Cloning

Definition. A clone is an organism (or cell, or DNA molecule) that is GENETICALLY IDENTICAL to another. Clones share 100% of nuclear DNA.

The common mechanism. All forms of cloning rely on MITOSIS — the cell division that produces genetically identical daughter cells. Since cloning involves no meiosis (no chromosome shuffling) and no fertilisation (no mixing of parental DNA), the offspring inherit identical DNA from the source.

Natural clones.

• Identical (monozygotic) twins — a fertilised egg divides by mitosis, and the early embryo splits into two. Both halves develop into separate individuals with identical nuclear DNA.
• Plant asexual reproduction — runners (strawberries), bulbs (onions), tubers (potatoes), rhizomes — all natural cloning.
• Bacterial binary fission — one cell divides into two identical daughter cells.

Artificial cloning techniques:

Why plant cloning is easier than animal cloning.

• Plant cells are TOTIPOTENT — most adult plant cells can dedifferentiate and develop into any plant tissue (or whole plant). So a leaf cell can be made to grow into a new plant.
• Animal cells lose totipotency early — adult animal cells are differentiated and committed. They cannot easily be reprogrammed (SCNT is the workaround — it uses the egg's cytoplasm to do the reprogramming).
• Plants reproduce asexually naturally; animals (especially mammals) reproduce sexually almost exclusively → no natural asexual machinery in animals.

Taking cuttings is the simplest and oldest form of artificial cloning — practised by gardeners for thousands of years.

Method:

1. Cut a stem from a parent plant — usually 15-30 cm long, with several leaves. Cut diagonally just below a leaf node (where there is more tissue available for rooting).
2. (Optional) dip the cut end in rooting hormone powder — contains AUXIN, which stimulates root formation. Improves success rate.
3. Plant in moist soil or water. Keep warm and humid (e.g. cover with plastic to retain moisture).
4. Cells at the cut end divide by MITOSIS — plant cells are totipotent, so they can dedifferentiate and grow new roots.
5. Once rooted, the cutting is a new plant — genetically identical to the parent → a CLONE.

Why this works. Plant cells retain TOTIPOTENCY — they can divide and form any tissue. Cells at the cut surface can become root-forming cells. Mitosis ensures all daughter cells have identical DNA to the parent.

Where cuttings are used:

• Garden plants (geraniums, fuchsias, ivy, roses).
• Many crop plants (e.g. sugar cane, cassava).
• Hedging plants (privet, box).

Advantages:

• Simple — anyone can do it.
• Cheap — no specialised equipment.
• Quick — a few weeks from cut to rooted plant.
• Preserves desirable traits exactly (no genetic shuffling).

Disadvantages:

• Low scale — only a few cuttings per parent.
• All clones share the same vulnerability to disease.
• Works for some plants but not others (e.g. orchids don't root well from cuttings — need micropropagation instead).

Micropropagation (also called plant tissue culture) is the modern, lab-based method of cloning plants on a massive scale.

The 6-step process:

1. Take a small EXPLANT. Cut a few mm of tissue from a parent plant with desirable characteristics. Common sources: shoot tip, leaf, stem segment, or root.
2. Surface-sterilise. Dip the explant briefly in dilute bleach or ethanol to kill surface microbes. All apparatus is autoclaved, and the work is done in a sterile laminar-flow cabinet. Sterility is essential — contaminating bacteria or fungi can outcompete the plant cells.
3. Place on nutrient agar with hormones. The agar contains sugar (energy source), mineral nutrients, vitamins, and PLANT HORMONES — initially a high ratio of AUXIN to CYTOKININ to stimulate cell division.
4. Cells divide → CALLUS forms. Cells in the explant dedifferentiate and divide by mitosis to form a CALLUS — a lumpy mass of undifferentiated cells. The callus can be divided into many small pieces.
5. Change hormone balance → plantlets. Move callus pieces to fresh agar with a different hormone balance:
   • High CYTOKININ → shoots form.
   • High AUXIN → roots form.
   • Each callus piece develops into a PLANTLET (tiny plant with shoot + roots).
6. Acclimatise + transplant. Gradually move plantlets from sterile, humid conditions to normal soil in a glasshouse. They grow into mature plants — all genetically IDENTICAL clones of the original parent.

