6.4- Cloning and Biotechnology

vegetative propagation

• form of asexual reproduction

• genetically identical individuals develop from non-reproductive tissues of parent plant such roots, stems, leaves

methods of natural vegetative propagation

• rhizomes

• stolons (runners)

• Suckers

• Tubers

• Bulbs

rhizomes

• specialised horizontal underground stems

• store food and can produce new vertical shoots and roots from nodes along rhizome

• e.g. marram grass

stolons

• horizontal stems that grow along soil surface away from parent plant

• nodes or stem tips that can root to form new plant upon contact with ground

• e.g. strawberries

suckers

• shoots that emerge from shallow root bud of parent plant

• e.g. elm trees

tubers

• form when tip of stem becomes swollen with food

• buds on tuber surface can develop into new shoots

• e.g. potatoes

bulbs

• form when leaf base becomes swollen with stored food

• bud inside bulb can form new shoots

• e.g. daffodil

what is a cutting

a section of stem, root or leaf taken from parent plant and planted in soil→ grows into clone of parent

taking stem cuttings

1. cut 5-10 cm piece of parent’s plant stem using sharp, sterile tool

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

3. dip cut end in rooting powder- contains plant hormones to encourage root growth

4. plant cutting in a suitable growth medium e.g. compost

5. place in warm, moist conditions to promote root development

6. transplant new clone once rooted

cuttings from roots or leaves

• root cuttings → take section of root and make angled cut on one end before treating it as you would a stem cutting

• leaf cuttings→ remove entire lead, score veins and place in growing medium with scored veins facing down

advantages of vegetative propagation

• fast

• high yield ensured

• cost effective

• maintains quality of crop

• allows plant to survive adverse conditions and regenerate each season

disadvantages of vegetative propagation

• results in lack of genetic variation in offspring

• plants more susceptible to diseases, pests and climate change

what is micropropagation

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

• type of asexual reproduction

steps in making a tissue culture for micropropagation

1. explant collection:

   • small tissue samples taken from parent plant to start micropropagation

   • typically taken from stem and root tips→ meristem cell which are totipotent

2. sterilisation

   • explant’s cells sterilised to remove and inhibit growth of contaminants e.g. bacteria

   • reduces risk of widespread infection

3. culture

   • sterilised explant cells cultured on nutrient rich medium→ supplies minerals, sugar, growth hormones etc

4. development

   • cells in each explant divide to form undifferentiated mass of cells→ callus

   • callus cells transferred to new medium with specific conditions to encourage shoot and root formation

   • allows callus cells to differentiate and develop into plantlets

5. transfer

   • fully formed plantlets moved to growth medium e.g. soil

   • can develop into mature plants- identical to parent plant

applications of micropropagation

• enables rapid, large scale propagation of plants that naturally reproduce slowly or are rare or endangered

• used for producing disease-free clones of crops and preserving valuable genetic resources

• allows mass production of GM plants

• can be used to produce seedless plants

advantages of micropropagation

• produces genetically identical plants→ reliable inheritance of traits

• can be carried out at all times of the year

• more space-efficient compared to conventional propagation methods

• rapidly produces large number of mature plants

disadvantages of micropropagation

• All plants genetically identical (monoculture)→ vulnerable

• may unintentionally propagate undesirable traits

• expensive and requires skilled technicians- less feasible on small scale

• explants and plantlets vulnerable to infection

natural animal cloning in invertebrates

• some invertebrates undergo regeneration or fragmentation

• forms new, genetically identical offspring from parts of bodies that have broken off

natural cloning in vertebrates

• can occur naturally when early embryo splits into 2 genetically identical embryos

• each embryo grows independently, resulting in genetically identical offspring i.e. identical twins

what is artificial embryo twinning

• single embryo manually split

• produces multiple identical offspring from single embryo

process of artificial twinning

1. female organism treated with hormones to produces multiple ova

2. ova extracted and fertilised in petri dish to produce embryo

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

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

5. embryos implanted into uteruses of surrogate mothers

what is somatic cell nuclear transfer

• nucleus transferred from somatic (body) cell of one animal into ovum of another to form embryo

steps in somatic cell nuclear transfer

1. somatic cell nucleus removed from adult animal

2. ovum of different female animal of same species is enucleated

3. nucleus from somatic cell transferred into enucleated ovum

4. somatic nucleus fused with enucleated ovum→ stimulated by electric shock

5. fused cell begins dividing→ forming embryo

6. embryo implanted into uterus of surrogate mother

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

applications of animal cloning

• medical research→ drug testing and disease modelling

• Conservation→ can boost numbers of endangered species from limited gene pool

• Agriculture→ can replicate animals with desirable characteristics

• Pharming→ can produce therapeutic proteins

• Stem cells→ provide source of immunocompatible stem cells for tissue repair

arguments for animal cloning

• ensures transmission of desirable characteristics to multiple offspring

• enables reproduction of infertile animals

• helps preserve biodiversity

• can rapidly increase population size of species

• facilitates medical advancements that could alleviate suffering

arguments against animal cloning

• high costs and technically complex

• reduced genetic diversity increases disease risk

• potential for shorter lifespans in clones

• ethical concerns regarding destruction of embryos

• clones animals have health issues

• inefficient→ high failure rates so many ova used to produce 1 cloned offspring

what is biotechnology

• use of living organisms or their components e.g. enzymes to synthesise, break down or transform materials for human use

