5dCloning

Micropropagation (also called tissue culture) is a way of making very large numbers of identical plants from one parent plant. Small pieces of tissue called explants are grown in vitro — that is, in glassware in the laboratory rather than in soil.

Why it works

Plants contain meristem tissue (at shoot tips and root tips) whose cells are still able to divide and to differentiate into any plant cell type. A tiny piece of this tissue can therefore regenerate a whole plant.

The stages — learn these in order

1. Select a parent plant with the desirable characteristics you want to copy.
2. Cut small explants from the plant, usually from the fast-growing shoot tip (meristem).
3. Sterilise the explants, e.g. by dipping in dilute bleach or alcohol, to kill bacteria and fungi that would otherwise compete with the tissue and destroy it.
4. Transfer each explant to a sterile nutrient agar (growth) medium. The medium contains glucose (for respiration, because the explant cannot yet photosynthesise enough), amino acids, minerals and plant hormones (auxins and cytokinins) to stimulate cell division, shoot growth and root growth.
5. Keep the containers in aseptic conditions in a warm, controlled environment with suitable light.
6. The cells divide by mitosis to form a shapeless mass of cells called a callus, which then differentiates into small plantlets with shoots and roots.
7. Transplant the plantlets into compost/greenhouse conditions and grow them on. All of them are clones — genetically identical to the parent plant and to each other.

Key vocabulary you must actually write

• explant (not "bit of plant")
• in vitro
• sterile / aseptic
• nutrient medium containing hormones
• mitosis
• clones / genetically identical

Common mistake

Writing that the cells divide by meiosis. Meiosis halves the chromosome number and produces genetic variation — the exact opposite of what cloning needs. Cloning of any kind is always mitosis.

Examiner tip. Two marks are routinely thrown away here. (1) Examiners repeatedly report candidates saying cell division is meiosis — it is mitosis, because the plants must be genetically identical. (2) If the command word is describe, give the sequence of steps; if it is explain, you must give reasons (e.g. why the explant is sterilised, why hormones are added). Reports say 'describe' and 'explain' are the most commonly confused command words.

Growers use micropropagation when they have one plant with desirable characteristics — high yield, disease resistance, attractive flowers, a particular fruit flavour — and want commercial quantities of plants that are exactly the same.

Why not just use seeds?

Seeds are produced by sexual reproduction, which involves meiosis and fertilisation, so the offspring show variation. Many would not have the desirable characteristic. Micropropagation uses mitosis only, so every plant is a clone of the parent and every plant carries the desirable characteristic.

Advantages

Disadvantages

Common mistake

Saying micropropagated plants "cannot evolve" or "will die out". Keep it precise: the population has no genetic variation, so there is no resistant variant to survive a new disease — the whole crop is vulnerable.

Examiner tip. Examiner reports warn about vague answers that lack depth and key terms. Do not write 'the plants are all the same' — write 'the plants are genetically identical clones, so all have the desirable characteristic'. And when asked to evaluate or discuss the use of micropropagation, you must give both advantages and disadvantages and then reach a judgement; simply listing advantages answers a different command word.

Dolly the sheep (1996) was the first mammal cloned from a mature (adult) body cell. The technique is nuclear transfer.

The stages — learn the sequence

1. A mature cell (an udder cell) is taken from sheep A — the sheep to be cloned. This cell contains a diploid nucleus, i.e. the full set of chromosomes.
2. An egg cell is taken from sheep B. Its nucleus is removed — the egg cell is now enucleated. (An egg nucleus is haploid, which is why it must go.)
3. The diploid nucleus from the udder cell is inserted into the enucleated egg cell, usually by fusing the two cells together with a small electric shock. The shock also stimulates the new cell to start dividing.
4. The cell divides by mitosis to form a ball of cells — an embryo.
5. The embryo is implanted into the uterus of a surrogate mother (sheep C), where it develops to term.
6. The lamb born — Dolly — is a clone of sheep A, because all her nuclear DNA came from sheep A's udder cell. She resembles neither sheep B nor the surrogate mother.

Which sheep contributes what?

Words that must appear in your answer

diploid nucleus · mature/adult body cell · enucleated egg cell · electric shock / fusion · mitosis · embryo · surrogate mother · clone / genetically identical

Common mistake

Writing just "the nucleus" instead of "the diploid nucleus", and writing that the embryo forms by meiosis. Both are on the examiners' list of errors and both lose marks. Meiosis would halve the chromosome number and create variation — impossible in cloning.

Examiner tip. Examiners specifically report: 'Common errors included not referring to a diploid nucleus and stating that the cell division is meiosis.' Put the word diploid in front of nucleus every single time, say the egg cell is enucleated (nucleus removed), and say the embryo develops by mitosis. Also state that the clone is genetically identical to the body cell donor, not to the surrogate mother.

A transgenic animal is one that has had a gene from another species inserted into its DNA. If that gene is a human gene, the animal can make a human protein — and cloning lets you copy that valuable animal many times over.

Why bother?

Some human proteins are needed as medicines: antitrypsin (for a lung condition), blood clotting factors (for haemophilia), insulin, antibodies. Extracting them from human donors is slow, expensive and risky. A transgenic animal can produce them in large amounts, usually in its milk, from which the protein is easily collected and purified.

The process

1. The human gene for the required protein is cut out of human DNA using a restriction enzyme.
2. The gene is inserted into the nucleus of an animal cell (e.g. a sheep body cell), often next to a section of DNA that switches the gene on only in the mammary glands, so the protein appears in the milk.
3. This transgenic cell is now used exactly as in the Dolly method: its diploid nucleus is transferred into an enucleated egg cell, given an electric shock, divides by mitosis into an embryo, and the embryo is implanted into a surrogate mother.
4. The offspring is transgenic and a clone. It produces the human protein in its milk.
5. Nuclei from this animal's cells are used to produce many identical clones, so a whole flock produces the protein.
6. The milk is collected and the human protein is separated and purified from it.

Why cloning is needed as well as genetic modification

Issues to mention if asked to evaluate

• For: large, reliable supply of a life-saving medicine; cheaper than existing methods; protein is genuinely human so less likely to cause an immune reaction.
• Against: animal welfare concerns; cloned animals may be unhealthy or have a shortened life; success rate is low, so many embryos are lost; ethical objections to modifying animals.

Examiner tip. Keep 'transgenic' and 'clone' as two separate ideas and say both: the gene gives the human protein, the cloning gives many genetically identical animals that all make it. Examiners note that answers lose marks for lacking depth and key terms — name the human gene, the restriction enzyme, the diploid nucleus, the enucleated egg cell, the surrogate mother and the milk. And check the command word: describe the stages means the sequence; explain why cloning is used means give reasons, not the method again.