Cloning and Biotechnology.


Natural and artificial cloning.

Cloning - production of genetically identical organisms.

Asexual reproduction - production of clones via mitosis.

Types of cloning:

  • Reproductive - produces a whole organism
  • Non-reproductive - produces cells such as embryonic stem cells
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Natural cloning in plants.

Occurs in flowering plants from structure called a perennating organ, which stores enough nutrients to sustain plant during non-growing season and develop into 1 or more new plants following year.

Produce clones by vegetative propagation - production of structures in a plant from non-productive tissues which are able to grow into genetically identical offspring.

Vegetative propagation in holticulture and agriculture:
-manipulate plant growth to induce vegetative progagation e.g. taking cuttings, splitting off bublets runners and rhizomes, grafting- joining shoot of one plant to growing stem and root of another plant, layering - bending stem of growing plant downwards so enters soil and grows into new plant
-shorter time to flower/crop than seeds
-guaranteed phenotype but lack of genetic diversity

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Vegetative propagation.

Examples of vegetative propagation:

  • bulbs - underground food store used by some plants e.g. onions where leaf base swells with stored food and buds form internally and develop into new shoot
    rhizomes - specialised stem strucures that grown horizontally underground away from parent plant e.g. bamboo where nodes from which new shoots and roots can develop
  • runners/stolons - specialised lateral stem structures that grow above ground on soil surface away from parent plant e.g. strawberry where new roots and shoots develop from nodes formed at end of stolen and runner eventually withers away leaving independent individual
  • stem tubers- large underground plant structures that act as food stores e.g. potatoes which are covered in 'eyes' that sprout and form new plant when tip becomes swollen with stored food
  • suckers - shoots that grow away from sucker buds on shallow root of parent plant e.g. elm tree which spread roots from parent tree allowing new tree to grown
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Taking plant cuttings to produce natural clones.

1. Use scalpel to take cutting from end of stem about 5cm-10cm long.

2. Remove leaves from lower end of cutting, leaving just one on tip.

3. Dip lower end of cutting in a pot containing hormone rooting powder to induce root formation.

4. Plant cutting in pot with suitable growth medium, e.g. well drained compost.

5. Provide cutting with warm and moist environment by covering whole pot with a plastic bag or putting in a propagator.

6. Plant elsewhere when roots have formed.

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Artificial cloning in plants.

Tissue culture - separation and subsequent growth of meristematic cells from tissue in vitro using nutritent mediums (used to clone plants that don't readily produce, that are endangered or rare, and to grow whole plants from genetically engineered plants).

1. Small piece of tissue, containing totipotent cells, is taken from plant shoot or roots using aseptic techniques to avoid contamination (sample called the explant).
2. Explant is sterilised to kill microorganisms by immersing in fluid e.g. ethanol.
3. Explant is placed on sterile culture medium containing organic nutrients and plant hormones e.g. cytokines and auxins.
4. Cells in explant divide by mitosis to form mass of identical cells called callus.
5. Callus is divided up into individual cells or clumps and transferred to new medium containing shoot-stimulating hormones to stimulate growth of identical plantlets.
6. Plantlets are transferred to medium of root-stimulating hormone.
7. Growing plants potent into compost, the outside, to grow and produce a crop.

Microprogation - form of large scale tissue culture, where cells are taken from developing cloned plants or subcultures plants with a fresh culture medium (e.g. used to produce fields full of crops that are pest resistant.

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Advantages and disadvantages of micropropagation.

-rapid production - high yield in short space of time
-produce plants following genetic modification
-known pheno/genotype - used in selective breeding
-culturing meristematic tissue produces disease free plants
-alternate method to grow plants naturally difficult to propagate or are infertile
-plants can be reproduced at any time of year as controlled conditions maximise growth and prevent disease
-less space needed
-produce seedless varieties of plants that consumers prefer
-reliable fast method to increase number of endangered species
-high production costs - labour intensive, skilled workers, high energy
-microbial contamination leads to large numbers of plants lost
-explants and plantlets vulnerable to mould during production
-no genetic variability - single disease or changing growing conditions could fail whole batch
-undesirable characteristics always passed on

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Natural cloning in animals.

Only embryonic cells which are totipotent stem cells are naturally capable to generate a new individual.

