Cloning in plants and animals

Clones without a laboratory

A clone is a group of genetically identical organisms, or a genetically identical cell produced from another cell. Cloning does not need a laboratory. As the cell-cycle lesson explained, mitosis produces daughter cells genetically identical to the parent, so any organism that reproduces by mitosis alone produces clones of itself. Bacteria do it by binary fission, and a great many plants do it routinely.

Cloning in plants is called vegetative propagation: a new plant grows from a vegetative part of the parent, a stem, a root or a leaf, rather than from a seed. Seeds come from fertilisation and carry two parents' alleles shuffled by meiosis, so a seedling is not a clone of anything. A plant grown from a runner is.

All of these depend on the same property. Plant tissues retain populations of unspecialised cells in their meristems throughout life, and a mature plant cell can often be induced to divide and differentiate again, a degree of potency an adult animal cell does not have, as the stem cells lesson explains.

The advantages and disadvantages are those of asexual reproduction generally, stated here for the plant, not for a grower. In favour: no pollinator and no second plant are needed, so a single arrival can colonise a site. The offspring inherit a genotype that has already proved itself where the parent is growing. Each new plant is supplied by the parent while it establishes, so it grows away faster than a seedling can.

Against: the offspring carry no new combinations of alleles. A population of them meets a new disease or a changed climate with no variation for selection to work on. They are crowded around the parent and compete with it for light, water and minerals. And they are not dispersed, which seeds are.

Clone

A group of genetically identical organisms, or a cell genetically identical to the cell it was produced from.

Vegetative propagation

Asexual reproduction in plants, in which a new plant develops from a stem, root or leaf of the parent and is genetically identical to it.

Meristem

A region of a plant in which the cells remain unspecialised and continue to divide by mitosis.

Cuttings and micropropagation

Growers have used vegetative propagation for centuries. A cutting is a piece of stem cut from a parent plant just below a node. The lower leaves are removed and the cut end is dipped in a rooting powder containing auxin. The cutting is pushed into moist compost and kept humid, so that water loss does not exceed what it can take up before roots form. Cells near the cut divide, roots develop, and the result is a new plant genetically identical to the parent. The method is cheap and requires no equipment, but the scale is limited: one parent plant yields a few dozen cuttings a season at most.

Micropropagation, also called tissue culture, applies the same principle in a laboratory at a much larger scale.

Describe the drawing

Six numbered steps running down the page and joined by arrows. One: cut an explant, a few millimetres of shoot tip or node, from the chosen parent plant. Two: sterilise its surface, because any surviving microbe would outgrow the tissue. Three: grow a callus, an undifferentiated lump of dividing cells, on agar. Four: transfer to media whose ratio of auxin to cytokinin makes the callus grow shoots and then roots. Five: divide the material and repeat, so one callus becomes hundreds of plantlets. Six: acclimatise the plantlets from agar to compost. A note beneath says every division was mitosis, so every plantlet is genetically identical to the parent.

The sequence runs like this. A small piece of tissue, a few millimetres of shoot tip or a node, is cut from the chosen parent plant; this piece is the explant. Its surface is sterilised, usually in dilute bleach or ethanol, because any bacterium or fungal spore left on it will outgrow the plant tissue on the medium provided. The explant is then placed on sterile nutrient agar containing sugar, mineral ions, vitamins and plant hormones, in a sealed vessel, and its cells divide into an undifferentiated lump called a callus.

The auxin-to-cytokinin ratio in the medium influences whether the callus forms roots or shoots. A medium with a high ratio of cytokinin to auxin induces shoot formation; transferring those shoots to a medium with a higher proportion of auxin induces root formation. These are the same growth regulators examined under plant responses. The callus can also be divided repeatedly before this stage, so one explant becomes many calluses and one parent plant becomes thousands of plantlets.

Finally the plantlets are moved out of their sterile vessels into compost and acclimatised, gradually, under high humidity that is reduced over days or weeks. Plants grown on agar have thin cuticles and stomata that do not close properly, so a plantlet moved straight to a greenhouse bench wilts and dies.

Explant

The small piece of tissue cut from a parent plant to start a tissue culture, usually a shoot tip or a node.

Callus

The mass of undifferentiated dividing cells produced from an explant on a nutrient medium, from which whole plantlets are regenerated.

Micropropagation

The production of very large numbers of genetically identical plants from a small piece of tissue, grown on sterile nutrient medium containing plant hormones.

The case for and against cloning plants

Micropropagation is expensive, so its use has to be justified commercially. Four arguments are usually given. It produces very large numbers from one parent, quickly, and at any time of year, whatever the flowering season. It produces plants that are uniform, which matters when a supermarket wants a crop that matures together and packs to a standard size. It can be started from meristem tissue and so clear a valuable variety of a virus it carries. And it works for plants that are difficult or slow to grow from seed, including many orchids, and for rare or endangered species and for genetically modified lines whose seed would not breed true.

Describe the drawing

Plant number against time through one year, drawn on a logarithmic vertical axis so that eight decades fit on one picture. The cuttings trace climbs by a fixed thirty plants over one season from a single parent and then stops, which bends on this axis because thirty added is a smaller multiple each time. The micropropagation trace is a staircase of equal steps, one explant divided four ways every four weeks, and equal steps in log space make it a straight line: thirteen subcultures in a year is four to the thirteenth, or sixty-seven million plants. Cuttings lead for the first two subcultures and are overtaken at twelve weeks, which is marked. The difference is not that one method is better but that one compounds and the other does not, so micropropagation earns its cost only when thousands are wanted. The factor and the interval are typical values rather than measurements, and a real culture meets contamination and bench space long before the arithmetic runs out, while the uniformity that suits a supermarket is what leaves a field with no variation in its resistance.

