6.4 - Cloning and Biotechnology

vegetative propagation

a form of asexual reproduction where new, genetically identical individuals develop from non-reproductive tissues of a parent plant such as its roots, stems, and leaves

asexual reproduction

the production of offspring that are genetically identical to the parent

methods of vegetative propagation

rhizomes, runners, suckers, tubers, bulbs

rhizomes

Specialised horizontal underground stems that store food and can produce new vertical shoots and roots from buds on nodes along the rhizome

runners

Horizontal stems that grow along the soil surface away from the parent plant, with nodes or stem tips that can root to form a new plant upon contact with the ground

suckers

Shoots that emerge from the shallow root buds of the parent plant

tubers

Form when the tip of a stem becomes swollen with food, with buds on the tuber surface that can develop into new shoots

bulbs

Form when a leaf base becomes swollen with stored food, and the bud inside the bulb can form new shoots

how are plants artificially propagated from stem cuttings?

1. Cut a piece from the end of a parent plant's stem using a sharp, sterile tool.

2. Remove the lower leaves, leaving only one leaf at the top.

3. Dip the cut end in rooting powder, which contains plant hormones that encourage root growth.

4. Plant the cutting in a suitable growth medium, such as compost.

5. Place it in warm, moist conditions to promote root development.

6. Once rooted, transplant the new clone.

how are plants artificially propagated from root cuttings?

Take a section of root and make an angled cut on one end before treating it as you would a stem cutting

how are plants artificially propagated from leaf cuttings?

Remove an entire leaf, score the veins, and place it in a growing medium with the scored veins facing down

Advantages of vegetative propagation:

• fast

• ensures a high yield

• cost effective

• maintains the quality of the crop because the new plants have the same genetic traits as their parents

• allows plants to survive adverse conditions and regenerate each season

Disadvantages of vegetative propagation

1. it results in a lack of genetic variation in offspring

2. The plants are more susceptible to diseases, pests, and climate change

micropropagation

• a technique for producing many identical plant clones from a single parent plant through a tissue culture

• a type of asexual reproduction on a very large scale

explant

a small sample of tissue

stages of making a tissue culture for micropropagation

explant collection, sterilisation, culture, development, transfer

explant collection

• explants are taken from the parent plant, usually from the stem and root tips as they have meristem cells

• meristem cells are totipotent so can differentiate into any type of plant cell

sterilisation

• explant cells are sterilised to remove and inhibit the growth of contaminants

• this reduces the risk of widespread infection

culture

• sterilised explant cells are cultured in a nutrient-rich medium, which supplies minerals sugars, vitamins and growth hormones

development

• cells in each explant divide to form an undifferentiated mass of cells (callus)

• the callus cells are transferred to a new medium with specific conditions to encourage root and shoot formation, which allows them to differentiate and develop into plantlets

transfer

• fully formed plantlets are moved into a growth medium like soil, where they can develop into mature plants that are identical to the parent plant

Applications of micropropagation

• enables the rapid and large-scale propagation of plants that naturally reproduce slowly

• produces disease-free clones of crops

• allows for mass production of GMO plants

• can be used to produce seedless plants

advantages of micropropagation

• Produces plants that are genetically identical so there is a reliable inheritance of traits

• can be carried out at all times of year

• more space-efficient compared to conventional propagation methods

• rapidly produces of a large number of mature plants

disadvantages of micropropagation

• all plants are genetically identical (monoculture) so crops are vulnerable to diseases and environmental changes

• It may unintentionally propagate undesirable traits

• expensive and requires skilled technicians

• Explants and plantlets are vulnerable to infection, increasing the risk of total crop loss

natural cloning in invertebrates

• Some undergo regeneration or fragmentation, which forms new, genetically identical offspring from parts of their bodies that have broken off

natural cloning in vertebrates

• can occur naturally when an early embryo splits into two genetically identical embryos

• Each embryo grows independently, resulting in genetically identical offspring, known as identical or monozygotic twins

artificial embryo twinning

a process in which a single early embryo is manually split, separating its cells before they start to differentiate. This produces multiple identical offspring from a single embryo.

process of artificial twinning

1. A female organism is treated with hormones to produce multiple egg cells

2. the egg cells are extracted and fertilised in a petri dish to produce an embryo

3. the embryo divides into several cells and, while the cells are still totipotent, the embryo is split

4. each cell is placed into its own petri dish to develop into individual embryos

5. the embryos are implanted into the uteruses of surrogate mothers for development

somatic cell nuclear transfer (SCNT)

a process in which a nucleus is transferred from a somatic (body) cell of one animal into an ovum of another animal to form an embryo

• develops a clone of the organism from which the nucleus was extracted

steps of SCNT

1. a somatic cell nucleus is removed from an adult animal.

