cell (usually E. coli)
- Methods: Heat shock (briefly heating bacteria in presence of calcium chloride), electroporation (brief electric pulse opens cell membrane pores), microinjection
Step 5: Selection of Transformed Cells
- Not all host cells take up the recombinant DNA; need to identify which ones did
- Methods using selectable markers:
- Antibiotic resistance genes on the plasmid (e.g., ampicillin resistance): transformed cells survive on antibiotic media; untransformed cells die
- Blue-white screening: The gene is inserted into the lacZ gene of the plasmid; colonies with the gene are white (lacZ disrupted, can't produce blue colour); colonies without the insert are blue
Step 6: Propagation (Cloning)
- Selected transformed bacteria are grown in large fermenters (bioreactors)
- As bacteria replicate, they copy the recombinant plasmid → billions of copies of the gene
Step 7: Expression
- The gene is expressed — bacteria produce the desired protein
- Conditions are optimised for maximum protein production
Step 8: Extraction and Purification
- The desired protein is extracted from the bacterial culture
- Purified using chromatography techniques
- Quality control testing before use
Production of Human Insulin Using Recombinant DNA Technology:
Before 1982, insulin for diabetes treatment was extracted from the pancreas of pigs and cattle (animal insulin), which caused allergic reactions in some patients and was expensive.
Step 1: Identify the human insulin gene. Human insulin is encoded by a gene on chromosome 11. It codes for preproinsulin, which is processed into two chains: A chain (21 amino acids) and B chain (30 amino acids) connected by C peptide.
Step 2: Synthesise cDNA for each insulin chain (A and B) from the mRNA, using reverse transcriptase.
Step 3: Insert each cDNA into a separate plasmid vector (in the USA, Genentech used E. coli; modern methods also use yeast).
Step 4: Transform E. coli with the recombinant plasmids. The bacteria produce the A and B chain proteins separately as fusion proteins with beta-galactosidase.
Step 5: Extract the fusion proteins, cleave the insulin chains chemically (cyanogen bromide treatment).
Step 6: Mix the A and B chains under conditions that allow the correct disulfide bonds to form between them → active human insulin.
Step 7: Purify and quality-test.
Answer:
PCR (Polymerase Chain Reaction):
Definition: PCR is an in vitro (in a test tube) technique used to amplify (make millions or billions of copies of) a specific target DNA sequence from a small starting sample. It was invented by Kary Mullis in 1983 (Nobel Prize in Chemistry, 1993).
Components Required for PCR:
1. Template DNA: The DNA containing the target sequence to be amplified (even tiny amounts from a single cell are sufficient) 2. Primers: Two short, single-stranded DNA oligonucleotides (typically 15–25 bases long), one complementary to each strand of the template at the boundaries of the target sequence. They define what is amplified. 3. Taq DNA Polymerase: A heat-stable DNA polymerase from the thermophilic bacterium Thermus aquaticus (lives in hot springs). Remains active even at the high temperatures used in PCR. 4. dNTPs (Deoxynucleotide Triphosphates): The building blocks of DNA (dATP, dGTP, dCTP, dTTP) 5. Buffer solution: Optimal ionic conditions for the enzyme 6. MgCl2: Cofactor for Taq polymerase
Steps of PCR (One Cycle = Three Steps):
Step 1: Denaturation (94–96°C, ~30 seconds)
- The reaction mixture is heated to high temperature (94–96°C)
- The hydrogen bonds between the two strands of double-stranded template DNA break
- The DNA strands separate (denature) into two single-stranded templates
Step 2: Annealing (50–65°C, ~30 seconds)
- The temperature is lowered to allow primers to bind (anneal) to their complementary sequences on the single-stranded template DNA
- The annealing temperature depends on the primer sequence (usually 5°C below the melting temperature of the primers)
- The two primers bind to opposite strands, flanking the target sequence
- This defines the region of DNA to be amplified
Step 3: Extension / Elongation (72°C, ~1 minute per kb)
- Temperature is raised to 72°C — the optimal temperature for Taq polymerase activity
- Taq DNA polymerase binds to the primer-template junction and synthesises a new complementary DNA strand
- It adds dNTPs in the 5' to 3' direction, extending from each primer
- Each template strand produces a new complementary strand
PCR Cycles:
- The cycle (denaturation → annealing → extension) is repeated 25–40 times
- After each cycle, the number of copies doubles
- After n cycles: 2n copies are produced
- After 30 cycles: approximately 1 billion (2^30 = 1.07 × 10^9) copies of the target sequence
PCR Machine:
- A thermocycler is used — it automatically and precisely controls temperature changes through the programmed cycles
Applications of PCR:
Answer:
Animal Cloning:
Definition: Cloning is the production of genetically identical copies of an organism. In animals, it is achieved through Somatic Cell Nuclear Transfer (SCNT) — transferring the nucleus from a somatic (body) cell into an enucleated egg cell.
Types of Animal Cloning:
Dolly the Sheep — The First Mammal Cloned from an Adult Somatic Cell:
Scientists: Ian Wilmut, Keith Campbell, and colleagues at the Roslin Institute, Edinburgh, Scotland.