-fragmentation - from fragments of original
-budding - small buds on side of body
-binary fission - splitting in two

-monozygotic twins - embryo splits at early stages after conception
-pathenogenesis in amphibians - no male present to fertilise eggs

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Artificial cloning in animals - artificial twinnin

Artificial twinning - young embryo, ball of totipotent cells, is split to create 'articulate identical twins', using the same principle of natural twinning.
Uses include rapidly increasing herd size of farmed animals with favourable characteristics.

1. Cow with desirable characteristics treated with hormones to cause superovualtion.
2. Ova is either fertilised naturally or artificially (artificial insemination and extraction or ova is extracted and fertilised in vitro).
3. Fertilised egg left to divide at least once to form an embryo in vitro.
4. Before day 6, the individual cells from embryo are separated and each put into a different Petri dish to allow each cell to divide and develop.
5. Embryos are implanted to surrogate cows, where they continue to develop and eventually genetically identical cows are born.

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Artificial cloning in animals - nuclear transfer.

Nuclear transfer - differentiated cells from an adult planted in an enucleated egg which is implanted in placenta of surrogate mother.
Somatic cell nuclear transfer (SCNT) was first successful in 1996 to produce Dolly the sheep. Modified transgenic animals can be used to grow organs, pharming for therapeutically useful molecules and reproductive cloning that can be used for embryo twinning.

1. Somatic cell taken from animal A and the nucleus is extracted.
2. Mature ovum or occyte (immature egg cell) is taken from animal B and the nucleus is ectracted to from an enucleated occyte.
3. Nucleus from animal A is inseted into enucleated occyte.
4. Nucleus and enucleated occyte are fused together by electrofusion which induces cell division.
5. Embryo is then implantedd into surrogate mother animal C, where it develops.
6. Baby animal genetically identical to animal A is born (contains some mitochondrial DNA from egg cell donor).

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Pros and cons of artificial cloning in animals.

-AT - produces many offspring from high yielding farm animals
-SCNT - enables GM embryos to be relicated and grown to give multiple embryos from single engineering procedures
-helped develop new treatments
-increase population of enedangered species
-clones animals anytime and anywhere
-clone infertile animals
-lower life expectancy
-very difficult, time consuming and expensive
-no gentic diversity so undesirable characteristics past on
-low success rate in increasing population of rare organism or allowing extinct animals back to life
-cloned embryos often miscarry or form malformed offspring
-inefficient - many eggs produce single offspring
-controversial using human embryos
-animal welfare issues

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Ethical concerns of gene manipulation.

GM soya bean - inserted gene to products Bt protein which is toxic to many pest insects and be resistant to weed colour:
-reduced chemical pesticide used and increases yield
-insects may become resistant to pesticides in crops
-encourages monoculture
-transferred genes spread to wild producing super weeds
-people in LEDCs prevented from using them by patents and issues of technology transfer
Pathogens for research - genetically modified to find treatments for disease:
-making untreatable diseases treatable reducing suffering
-protenital infection with life pathogen leading to outbreak of disease
-worry it gets into wrong hands and used by wrong hands to create agents for biowelfare
Pharming - genetically modified animals produce pharmaceuticals by creating animal models (additions or removal of genes so animals develop certain diesease and creating human proteins (intro of human gene coding for medically required protein):
-drugs made in larvae quantities
-harmful animal side effects and enforces idea that animals are just asssests

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Gene therapy.

Cures genetic diseases caused by inherited abnormal genes by altering genes inside cells by either supplementing faulty genes with a dominant allele or silencing a dominant allele by inserting DNA in the middle. New alleles inserted into cell by vectors.

Somatic therapy:
-replacing mutant allele with healthy allele in affected somatic cell
-viral vectors often used
-treating retinal diseases, immune diseases, leukaemias, myeloma and haemophilia
-temporary solution - somatic cells have limited life so are replaced with stem cells and faulty alleles still placed to children

Germ line therapy:
-inserting healthy allele into germ cell or into embryo immediately after fertilisation as part of IVF
-pass of healthy genes to offspring
-illegal for human embryo as violates right of unborn baby and runs risk of 'designer babies'

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Using enzymes in industry.