Why micropropagation is used commercially.

• Mass production — one explant → thousands or millions of clones.
• Works for plants that don't root well from cuttings — orchids, banana, oil palm, ferns, many rare ornamentals.
• Disease-free — sterility means no virus or fungus transferred.
• Year-round — not dependent on growing season.
• Preservation of rare/endangered species — a tiny piece of tissue can be preserved and propagated.

Example: Cavendish banana. Almost ALL commercial bananas in supermarkets are Cavendish — propagated by tissue culture (the variety is sterile and cannot reproduce sexually). All Cavendish bananas worldwide are GENETICALLY IDENTICAL CLONES. This is why Panama disease (a fungal disease) is such a threat — one disease strain could affect every plant.

Disadvantages:

• Expensive — needs lab, sterile cabinet, hormones, agar.
• Slow per stage — months from explant to mature plant.
• No genetic variation → vulnerability to disease (as in Cavendish banana).
• Requires technical skill.

Embryo splitting (also called embryo twinning) is the simpler of the two animal cloning techniques.

Method:

1. Fertilise an egg in vitro (or take a naturally fertilised early embryo from a donor animal).
2. At the EARLY STAGE (e.g. 2-, 4- or 8-cell embryo), the cells are still TOTIPOTENT — each cell can develop into a whole organism.
3. Use a micromanipulator to SPLIT the embryo into individual cells (or small clumps).
4. Allow each separated cell to continue developing → each becomes a separate embryo.
5. Implant each embryo into a different surrogate mother → each gives birth to a clone.

Result: multiple offspring genetically identical to each other (and to the original fertilised egg).

Used for: mass-producing high-value cattle from a prize cow's single embryo. A single embryo can produce 4-8 identical calves.

Important — these are CLONES OF EACH OTHER, not of an adult animal. The DNA comes from the original sperm + egg fertilisation event. Embryo splitting cannot replicate a known adult animal — it just produces multiple identical siblings.

To clone a known adult animal, you need a different technique: somatic cell nuclear transfer (SCNT).

Somatic cell nuclear transfer (SCNT) — also called adult cell cloning — is the technique used to produce DOLLY THE SHEEP in 1996 (Roslin Institute, Edinburgh). It was a landmark discovery because it showed that an adult differentiated cell could be reprogrammed back to a totipotent state.

The 7-step process:

1. Take an adult body cell (somatic cell) from the animal to be cloned. For Dolly: a mammary gland cell from a Finn Dorset ewe. This cell contains the DIPLOID nucleus (full chromosome set) of the donor.

2. Take an unfertilised egg cell from a different sheep. For Dolly: a Scottish Blackface ewe. The egg has a HAPLOID nucleus.

3. Remove the egg's nucleus (ENUCLEATION). Using a fine micropipette, the haploid nucleus is sucked out → leaves an ENUCLEATED EGG (no nucleus, but cytoplasm + mitochondria intact).

4. Insert the adult diploid nucleus into the enucleated egg. The body-cell nucleus is transferred into the empty egg. A brief ELECTRIC SHOCK fuses the cell membranes and STIMULATES DIVISION (mimicking the trigger that fertilisation normally provides).

5. Egg divides → embryo. The reconstructed egg now contains a diploid nucleus in egg cytoplasm. It begins dividing by MITOSIS → becomes a BLASTOCYST embryo. The egg's cytoplasm reprograms the adult nucleus back to a totipotent state — the magic step.

6. Implant in surrogate mother. The early embryo is implanted into the uterus of another sheep (another Scottish Blackface, the surrogate). She carries the pregnancy normally.

7. Birth and identification. ~150 days later the surrogate gives birth to a lamb. Although the surrogate gave birth, the lamb is GENETICALLY IDENTICAL to the body-cell donor (Finn Dorset) — NOT to the surrogate or the egg donor. Dolly was a Finn Dorset, instantly identifiable by her white face (Scottish Blackface have black faces).

