Cloning

Question 1

What is vegetative propagation?

Answer 1

Asexual reproduction where new genetically identical individuals develop from non-reproductive tissues of a parent plant e.g roots, stems, and leaves

Question 2

What are methods of natural vegetative propagation?

Answer 2

• rhizome
• stolons (runners)
• suckers
• tubers
• bulbs

Question 3

Outline the process of taking a stem cutting?

Answer 3

1) cut 5-10 cm piece from end of parent plant’s stem using sharp sterile tool

2) remove lower leaves leaving only 1 leaf at top

3) dip cut end in rooting powder containing plant hormones encouraging root growth

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

5) place in warm moist conditions promoting root development

6) once rooted transplant new clone

Question 4

How do you take a root cutting?

Answer 4

Take section of root and make angled cut on 1 end before treating as you would stem cutting

Question 5

How do you take a leaf cutting?

Answer 5

Remove entire leaf, score veins, and place in growing medium with 2nd veins facing down

Question 6

What are advantages of vegetative propagation?

Answer 6

• fast
• ensures high yield
• cost effective
• maintains quality of crop because new plants have same genetic traits as parents
• allows plants to survive adverse conditions and regenerate each season

Question 7

What are disadvantages of vegetative propagation?

Answer 7

• results in lack of genetic variation in offspring
• plants more susceptible to diseases, pests, and climate change

Question 8

What is micropropagation?

Answer 8

Technique producing many identical plant clones from single parent plant through tissue culture asexually (large scale)

Question 9

Outline the process of making a tissue culture for micropropagation?

Answer 9

1) small tissue sample (explants) taken from parent plant to typically from stem and root tips as have meristem cells

2) explant’s cells sterilised to remove and inhibit growth of contaminants e.g bacteria and fungi reducing risk of widespread infection

3) sterilised explant cells cultured on nutrient-rich medium supplying minerals, sugars, vitamins, and growth hormones

4) cells in each explant divide to form undifferentiated mass of cells called callus, transferred to new medium with specific conditions to encourage shoot and root formation

5) callus cells differentiate and develop into plantlets

6) fully formed plantlets complete with shoots and roots moved to growth medium e.g soil where they can develop into mature plants identical to parent plant

Question 10

What are the applications of micropropagation?

Answer 10

• enables rapid large-scale propagation of plants that naturally reproduce slowly or rare or endangered
• used for producing disease-free clones of crops and preserving valuable genetic resources
• allows mass production of genetically modified plants, such as those engineered for herbicide resistance
• can be used to produce seedless plants or those difficult to cultivate from seeds

Question 11

What are advantages of micropropogation?

Answer 11

• produces genetically identical plants so reliable inheritance of traits e.g high yields
• can be carried out all times of year
• more space-efficient compared to conventional propagation methods
• rapidly produces large number of mature plants

Question 12

What are disadvantages of micropropogation?

Answer 12

• all plants genetically identical (monoculture) so vulnerable to diseases and environmental changes
• may unintentionally propagate undesirable traits
• expensive and requires skilled technicians making it less feasible on small scale
• explants and plantlets vulnerable to infection increasing risk of total crop loss

Question 13

How do invertebrate naturally clone?

Answer 13

Regeneration or fragmentation

forms new genetically identical offspring from parts of body broken off

Question 14

How can vertebrae naturally clone?

Answer 14

Early embryo splits into 2 genetically identical embryos

each embryo grows independently resulting in genetically identical offspring

Question 15

Outline the process of artificial twinning?

Answer 15

1) female organism treated with hormones to produce multiple ova (egg cells)

2) ova extracted fertilised in Petri dish to produce embryo

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

4) each cell placed into own Petri dish to develop into individual embryos

5) embryos implanted into uteruses of surrogate mothers for development

Question 16

What is somatic cell nuclear transfer?

Answer 16

Process which nucleus transferred from somatic (body) cell of 1 animal into ovum of another animal to form embryo

Question 17

Outline the stages of somatic cell nuclear transfer?

Answer 17

1) somatic cell nucleus removed from adult animal

2) ovum of different female animal of same species enucleated (nucleus removed)

3) nucleus from somatic cell transferred into enucleated ovum

4) somatic nucleus fused with enucleated ovum often stimulated by electric shock through electrofusion

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

Question 18

What are applications of animal cloning?

Answer 18

Medical research = cloning produces genetically identical animals for drug testing and disease modelling

Conservation = offers method to boost numbers of endangered species from limited gene pool

Agriculture = can replicate animals with desirable characteristics for selective breeding to improve product quality

Pharming = genetically engineered animal clones can be used to produce therapeutic proteins

Stem cells = cloned embryos provide source of immunocompatible stem cells for tissue repair

Question 19

What are arguments for animal cloning?

