6.4 - Cloning and Biotechnology

Question 1

What is a clone? How is it derived and as a result of what type of reproduction?

Answer 1

Genetically identical organisms or cells. Derived by mitosis. Produced by asexual reproduction.

Question 2

What is vegetative propagation?

Answer 2

Asexual reproduction in plants.

Question 3

Describe 7 different examples of the production of natural clones in plants that use vegetative propagation.

Answer 3

Runner (or stolen): Horizontal stems that grow on the ground surface & form roots at certain points, e.g. strawberry plant.
Rhizome: Horizontal stems that grow underground. Often used as overwintering organs from which new stems grow from in the spring, e.g. ginger.
Sucker: New stems that grow from the roots of a plant e.g. tomato.
Bulb: An underground swollen stem with stored food and a bud (fleshy), e.g. onion.
Corm: An underground stem with scaly leaves & buds (solid), e.g. crocus.
Leaves: Clones grow on leaf margins. Immature plants drop off & take root, e.g. kalanchoe leaf.
Tuber: Underground stem, often used as a storage organ for nutrients, e.g. potato.

Question 4

Describe how to take plant cuttings as an example of a simple cloning technique.

Answer 4

Cut a stem between two leaf joints (nodes) at an angle.
Dipping cut end in rooting hormone (IAA) to stimulate root growth.
Place cut end in moist soil.
New roots will grow from stem tissues.

Question 5

What is grafting? What are the advantages of grafting?

Answer 5

Taking a cutting from one plant and grafting it onto the stem of another. Allows the features of both plants to be available.

Question 6

What is micro-propagation (tissue culture)?

Answer 6

Growing large numbers of new plants from meristem tissue taken from a sample plant. It is a form of large scale artificial cloning by separating cells and growing them on a growth medium.

Question 7

Describe the production of artificial clones of plants from tissue culture.

Answer 7

Small pieces of meristematic plant material selected (growing shoot or root) = explants.
Explants are sterilised, aseptic techniques used.
Explants placed on sterile growth medium, containing nutrients & plant growth substances auxin & cytokinin.
Auxin & cytokinin stimulate mitosis and explants form a callus (a mass of undifferentiated, totipotent cells).
Callus is sub-divided.
Small clumps grown on different growth media to encourage roots & then shoots to form.
(100 auxin:1 cytokinin stimulates roots / 4 auxin: 1 cytokinin stimulates shoots)
(auxins stimulate root growth and cytokinins predominantly stimulate shoot growth)
Tiny plantlets form & transferred to a greenhouse to be grown in compost or soil & hardened off.

Question 8

What are the constituents of the culture medium other than auxin & cytokinin?

Answer 8

Sucrose; amino acids; vitamins; ions / named ions; water; agar.

Question 9

One disadvantage of micro-propagation is that it can be more expensive than traditional methods. Suggest some factors which may contribute to this extra cost.

Answer 9

Requires lots of skilled workers - labour intensive.
Requires sterile conditions & use of aseptic techniques.
Requires expensive equipment.
Facilities & space to grow & mature plantlets.

Question 10

Evaluate the production of natural clones.

Answer 10

Advantages: relatively quick so can take advantage of favourable conditions; no reproductive partner has to be found.
Disadvantages: no genetic variation; all susceptible to the same selection pressures; disease, environmental changes.

Question 11

Evaluate the production of artificial clones.

Answer 11

Advantages: all are clones & so all have same desirable characteristics; high yield, disease resistance; plants that have lost their ability to breed sexually can be reproduced (e.g commercially grown bananas).
Disadvantages: tissue culture is labour intensive + expensive to set up & maintain; tissue culture can fail due to microbial contamination; no genetic variation - all are clones & so all susceptible to the same pests &/or diseases.

Question 12

How could genetic variation be introduced to both natural & artificial clones?

Answer 12

By a mutation.

Question 13

Give examples of natural animal clones.

Answer 13

Twins, waterfleas & greenfly.

Question 14

What natural process does embryo twinning mimic?