2. an ovum of a different female animal of the same species is enucleated (the nucleus is removed)

3. the nucleus of the somatic cell is transferred into the enucleated ovum

4. the somatic nucleus is fused with the enucleated ovum, often stimulated by an electric shock via electrofusion

5. the fused cell begins dividing, forming an embryo

6. the embryo is implanted into the nucleus of the surrogate mother

7. the surrogate mother eventually gives birth to a clone of the somatic cell donor

applications of animal cloning

• medical research - cloning produces genetically identical animals for drug testing and disease modelling

• conservation - its a method that can boost the number of endangered species

• agriculture - can replicate animals with desirable characteristics for selective breeding

• pharming - can produce therapeutic proteins

• stem cells - they provide a source of immunocompatible stem cells for tissue repair

arguments for animal cloning

• Helps to preserve biodiversity

• Enables the reproduction of infertile animals

• Facilitates medical advancements that could alleviate suffering

arguments against animal cloning

• Reduced genetic diversity increases disease risk

• Ethical concerns regarding the destruction of embryos

• Cloned animals often have health issues.

biotechnology

a technique in which living organisms or their components are used to to synthesise, breakdown, or transform materials for human use

major applications of microbes in biotechnology

• brewing - yeasts ferment sugars anaerobically to produce ethanol and carbon dioxide to make alcoholic drinks

• baking - Carbon dioxide produced by yeast during sugar fermentation, makes bread dough rise

• yoghurt - certain bacteria ferment lactose into lactic acid, which sours and solidifies milk into yogurt

• medicines - bioengineered fungi and bacteria produce drugs, like penicillin (antibiotic)

in what conditions is penicillin made?

1. small fermenters with constant stirring to ensure high oxygen levels

2. a nutrient-rich medium for optimum growth

3. a buffer to keep the pH stable at around 6.5

4. a constant temperature of about 25-27°C for ideal fungal activity

5. penicillin can then be extracted and purified for medical use

bioremediation

a technique that uses microbes to decompose pollutants and contaminants in soil or water

two main approaches to bioremediation

1. use of natural organisms - uses microbes' natural ability to digest organic materials, such as in sewage, along with the addition of certain nutrients

2. Develop genetically modified organisms for specific contaminants - uses bacteria to break down or accumulate specific pollutants like mercury

advantages of using microorganisms in biotechnology

• No ethical issues related to animal welfare

• Rapid reproduction rates enable fast, large-scale production

• Independent of weather or breeding cycles, allowing for year-round production regardless of climate

disadvantages of using microorganisms in biotechnology

• Risk of contamination by unwanted microbes

• Social concerns about genetically modified foods or microbes grown on waste products

• Sterile conditions are necessary, which can increase operational costs

what does biotechnology involve?

growing cultures of microorganisms, such as bacteria or yeasts, under controlled conditions

main reasons to culture microorganism

• To generate biomass of the microorganisms

• To manufacture compounds the microbes synthesise

Primary metabolites

substances that are produced in processes that are essential for normal microbial functioning.

• e.g. Ethanol from anaerobic respiration in yeast

Secondary metabolites

substances produced in non-essential processes.

• e.g. Antibiotics or plant defence chemicals

what are bioreactors used for?

large, commercial-scale production of microbial cultures

Typical components of a bioreactor

• A metal or plastic tank with inputs and outputs for liquids and gases.

• Paddles for mixing the culture

• Probes to monitor pH, temperature, and dissolved oxygen

• Ports for adding ingredients and removing products

• A sterilisation system

importance of a nutrient medium

provides the essential nutrients for microbial growth, in either a liquid form (broth) or a solid form (agar)

how is nutrient availability controlled inside bioreactors?

• Fresh medium circulated by paddles

• As population size increases, nutrient demand may exceed nutrient supply, so a constant supply ensures microbes have the nutrients they need

how is temperature controlled inside bioreactors?