Answer 19

• ensure transmission of desirable genetic traits to multiple offspring
• enables reproduction of infertile animals
• helps preserve biodiversity
• rapidly increase population size of certain species
• facilitates medical advancements that could alleviate suffering

Question 20

What are arguments against animal cloning?

Answer 20

• high costs and technically complex
• reduced genetic diversity increases disease risk
• potential for shorter lifespans in clones
• ethical concerns regarding destruction of embryos
• cloned animals often have health issues
• inefficient as can take many ova to produce single cloned offspring due to high failure rates

Question 21

What is biotechnology?

Answer 21

Uses living organisms or components e.g enzymes, to synthesise, breakdown, or transform materials for human use

Question 22

What are applications of microbes in biotechnology?

Answer 22

Brewing = yeasts ferment sugars anaerobically to produce ethanol and CO2 to make alcoholic drinks e.g wine and beer

Baking = CO2 produced by yeast during sugar fermentation makes bread dough rise

Cheese making = enzyme from genetically modified yeast coagulates milk into curds while bacteria and moulds contribute to acidification, thickening, and flavour

Yoghurt = certain bacteria ferment lactose into lactic acid which sours and solidifies milk into yoghurt

Medicines = bioengineered fungi and bacteria produce drugs e.g antibiotic penicillin and insulin for diabetes treatment

Bioremediation = microbes speed up degradation of pollutants e.g oil spills

Question 23

What is bioremediation?

Answer 23

Uses microbes to decompose pollutants and contaminants in soil or water, making it critical environmental restoration technique

Question 24

What are the 2 main approaches to bioremediation?

Answer 24

Natural organisms = uses microbes’ natural ability to digest organic materials e.g sewage or oil spill along with addition of certain nutrients

Develop GM organisms for specific contaminants = uses bacteria to break down or accumulate specific pollutants e.g mercury

Question 25

What are advantages of using microorganisms in biotechnology?

Answer 25

- cost-effective cultivation and mass production lead to lower consumer prices - no ethical issues related to animal welfare - rapid reproduction rates enable fast large-scale production - simple and versatile nutrient requirements including ability to use waste materials lowering costs - high protein and low fat content make them efficient food source offering a sustainable alternative to meat - genetic modification can enhance nutrient profiles or optimise production - year round production regardless of climate

Question 26

What are disadvantages of using microorganisms in biotechnology?

Answer 26

- sterile conditions necessary increasing operational costs - risk of contamination by unwanted microbes - potential toxin production by some microbe necessitates removal processes - separation of microorganisms from nutrient broth required for food production - differences in texture and taste from traditional food sources may require addition of flavour enhancers - social concerns about genetically modified foods or microbes grown on waste products

Question 27

Why are microorganisms cultured?

Answer 27

- generate biomass of microorganisms e.g for use in producing single-cell protein as animal feedstock - manufacture compounds microbes synthesise e.g antibiotics, vitamins, or enzymes

Question 28

What are primary metabolites and give an example?

Answer 28

- substances produced in processes essential for normal microbial functioning e.g. ethanol from anaerobic respiration in yeast

Question 29

What are secondary metabolites and give an example?

Answer 29

- substances produced in non-essential processes e.g. antibiotics or plant defence chemicals

Question 30

What are bioreactors?

Answer 30

Large fermentation tanks optimised for microbial growth and are used for large commercial-scale production of microbial cultures

Question 31

What are typical components of a bioreactor?

Answer 31

- metal or plastic tank with inputs and outputs for liquids and gases - paddles for mixing culture to ensure even distribution of food, O2, and temp throughout - probes to monitor pH, tempe, and dissolved O2 - ports for adding ingredients and removing products - sterilisation system e.g steam injection - nutrient medium of either liquid broth or solid agar

Question 32

What are limiting factors in a bioreactor?

Answer 32

- nutrient availability - temp - PH - O2 levels - contamination and waste

Question 33

How and why is nutrient availability regulated in a bioreactor?

Answer 33

Fresh medium circulated by paddles as population size increases nutrient demand may exceed nutrient supply so constant supply ensures microbes have nutrients needed

Question 34

How and why is temperature regulated in a bioreactor?

Answer 34

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

Question 35

How and why is PH regulated in a bioreactor?