Answer 14

The process that results in identical twins.

Question 15

Describe the process of embryo twinning.

Answer 15

A zygote is created by IVF.
Zygote divides by mitosis to 16 cell embryo.
Individual cells are separated into petri dishes and continue to divide.
Each small mass of cells is then placed into the uterus of a surrogate mother.
Embryos continue to develop & eventually genetically identical offspring are born.

Question 16

Describe the process of somatic cell nuclear transfer (SCNT).

Answer 16

A somatic cell (non-reproductive cell) is taken from sheep A. The nucleus is extracted and kept.
An egg cell from sheep B has its nucleus removed (encleated).
The nucleus from sheep A and the enucleated egg cell from sheep B are fused using an electric shock and begin dividing normally to form an embryo.
The embryo is implanted into a surrogate mother – the offspring will be a clone of sheep A (as it contains the genetic information from sheep A).

Question 17

What are the arguments for animal cloning?

Answer 17

Desirable characteristics are always passed on to clones.

Increasing the population of endangered species to help preserve biodiversity.

Question 18

What are the arguments against animal cloning?

Answer 18

The techniques are very difficult, time-consuming & expensive.
No genetic variability in cloned populations – so all susceptible to the same diseases.

Question 19

What is biotechnology?

Answer 19

It is the large-scale industrial use of living organisms to produce food, drugs or other products.

Question 20

Name some industrial processes that involve microorganisms.

Answer 20

Brewing: Yeast added to barley. Respired anaerobically using the glucose from the barley & produces ethanol and carbon dioxide (fermentation).
Baking: Fermentation of yeast makes bread rise. Flatbreads do not use yeast!
Cheese making: Instead of using rennet from the lining of a calves’ stomachs, yeast cells have been genetically modified to produce the enzyme present in rennet called chymosin, which clots the milk. Lactic acid bacteria used to convert lactose in milk to lactic acid, making it turn sour & contributes to it solidifying. Fungi are added to make blue vein cheese.
Yoghurt making: Also uses lactic acid bacteria (Lactobacillus) to clot the milk.
Penicillin production: When stressed, Penicillium fungi produce an antibiotic to stop bacteria from growing & competing for resources. Fungus is grown under stress in industrial fermenters and the penicillin is collected & processed to be used in medicine.
Insulin production: Produced by GM bacteria which have the human insulin gene inserted into DNA. Grown in an industrial fermenter and the insulin produced is collected and purified.
Bioremediation: Microorganisms are used to remove pollutants, like oil & pesticides from contaminated sites.

Question 21

Why are microorganisms used in biotechnological processes?

Answer 21

Ideal growth conditions can easily be created.
Fast growth & reproduction (asexual).
Can be grown on waste materials from industry.
No animal welfare issues.
Can be grown at any time of year.

Question 22

What are the advantages of using microorganisms to make food for human consumption?

Answer 22

The fungus that makes single-cell protein (SCP / Quorn) can be grown on waste materials from industry. E.g. Molasses a by-product of sugar processing.
Many can be grown rapidly, easily and cheaply as have simple growth requirements.
Can be cultured anywhere if you have the right equipment – so could be used to tackle malnutrition in developing countries where growing crops & rearing livestock is difficult.

Question 23

What are the disadvantages of using microorganisms to make food for human consumption?

Answer 23

Food can be easily contaminated as they are grown under conditions favourable to all microorganisms – unwanted bacteria could be dangerous to humans or spoil food.
Some people do not like the idea of eating food grown on waste products.
SCP does not have same texture as meat.

Question 24

How are growing conditions inside fermenters manipulated in order to maximise yield?

Answer 24

pH & temperature are monitored & kept at optimum level, to allow enzymes to work efficiently, so rate of reaction is kept high.
Access to nutrients - paddles constantly circulate fresh medium around the vessel.
Volume of oxygen - to maintain aerobic conditions for respiration.
Vessel kept sterile to prevent contamination & competition for resources.

Question 25

Describe a batch fermenter.