• Heating/cooling water jacket surrounds vessel

• Too low and bacterial enzymes won't work so bacteria won't grow, too high and bacterial enzymes denature

how is pH controlled inside bioreactors?

• pH probe

• A build up of carbon dioxide may reduce pH, which can inhibit enzyme activity so keeping optimal pH allows microbial enzymes to function efficiently

how is oxygen levels controlled inside bioreactors?

• Sterile air pumped in

• As population size increases, oxygen demand may exceed oxygen supply as aerobically respiring microbes require oxygen

how is contamination and waste controlled inside bioreactors?

• Steam sterilisation between batches and removal of waste products

• Unwanted microbial contamination creates competition from other microbes, and a build up of toxic waste may kill the culture

Batch fermentation

• Microbes are grown in a fixed volume in individual batches until nutrients deplete and waste accumulates.

• Each batch is followed by emptying and cleaning of the vessel before starting the next batch.

continuous fermentation

• This involves continuously supplying fresh nutrients and removing the culture broth.

• This maintains the growth of the culture indefinitely

phases of Microbial growth curves in batch cultures

1. Lag phase - Cells have slow initial growth as they adapt to their environment and produce essential enzymes.

2. Log phase (exponential phase) - Rapid doubling of cell numbers occurs under ideal conditions, and growth rate is at its maximum.

3. Stationary phase - Growth rate plateaus as nutrients diminish and waste accumulates, and cell growth is equal to cell death.

4. Death phase - Cell death rate exceeds cell growth rate due to resource limitation and build up of toxins.

How to grow microbes on agar plates

1. Sterilise all equipment before use

2. Dip the sterilised inoculating loop into a starter culture, like broth that contains a bacterial suspension.

3. Transfer the microbes to a Petri dish containing a sterile nutrient medium by lightly zig-zagging the loop across the agar.

4. Close the plates and lightly tape them so they are not completely sealed (to prevent growth of anaerobic microbes).

5. Label the plates with relevant information, such as the type of microbe

6. Incubate the plates upside down under the required conditions.

7. Repeat steps 1 to 6 for a control agar dish with no bacteria.

8. Assess microbial growth by observing colony formation on the agar.

Factors that may affect microbial growth and how they can be investigated

• Temperature - Incubate duplicate plates at different temperatures.

• pH - Add buffer solutions to the agar to maintain different pH levels.

• Nutrient availability - Prepare agar with varying nutrient concentrations.

• Antimicrobial substances - Add different antimicrobial compounds to the agar plates.

enzyme immobilisation

a method which involves attaching or enclosing an enzyme onto a solid support or matrix. This allows for the reuse of the enzyme and increases its stability.

main methods of enzyme immobilisation

1. Binding - Enzymes may be bound to insoluble support materials like cellulose or collagen fibres by covalent or ionic bonds.

2. Adsorption - Enzymes may be adsorbed onto the surface of insoluble support materials.

3. Entrapment - Enzymes may be trapped in a matrix (e.g. silica gel) or a microcapsule.

4. Encapsulation - Enzymes may be isolated by a partially permeable membrane (e.g. within semi-permeable alginate beads)

advantages of using immobilised enzymes

• Cost-effective - Immobilising enzymes allows for the reuse of enzymes, reducing the need to purchase new enzymes.

• Product purity - Immobilisation produces enzyme-free products, avoiding contamination of the product with the enzyme.

• Improved stability - Immobilised enzymes are more tolerant of temperature and pH changes than enzymes in solution, making them more stable and less likely to denature

disadvantages of using immobilised enzymes

• Higher initial costs - Materials and bioreactors are more expensive than free enzymes and traditional fermenters, so are not always cost effective for small-scale production.

• Reduced enzyme activity - Immobilisation may reduce enzyme efficiency.

• Technical problems - The reactor systems are complex and prone to more technical problems.

Immobilising lactase to produce lactose-free milk

1. The lactase enzyme is attached to alginate beads to immobilise it.

2. The lactase-containing beads are packed into a column.

3. Milk is allowed to flow through the column.

4. Lactase hydrolyses the lactose in the milk into glucose and galactose, producing lactose-free milk.

5. The lactase remains in the column, allowing more milk to be processed continually.

6. The lactose-free milk can then be used to make dairy products for lactose-intolerant individuals