Three sheep involved in Dolly's cloning:

Important — mitochondrial DNA. SCNT clones get nuclear DNA from the body-cell donor, but MITOCHONDRIAL DNA from the egg-cell donor (mtDNA is in the egg cytoplasm and is NOT removed during enucleation). So Dolly was not 100% genetically identical to the body-cell donor — but ~99.9% (mtDNA is a tiny fraction of total DNA).

Outcome and significance.

• Dolly was the FIRST mammal cloned from an adult cell.
• Took 277 attempts to produce one successful clone.
• Dolly lived 6.5 years (typical sheep lifespan ~10-12); died of lung disease (probably unrelated to cloning, but raised questions).
• Showed that adult cells could be reprogrammed → opened the door to stem cell research, induced pluripotent stem cells (iPSCs), and potential therapeutic applications.
• Ethical debate around human cloning was triggered — reproductive human cloning was rapidly BANNED in essentially all countries.

Other cloned animals. Since Dolly: mice, cattle, pigs, cats, dogs, horses, monkeys (2018). All require SCNT or variants.

Uses of cloning:

1. Mass-producing high-yield livestock — clones of prize dairy cows, racehorses, working dogs. Limited commercial use due to cost + welfare concerns.

2. GM-animal pharmaceutical production. A GM animal producing a useful protein (e.g. AAT, anti-thrombin, human serum albumin) in its milk can be cloned to produce many identical individuals → uniform drug supply. Link to T5.3 (GM).

3. Preservation of endangered species.

   • The black-footed ferret was cloned in 2020 from cells frozen in 1988 — increasing genetic diversity of the surviving population.
   • The bucardo (Pyrenean ibex) was cloned in 2003 but the clone lived only 7 minutes — the first 'de-extinction' attempt.
   • Ongoing efforts to clone the woolly mammoth (using elephant surrogates) — controversial.

4. Research animals. Clones make ideal experimental subjects — any difference in outcome can be attributed to the treatment, not to genetic variation.

5. Therapeutic cloning. Creating cloned embryos to harvest stem cells — provides patient-matched cells for medical treatment. Distinct from reproductive cloning. Heavily regulated; allowed in some countries.

6. Pet cloning. Wealthy individuals can clone beloved pets (~$50 000-$100 000 per clone). Controversial — the clone has identical DNA but a different personality (environment + epigenetics shape behaviour).

Ethical concerns:

1. Animal welfare.

• Very LOW SUCCESS RATE — most cloned embryos die in utero or are born with abnormalities. Dolly took 277 attempts.
• Cloned animals often have health problems (large-offspring syndrome, organ defects).
• Surrogate mothers undergo invasive procedures.
• Critics argue this is unacceptable animal suffering for the limited benefits.

2. Premature ageing.

• Dolly died at 6.5 years (typical sheep ~10-12).
• Some studies suggest cloned animals have SHORTENED TELOMERES (the chromosome end-caps) inherited from an aged adult cell.
• Epigenetic problems from incomplete reprogramming may also occur.

3. Reduced genetic diversity.

• A herd of clones has very low diversity → vulnerability to one disease wiping out the lot.
• Same problem as in highly selectively-bred populations (link to T5.2).

4. Religious / philosophical objections.

• Some traditions view all life as sacred and oppose 'creating' life artificially.
• Particularly strong opposition to human cloning.

5. Human cloning.

• Reproductive human cloning is BANNED globally — concerns about safety (same as animals); psychological identity (a clone has the same DNA as someone alive or dead); consent (a clone cannot consent to being created); potential for 'designer babies' or eugenics.
• Therapeutic human cloning (cloning embryos for stem cells) is allowed in some countries under strict regulation — but creating cloned embryos and destroying them for cells raises its own ethical issues (the embryo question).

Balanced view. Cloning has real, legitimate uses — particularly in conservation and pharmaceutical production — but raises serious welfare and ethical concerns. Regulation should evaluate each application on its own merits rather than rejecting or accepting cloning uniformly. Reproductive human cloning is universally agreed to be off-limits with current science.