Advantages of using isolated enzymes instead of whole organisms:
-less wasteful - microorganisms use up substrate growing and reproducing, producing biomass rather than product
-more efficient - isolated enzymes work at higher concentrations than is possible when they are part of a whole microorganisms
-maximise efficiency - give isolated enzymes ideal conditions for maximum product formation which may differ from those of whole microorganism
-less downstream processing - pure product is produced by isolated enzymes whereas whole microorganisms give variety of products
Extra cellular enzymes are generally easier because:
-they are secreted so easier to isolate
-microorganisms produce relatively few extracellular enzymes so easier to identify
-tend to be more robust as conditions outside cell are less tightly controlled than conditions in cytoplasm
However, benefits of using very specific enzymes outweighs disadvantages of expensive extraction and isolation process, and need for more tightly controlled conditions.

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Using immobilised enzymes.

Free enzymes are often very wasteful and not cheap because at the end of the process they cannot usually be recovered and are lost.
Immobilised enzymes - enzymes attached to an inert and insoluble support system over which substrate passes and is converted to product (a case of technology mimicking nature - enzymes in cells usually bound to membranes to carry out repeated cycles of catalysis).
-reusable - cheaper
-easily separated from reactants and products of reaction so reduced downstream processing so is cheaper
-more stable - less likely to denature as greater temperature tolerance so bioreactor is cheaper to run
-more reliable - more control as support provides stable microenvironment
-ease of manipulation - catalytic properties can be altered to fit process more easily
-reduced efficiency - reduced rate of activity as not mixing directly with substrate
-higher initial material costs - more expensive than microbes and free enzymes
-high initial bioreactor costs - conditions different from traditional fermenter
-more technical issues - more complex reactors

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Methods of immobilising enzymes.

Surface immobilisation - adsorption to inorganic carriers e.g. silica and carbon nanotubes with partially permeable membrane for substrate.
+simple, cheap and can be used with many different processes
+enzymes very accessible to substrate so activity virtually unchanged
-enzymes easily lost from matrix
Surface immobilisation - covalent or ionic bonding to inorganic carries.
+variable cost and enzymes strongly bound so not lost
+enzymes accessible to substrate
+pH and [substrate] has little effect
-variable cost and active site of enzyme may be modified so less effective
Entrapment/inclusion - in matrix e.g. gelatin, polysaccharides.
+widely applicable to different processes and enzyme in natural state
-expensive and difficult, may effect enzyme activity depending on matrix
-diffusion of substrate to and product from active site slows rate
Membrane entrapment - in micro capsules or behind semi-permeable membrane.
+simple and widely applicable with small effect on enzyme activity
-expensive and diffusion to and from active site can slow rate

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Immobilised microorganisms.

Sometimes, whole microorganisms are immobilised:

  • many of same advantages as immobilised enzymes
  • avoid time consuming expensive processes of extracting pure enzymes
  • microbes need food, oxygen and carefully controlled conditions to work at optimum
  • microorganisms have different optimums to their enzymes
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Uses of immobilised enzymes.

Immobilised penicillin amylase - used to make semi-synthetic penicillins from naturally produced penicillins to treat bacteria treat those still vulnerable to semi-synthetic penicillin.
Immobilised glucose isomerase - used to producuce fructose from glucose as it is much sweeter and can be used in smaller quantities to produce the same level of sweetness (glucose produced from cheap, starch rich material).
Immobilised lactase - used to produce lactose free-milk for those who are intolerant to lactose as don't produce enough lactase, which hydrolyses lactose to glucose and galactose.
Immobilised glucoamylase - used to break down starch to glucose syrup, amylase enzymes break starch into small chain polymers called dextrin which break down to glucose by action of glucoamylase (used in food industry to sweeten and thicken).
Immobilised aminoacylase - used to produce pure sample of L-amino acids by separating L and D amino acid isomers that scientists have synthesised (used for food production, dietary supplements, pharmaceuticals, organic chemicals and cosmetics).
Immobilised nitrile hydratase - used to catalyse conversion of acrylonitrile to acrylamide in hydration reaction with moderate conditions so is cheaper, gives 99% yield and no unwanted products (used in production of many plastics).

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Thank you so much for this. You summarised everything perfectly. <3




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