Year: Dolly was born on 5 July 1996; the research was published in February 1997. She was named after the country singer Dolly Parton.
Procedure (Somatic Cell Nuclear Transfer — SCNT):
Step 1: Donor Nucleus
- A somatic (body) cell — specifically a mammary gland cell — was taken from a 6-year-old adult Finn Dorset white sheep (nuclear donor)
- These cells were cultured in the laboratory and then starved of nutrients (serum-deprived) to arrest cell division in the G0 phase — this synchronises the cell cycle, which is essential for successful nuclear transfer
Step 2: Enucleated Egg Cell
- An unfertilised egg cell (oocyte) was obtained from a Scottish Blackface sheep (egg donor)
- The nucleus was removed from the egg cell by micromanipulation (enucleation) — creating an enucleated egg (cytoplast)
Step 3: Cell Fusion / Nuclear Transfer
- The enucleated mammary gland cell was placed next to the enucleated egg cell
- They were fused together using an electric pulse (electrofusion) — this both fused the cells and activated the egg to begin development
- The nucleus of the mammary cell is now inside the egg cytoplasm — a "reconstructed embryo"
Step 4: Activation and Development
- The reconstructed embryo was stimulated to divide and develop (the egg cytoplasm reprogrammed the somatic cell nucleus back to a totipotent embryonic state)
- The embryo developed in culture to the blastocyst stage
Step 5: Implantation
- The blastocyst was implanted into the uterus of a surrogate mother (another Scottish Blackface sheep)
- The surrogate carried the pregnancy to term
- Dolly was born with white wool (like the Finn Dorset donor), not black-faced like the surrogate or the egg donor
- DNA testing confirmed she was genetically identical to the Finn Dorset mammary cell donor
- Dolly successfully reproduced naturally (had 6 lambs), demonstrating that she was a fully functional animal
Significance of Dolly: 1. First proof that the nucleus from an adult, fully differentiated somatic cell can be reprogrammed to be totipotent (can direct the development of a complete organism) 2. Overturned the previous belief that differentiation was irreversible 3. Opened the field of nuclear reprogramming and led eventually to iPSC technology
Dolly's health: Dolly was euthanised on 14 February 2003 at age 6.5 (Finn Dorset sheep normally live to 11–12 years). She had developed progressive lung disease and arthritis. Some scientists suggested she aged prematurely — the Finn Dorset donor was 6 years old, so her telomeres were already shortened.
Other Cloned Animals:
- Cattle, pigs, horses, cats, dogs, mice have all been cloned
- Endangered species cloning attempts: Gaur (cloned 2001, died of infections), Banteng (lived, 2003)
- No confirmed human cloning (major ethical, legal, and scientific barriers)
Ethical Issues:
- Reproductive human cloning is banned in virtually all countries
- Therapeutic cloning raises ethical debates about the status of human embryos
- Low efficiency of SCNT (Dolly required 277 attempts)
- Health problems in cloned animals (epigenetic abnormalities)
Answer:
Plant Tissue Culture (Micropropagation):
Definition: Plant tissue culture is the technique of growing isolated plant cells, tissues, or organs on an artificial nutrient medium under aseptic (sterile) conditions.
Basis: Plant cells are totipotent — each living cell contains the full genetic information to regenerate a whole plant.
History: F.C. Steward (1950s) grew a complete carrot plant from single root cells — demonstrated totipotency.
Basic Technique of Plant Tissue Culture:
Step 1: Selection of Explant
- An explant is the plant tissue used to initiate tissue culture
- Can be any part: leaf, root tip, stem, seed, embryo, pollen grain, bud
- Younger, rapidly dividing tissues give best results
- Must be free from disease
Step 2: Surface Sterilisation
- The explant is surface sterilised to remove all microorganisms
- Methods: 70% ethanol wash, sodium hypochlorite (bleach) solution, followed by multiple sterile water washes
Step 3: Nutrient Medium
- Murashige and Skoog (MS) medium — most commonly used
- Contains: macronutrients (N, P, K, Ca, Mg, S), micronutrients, vitamins, sugar (sucrose — energy source), plant growth regulators (phytohormones)
- Auxins (e.g., 2,4-D, IAA): Promote root formation and callus formation
- Cytokinins (e.g., Kinetin, BAP): Promote shoot formation and cell division
- Ratio of auxin to cytokinin determines the developmental pathway:
- High auxin:cytokinin → roots
- Low auxin:cytokinin → shoots
- Equal → callus
Step 4: Callus Formation
- The explant placed on nutrient medium with high auxin → dedifferentiates into an undifferentiated mass of cells called a callus
- Callus cells are actively dividing
Step 5: Organogenesis (Shoot and Root Formation)
- Callus on medium with high cytokinin → develops shoots (shoot organogenesis)
- Alternatively, single callus cells can be cultured in liquid medium → embryoids (somatic embryos) → plantlets
Step 6: Acclimatisation
- Test tube plantlets are gradually transferred from sterile conditions to greenhouse, then to field
- They need time to adjust from high humidity and artificial light to normal conditions