Answer 35

Monitored by pH probe and automatically adjusted by adding acids/bases build up of CO2 may reduce pH inhibiting enzyme activity so keeping optimal pH allows microbial enzymes to function efficiently

Question 36

How and why is O2 levels regulated in a bioreactor?

Answer 36

Sterile air pumped in as population size increases O2 demand may exceed O2 supply as aerobically respiring microbes require oxygen

Question 37

How and why is contamination and waste regulated in a bioreactor?

Answer 37

Steam sterilisation between batches and removal of waste products unwanted microbial contamination creates competition from other microbes and build up of toxic waste may kill culture

Question 38

Why must aseptic technique be used when culturing microorganisms?

Answer 38

Prevent contamination with unwanted microorganisms ensuring nutrient medium remains sterile until use and high yields of desired product are made

Question 39

What is batch fermentation?

Answer 39

- microbes grown in fixed volume in individual batches until nutrients deplete and waste accumulates - each batch is followed by emptying and cleaning of vessel before starting next batch

Question 40

What is continuous fermentation?

Answer 40

- involves continuously supplying fresh nutrients and removing culture broth - maintains growth of culture indefinitely

Question 41

What are the 4 key phases in microbial growth curves in batch cultures?

Answer 41

- lag phase - log phase (exponential phase) - stationary phase - death phase

Question 42

What happens in the lag phase?

Answer 42

Cells have slow initial growth as they adapt to environment and produce essential enzymes

Question 43

What happens in the log phase?

Answer 43

Rapid doubling of cell numbers occurs under ideal conditions and growth rate is at its maximum

Question 44

What happens in the stationary phase?

Answer 44

Growth rate plateaus as nutrients diminish and waste accumulates and cell growth is equal to cell death

Question 45

What happens in the death phase?

Answer 45

Cell death rate exceeds cell growth rate due to resource limitation and build up of toxins

Question 46

In continuous systems how is build up of inhibitory by-products overcome?

Answer 46

Constantly replenishing nutrients and removing wastes

Question 47

Outline how to grow microbes on an agar plate?

Answer 47

1) sterilise all equipment before use by holding wire inoculation loop in bunsen flame 2) dip sterilised wire inoculating loop into starter culture e.g broth containing bacterial suspension 3) transfer microbes to Petri dish containing sterile nutrient medium by lightly zig-zagging loop across agar 4) close plates and lightly tape them so not completely sealed (to prevent growth of anaerobic microbes) 5) label plates with relevant info e.g microbe, date, and conditions 6) incubate plates upside down under required conditions 7) repeat steps 1 to 6 for control agar dish with no bacteria 8) assess microbial growth by observing colony formation on agar

Question 48

What are examples of factors affecting microbial growth that can investigate?

Answer 48

Temperature = incubate duplicate plates at different temperatures pH = add buffer solutions to agar to maintain different pH levels Nutrient availability = prepare agar with varying nutrient concentrations Antimicrobial substances = add different antimicrobial compounds to agar plates

Question 49

What are immobilised enzymes?

Answer 49

Enzymes attached or enclosed onto a solid support or matrix allowing for reuse of enzyme and increasing its stability

Question 50

What are the 4 methods of enzyme immobilisation?

Answer 50

Binding = enzymes may be bound to insoluble support materials e.g cellulose or collagen fibres by covalent or ionic bonds Adsorption = enzymes may be adsorbed onto surface of insoluble support materials Entrapment = enzymes may be trapped in matrix (e.g. silica gel) or a microcapsule Encapsulation = enzymes may be isolated by partially permeable membrane (e.g. within semi-permeable alginate beads)

Question 51

What are advantages of using immobilised enzymes?

Answer 51

- cost-effective as immobilised enzymes can be reused reducing need to purchase new enzymes - product purity as immobilisation produces enzyme-free products avoiding contamination of product with enzyme - improved stability as immobilised enzymes more tolerant of temp and pH changes than enzymes in solution making them more stable and less likely to denature

Question 52

What are disadvantages of using immobilised enzymes?

Answer 52

- higher initial costs as materials and bioreactors more expensive than free enzymes and traditional fermenters so not always cost effective - reduced enzyme activity due to immobilisation reducing efficiency - technical problems as reactor systems are complex and prone to more technical problems

Question 53

How can lactase be immobilised to produce lactose free milk?

Answer 53

1) lactase enzyme attached to alginate beads to immobilise 2) lactase-containing beads packed into column 3) milk allowed to flow through column 4) lactase hydrolyses lactose in milk into glucose and galactose producing lactose-free milk 5) lactase remains in column allowing more milk to be processed continually