Answer 25

Microorganisms are grown in individual batches.
When one culture ends it is removed.
Fermentation tank emptied.

Question 26

Describe a continuous fermenter.

Answer 26

Microorganisms are continually grown in a fermentation vessel without stopping.
Nutrients are put in and waste products taken out at a constant rate.

Question 27

What is a closed culture?

Answer 27

In a closed culture the conditions are set at the start and there is no exchange with the external environment.
(Similar to batch culture but sometimes substances are added to keep population growing until the nutrients are used up e.g. oxygen)

Question 28

Explain the standard growth curve in a closed culture.

Answer 28

Lag phase - Bacteria adjusting to new conditions, takes a while for enzyme production.
Log phase - Number of bacteria increase rapidly.
Stationary Phase - Rate of growth is equal to rate of death.
Decline Phase - death rate is greater than “birth rate”.

Question 29

What is a primary metabolite & what culture technique would be used to produce it?

Answer 29

Substances produced during active cell growth e.g. Amino acids, Fermentation end products, many types of enzyme. Continuous culture.

Question 30

What is a secondary metabolite & what culture technique would be used to produce it?

Answer 30

Secondary metabolites accumulate during periods of nutrient limitation and waste build-up (stress). Produced after the main growth phase. Not essential for normal growth. Batch culture.

Question 31

Why would a continuous culture be unsuitable to produce penicillin?

Answer 31

It is a secondary metabolite that is produced at the end of the main growth phase (during the stationary phase).
Penicillin is produced (at maximum) when kept short of nutrients. Glucose sometimes added as a respiratory substrate to keep the culture alive.
Continuous culture is maintained in the growth phase and so penicillin would not be produced.

Question 32

What are the problems of unwanted microorganisms growing in cultures?

Answer 32

Compete with the culture microorganisms.
Reduce the yield of useful products.
Cause spoilage of the product.
Produce toxic chemicals.
Destroy the culture microorganism or its products.

Question 33

What are the 3 stages of growing microorganisms on agar?

Answer 33

Sterilisation - using an autoclave (high temp & pressure).
Inoculation - introducing the microorganisms to sterile medium.
Incubation - plates left in suitable warm environment.

Question 34

Why are the neck of culture bottles flamed?

Answer 34

It causes air to expand and pushes bacteria away so they are less likely to settle into the tube.
Also kills bacteria at the neck of the tube.

Question 35

Why is the lid held over the agar plate during inoculation?

Answer 35

It avoids infection with bacteria in the air.

Question 36

What other aseptic techniques are used other than flaming the neck of the bottle and holding the lid over the agar plate?

Answer 36

Work near a Bunsen flame, as hot air rises, bacteria in the air will be drawn away from the culture.
Sterilise the instrument used to transfer cultures both before and after inoculation.
Sterilise all equipment before and after each use.

Question 37

State the meaning of the term an ‘immobilised enzyme’ and describe how immobilisation can be achieved.

Answer 37

‘Immobilised enzyme’ means attached to an insoluble material.
Enzymes can be immobilised by:
Encapsulation - trapped in alginate beads.
Adsorption – stuck onto porous clay beads (ionic bonding).
Gel entrapment – trapped in silica gel.
Cross-linked to cellulose fibres (covalent bonding).

Question 38

What are the advantages of using immobilised enzymes?

Answer 38

Immobilised enzymes are reusable.
Product is enzyme free (no money or time wasted separating them).
Immobilised enzymes are more stable than free enzymes
(they are more tolerant to pH and temperature changes).

Question 39

What are the disadvantages of using immobilised enzymes?

Answer 39

Extra equipment is needed - can be expensive.
Immobilised enzymes are more expensive to buy than free enzymes - so not economical in small-scale production.
Immobilisation techniques can reduce enzyme activity as they are not free to mix with substrate.

Question 40

Give two examples of immobilisation in biotechnology.

Answer 40

Immobilised glucose isomerase converts glucose to fructose which is used to sweeten drinks.
Lactase converts lactose to sweeter sugars glucose & galactose. Used to make lactose free milk & condensed milk.