• often uses microorganisms

applications of microbes in biotechnology

• brewing

• baking

• Cheese Making

• Yoghurt

• medicines→ bioengineered fungi + bacteria produce drugs

• bioremediation→ microbes speed up degradation of pollutants like oil spills

bioremediation

• uses microbes to decompose pollutants and contaminants in soil or water

• two main approaches:

  • use natural organisms→ uses microbes’ natural ability to digest organic materials

  • develop GM organisms for specific contaminants→ uses bacteria to break down/ accumulate specific pollutants

advantages of using microorganisms in biotechnology (7)

• cost-effective mass production= lower consumer prices

• no ethical issues related to animal welfare

• rapid reproduction rates= large-scale production

• simple nutrient requirements

• high protein, low fat= efficient food source as meat alternative

• genetic modification= enhanced nutrient profiles

• independent of weather or breeding cycles

Disadvantages of using microorganisms in biotechnology (6)

• sterile conditions necessary- can increase costs

• risk of contamination by unwanted microbes

• potential toxin production

• separation of microorganisms from nutrient broth required for food production

• differences in taste and texture from traditional food sources

• social concerns about GM foods or microbes on waste products

why are microorganisms cultured in biotechnology

• to generate biomass of microorganisms→ use in producing single cell protein as animal feedstock

• to manufacture compounds the microbes synthesise e.g. antibiotics, vitamins, enzymes

primary metabolites

• substances that are produced in processes essential for microbial functioning

• e.g. ethanol from respiration of yeast

secondary metabolites

• substances produced in non-essential processes

• e.g. antibiotics or plant defence chemicals

components of bioreactors

• metal/ plastic tank with inputs and outputs for liquids and gases

• paddles for mixing culture to ensure even distribution of food, oxygen and temperature throughout

• probes to monitor pH, temperature and dissolved oxygen

• ports for adding ingredients and removing products

• sterilisation system e.g. steam injection

• nutrient medium

how are conditions optimised inside bioreactors

• fresh medium circulated by bioreactors→ constant supply of nutrients for microbes

• heating/cooling water jacket surrounds vessel→ ensures optimal temp. for microbes

• pH monitored by pH probe and adjusted automatically by adding acids and bases→ allows optimal pH for enzyme activity

• sterile air pumped in→ allows sufficient oxygen for aerobic respiration

• steam sterilisation between batches and removal of waste products→ prevents contamination which could kill the culture

batch fermentation

• microbes grown in a fixed volume in individual batches until nutrients deplete and waste accumulates

• each batch followed by emptying and cleaning of the vessel before starting next batch

continuous fermentation

• continuously supplying fresh nutrients and removing culture broth

• maintains growth of culture indefinitely

microbial growth curves in batch cultures

1. lag phase→ initial cell growth as they adapt to environment and produce essential enzymes

2. log 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- cell growth= cell death

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

factors affecting microbial growth

• temperature

• pH

• nutrient availability

• antimicrobial substances

what is enzyme immobilisation

• a way of reusing enzymes

• attaching or enclosing an enzyme onto a solid support or matrix

  • allow the reuse of the enzyme and increases its stability

main methods of immobilising enzymes

• binding→ enzymes bound to insoluble support materials e.g. cellulose/ collagen fibres by covalent or ionic bonds

• adsorption→ enzymes adsorbed onto the surface of insoluble support materials

• entrapment→ enzymes trapped in a matrix or microcapsule

• encapsulation→ enzymes isolated by partially permeable membrane

advantages of using immobilised enzymes

• cost effective→ allows reuse of enzymes= reduced need to purchase new enzymes

• product purity→ avoids contamination of products with the enzyme

• improved stability→ immobilised enzymes more tolerant of temp. and pH changes- less likely to denature

disadvantages of enzyme immobilisation

• higher initial costs

• reduced enzyme activity- may change shape of active or allosteric sites

• technical problems→ complex reactor systems more prone to technical problems

immobilising lactase to produce lactose-free milk

1. lactase enzyme attached to alginate beads to immobilise it

2. lactase- containing beads packed into a column

3. milk allowed to flow through the column

4. lactase hydrolyses lactose in milk into glucose and galactose→ produces lactose- free milk

5. lactase remains in column, allowing continual processing of milk

6. lactose-free milk can be used to make dairy products