Cloning and Biotechnology¶

Part of Module 6: Genetics, evolution and ecosystems.

Cloning — the production of genetically identical copies of an organism — occurs naturally in plants and is used commercially in horticulture and animal husbandry. Biotechnology is the broader industrial use of living organisms or their components to make products. This topic covers the techniques of plant and animal cloning, the use of microorganisms in industrial fermentation, and the immobilisation of enzymes for continuous industrial processes.

Learning Objectives¶

Natural Cloning¶

Plants: Vegetative Propagation¶

Many plants reproduce asexually by vegetative propagation — the generation of a new individual from part of the parent plant. Because no meiosis or fertilisation is involved, the offspring are genetically identical to the parent.

Examples: - Stolons / runners: horizontal above-ground stems that produce new plants at nodes (e.g. strawberry) - Bulbs: underground storage organs containing meristematic tissue that can develop into new plants (e.g. daffodil, tulip) - Rhizomes: underground horizontal stems (e.g. grass, iris) - Tubers: swollen underground stems (e.g. potato) - Suckers: shoots that arise from the root system of a parent plant (e.g. English Elm — adapted to asexual reproduction when damaged)

Perennating organs (e.g. tubers, bulbs, rhizomes) also function as food stores that allow plants to survive dormancy over winter, then use the stored resources to regenerate rapidly in spring.

Animals: Natural Twinning¶

Natural clones in animals arise by embryo splitting shortly after fertilisation. When the cells of an early embryo (up to about the 8-cell stage) separate, each can develop into a complete individual — producing identical (monozygotic) twins. Identical twins are natural clones because they have the same genotype.

Artificial Cloning: Plants¶

Plant Cuttings¶

A cutting is a simple, traditional cloning technique: 1. A section of stem is cut between a leaf node and the main stem 2. Leaves are removed from the lower end to reduce water loss 3. The cut end is treated with rooting powder (containing synthetic auxin, e.g. IBA — indole-3-butyric acid) to stimulate root development 4. The cutting is placed in moist compost; roots develop and the cutting grows into an independent plant

This produces plants genetically identical to the parent and is widely used in horticulture (roses, shrubs, houseplants).

Tissue Culture (Micropropagation)¶

Tissue culture allows the rapid, large-scale production of genetically identical plants from very small amounts of plant tissue.

Procedure: 1. Explant isolation: A small piece of tissue (the explant) is taken from the meristematic region of the parent plant (e.g. shoot tip, which contains actively dividing cells) 2. Surface sterilisation: The explant is sterilised to remove any contaminating microorganisms 3. Callus formation: The explant is placed on a nutrient medium containing: - Essential mineral ions - Sucrose (carbon source) - High auxin and low cytokinin ratio → stimulates cell division but not differentiation → a mass of undifferentiated cells called a callus is produced 4. Shoot induction: Small pieces of callus are transferred to a medium with low auxin and high cytokinin ratio → shoots begin to form (organogenesis) 5. Root induction: Developing shoots are transferred to a medium with high auxin → roots develop 6. Acclimatisation: Young plantlets are transferred to compost in a greenhouse and gradually acclimatised to less controlled conditions

Advantages of micropropagation: - Very large numbers of plants produced from a small amount of donor material - Season-independent and weather-independent production - Can propagate plants that are difficult to grow from seed (e.g. some orchids) - Useful for propagating genetically modified plants where all plants need to be identical - Can propagate endangered species for conservation

Disadvantages: - All plants are genetically identical (no genetic diversity) → susceptible to the same pathogens or environmental changes - More technically demanding and expensive than growing from seed - Risk of contamination with microorganisms

Artificial Cloning: Animals¶

Nuclear Transfer (Somatic Cell Nuclear Transfer, SCNT)¶

SCNT produces an individual genetically identical to a donor organism:

1. A differentiated somatic (body) cell is taken from the donor animal (e.g. an udder cell from Dolly the sheep)
2. An unfertilised egg cell is obtained from a donor female; its nucleus is removed by a micropipette (enucleation)
3. The donor nucleus is inserted into the enucleated egg cell (by direct injection or by fusing the two cells using an electric pulse)
4. The reconstructed cell is stimulated to divide (by electric pulse or chemical treatment)
5. The resulting embryo is implanted into the uterus of a surrogate mother
6. The surrogate gives birth to an animal genetically identical (in nuclear DNA) to the original donor

Note: The cloned animal is not 100% identical to the donor because the egg cell contains its own mitochondria (with mitochondrial DNA). Dolly the sheep (1996) was the first cloned mammal produced from a differentiated adult cell.

Embryo Splitting¶

An alternative approach: 1. An early embryo (4–16 cell stage) is obtained from a female after artificial insemination 2. The embryo is split into 2 or more pieces 3. Each piece is implanted into a different surrogate mother 4. Each develops into a genetically identical individual

This technique is used in livestock breeding to produce multiple offspring from a high-value female without the complexity of SCNT.

Applications of animal cloning: - Medical research: genetically identical animals for drug testing and disease modelling - Conservation: boosting numbers of endangered species from a limited gene pool - Agriculture: replicating animals with desirable traits (high milk yield, lean muscle) for selective breeding programmes - Pharming: genetically engineered cloned animals that produce therapeutic proteins in their milk - Stem cells: cloned embryos provide a source of immunocompatible stem cells for potential tissue repair

Advantages of animal cloning: - Allows rapid reproduction of high-value animals (prize livestock, rare breeds) - Can preserve endangered species; enables reproduction of infertile animals

Disadvantages: - Low success rates (SCNT still technically unreliable; many ova needed per successful clone) - Cloned animals may have compromised health or shortened lifespan - No genetic variation → vulnerability to disease - Significant ethical concerns about animal welfare and the ethics of producing clones

The Use of Microorganisms in Biotechnology¶

Biotechnology is the industrial application of living organisms or their components to produce useful products or processes.

Microorganisms are used extensively in biotechnology because: - They grow rapidly (short generation times — E. coli divides every 20 minutes) - They can grow at low temperatures on cheap substrates (including industrial waste products) - They can be grown in very large quantities in controlled conditions - They can be genetically engineered to produce specific products at high purity - The products they make (enzymes, fermentation products) are often purer and more consistent than those from chemical processes

Primary and Secondary Metabolites¶

The substances produced by microorganisms in biotechnology are classified as:

• Primary metabolites: substances produced in processes essential for normal microbial functioning. They are made throughout growth. Examples: ethanol (anaerobic respiration in yeast), lactic acid, amino acids.
• Secondary metabolites: substances produced in non-essential processes, often made at the end of the exponential phase or in the stationary phase, not during active growth. Examples: antibiotics (penicillin), many plant defence chemicals.

This distinction matters in industrial fermentation: a secondary metabolite like penicillin is produced most abundantly when growth slows (stationary phase), influencing how fermenters are operated.

Biotechnological Products from Microorganisms¶

Microbial Growth Curve¶

When a small number of microorganisms is inoculated into a nutrient medium in a closed system (batch culture), growth follows a characteristic S-shaped curve with four phases:

The maximum population size is determined by the carrying capacity — the maximum population that the environment can support.

Batch Culture vs Continuous Culture¶

Optimising Fermenter Conditions¶

To maximise product yield, fermenters are designed to maintain optimal conditions: - Temperature: Maintained at the optimum for the organism's enzymes (e.g. 37 °C for E. coli) using a water jacket; prevents denaturation or slowing of metabolic reactions - pH: Monitored and adjusted using acids or alkalis; prevents enzyme denaturation - Oxygen supply: Stirring and aeration ensure aerobic conditions (if the organism is aerobic); prevents anaerobic by-products - Nutrient supply: Continuously monitored; supplements added as needed - Aseptic conditions: All equipment and media sterilised before use; ongoing monitoring for contamination

Enzyme Immobilisation¶

Immobilised enzymes are enzymes fixed to an insoluble support material, allowing them to be used repeatedly and easily separated from products.

Methods of Immobilisation¶

Advantages of Immobilised Enzymes in Industry¶

• Product not contaminated with enzyme — no costly purification step needed
• Enzyme can be reused many times — economical
• Greater stability — immobilised enzymes are often more resistant to temperature and pH changes than free enzymes
• Continuous operation — substrate flows over or through the immobilised enzyme continuously (columns)

Industrial Examples¶

Key Terms¶

• Clone: genetically identical copy of a cell or organism.
• Vegetative propagation: natural asexual reproduction in plants using roots, stems, or leaves.
• Perennating organ: a plant organ (e.g. bulb, tuber, rhizome) that stores food enabling the plant to survive dormancy and regenerate each season.
• Micropropagation: plant tissue-culture method used to produce many clones from a small piece of tissue.
• Somatic cell nuclear transfer (SCNT): cloning technique in which a diploid nucleus from a somatic cell is inserted into an enucleated egg cell.
• Embryo splitting: production of clones by separating cells from an early embryo.
• Aseptic technique: working method that prevents contamination by unwanted microorganisms.
• Batch culture: microbial culture in a closed system with no fresh nutrient input once started.
• Continuous culture: microbial culture in an open system with nutrients added and product removed continuously.
• Fermenter: controlled vessel used to grow microorganisms on an industrial scale.
• Immobilised enzyme: enzyme fixed in place so substrate can pass over it while the enzyme is retained.
• Primary metabolite: substance produced by microorganisms during normal growth and metabolism (e.g. ethanol).
• Secondary metabolite: substance produced by microorganisms in non-essential processes, often during stationary phase (e.g. penicillin).
• Pharming: use of genetically engineered animals to produce therapeutic proteins in their milk or other tissues.
• Adsorption: immobilisation method in which an enzyme binds to the surface of a support material.
• Entrapment: immobilisation method in which an enzyme is trapped within a gel matrix.

Connected Pages¶

• 6.1.3 Manipulating genomes (genetic engineering of microorganisms)
• 5.1.5 Plant and animal responses (commercial use of plant hormones)
• 5.2.2 Respiration (fermentation — anaerobic respiration)
• 2.1.4 Enzymes (enzyme structure and function)
• Module 6: Genetics, evolution and ecosystems