The arguments against are as follows. The equipment, the sterile conditions and the skilled labour make it far more costly per plant than seed or cuttings. A contamination found late in the process costs the whole batch. Not every species will form a callus and regenerate from one, so the method is not universal.

And the uniformity that is an advantage in a supermarket is a hazard in a field. A crop of genetically identical plants has no variation in its resistance. A pest or a pathogen that can infect one plant can infect every plant, and a whole planting can be lost to a single new strain. The same point has a longer-term version: a breeding programme needs variation to select from, and a population of clones offers none.

When evaluating plant cloning, discuss the technique's scale, cost, disease status, genetic uniformity and effect on variation. Ethical and welfare issues belong to questions about animal cloning unless the question explicitly asks for a comparison.

Cloning animals: twinning, splitting and nuclear transfer

None of the plant methods applies to animals. An adult animal cell has a much narrower potency than a mature plant cell, so an animal cannot be grown from a cutting. Natural cloning in mammals occurs only very early in development, before the cells of an embryo have committed to particular fates.

Splitting at that stage produces identical twins. An early embryo splits in two, each half develops into a whole individual, and the two are genetically identical to each other because both came from one zygote by mitosis. They are not identical to either parent: the zygote they came from was produced by fertilisation, so its combination of alleles is new.

Describe the drawing

Two cards side by side. On the left, embryo splitting: a four-cell embryo is divided into two smaller embryos, and the text records that the twins produced are genetically identical to each other and to neither parent. On the right, somatic cell nuclear transfer: the nucleus of an adult body cell is moved into an egg cell whose own nucleus has been removed, and the text records that the clone's nuclear DNA comes from the adult donor while its mitochondrial DNA comes from the egg donor. Beneath both cards a note explains that implanting the embryo makes the cloning reproductive, while harvesting stem cells from it, without implantation, makes it therapeutic.

Artificial embryo splitting is that natural process carried out deliberately. An egg is fertilised, in the animal or in the laboratory, and the embryo is grown to a few cells and then divided, each part being implanted into a surrogate mother. It is used in cattle, where a mating between two valuable animals can be turned into several calves instead of one. Its limitation is the one above: the parents' qualities are known, but the calves' are not, because their genotype is new.

Somatic cell nuclear transfer answers that limitation, because it copies an animal whose phenotype is already known. A somatic cell is any body cell that is not a gamete. The steps are these. An egg cell is taken from a donor female and enucleated, its own nucleus removed with a fine pipette, leaving the cytoplasm intact. A somatic cell is taken from the animal to be cloned, often a skin or mammary gland cell, and is held in a resting state.

The somatic cell, or its nucleus, is placed beside the enucleated egg and the two are fused with a pulse of electricity. The pulse also stimulates the cell to begin dividing. Factors in the egg cytoplasm then reprogramme the transferred nucleus, restoring the pattern of gene expression of an embryo. That is possible because differentiation changes gene expression rather than gene content. The embryo is cultured to the blastocyst stage, after which the two purposes diverge.

If the blastocyst is implanted into a surrogate mother and allowed to develop into a whole animal, that is reproductive cloning. The first mammal cloned this way from an adult cell was a sheep, born in 1996, and the numbers are part of the topic: 277 reconstructed eggs gave 29 early embryos, a single pregnancy among 13 surrogate ewes and one surviving lamb. Success rates in the decades since have improved but remain low. Pregnancies that fail late, and offspring born oversized or unhealthy, are the basis of the welfare argument against the technique.

Longevity is the other question asked of cloned animals. Dolly, as that sheep was named, was put down at six and a half, about half the usual lifespan of her breed. She had arthritis and a lung tumour caused by a virus common in sheep kept indoors. Her telomeres were shorter than those of other sheep her age, as might be expected of a nucleus taken from a six-year-old ewe. A later study followed thirteen cloned sheep, four of them from Dolly's own cell line, and found them ageing normally at seven to nine years old. So clones that survive birth may have a normal lifespan, though the numbers studied are small.

A clone is genetically identical at the nuclear-DNA level but is not a duplicate of the donor. Its nuclear DNA is the donor's, but its mitochondrial DNA came with the egg cytoplasm and so belongs to the egg donor. It develops in a different uterus, is born at a different time, and grows up in different surroundings. And the pattern of gene expression a cell carries is not reset perfectly by the egg. A cloned cat with a coat pattern unlike its donor's is the standard illustration. The pattern depends on which X chromosome is inactivated in which patch of skin, and that is settled at random in each embryo.

Somatic cell

Any body cell of a multicellular organism other than a gamete or the cells that produce gametes.

Enucleation

The removal of the nucleus from a cell, usually an egg cell, leaving its cytoplasm intact.

Somatic cell nuclear transfer

The transfer of a nucleus from a body cell into an enucleated egg cell, which is then stimulated to divide as an embryo genetically identical to the nucleus donor.

Reproductive cloning

Cloning in which the embryo produced is implanted into a surrogate and develops into a whole new individual.

Therapeutic cloning

Cloning in which the embryo produced is used as a source of stem cells and is never implanted.

Reproductive against therapeutic cloning

The two purposes share every step up to the blastocyst and diverge there, so evaluate them separately.

Reproductive cloning is defended on the ground that it copies an animal of proven value. That might be a bull whose daughters yield well, a genetically modified sheep whose milk carries a human protein, or a working animal of exceptional ability. It has also been proposed for conservation, to produce individuals of a species too rare to breed in the ordinary way.

Against it stand the success rates and what they cost in animal welfare, the health of the offspring that are born, and the loss of genetic variation in a herd bred from few genotypes. When the subject is raised, there is also the argument that no case has been made for cloning a human being at all. That is why human reproductive cloning is prohibited by law in the United Kingdom and in many other countries.

Therapeutic cloning is a different proposition, because no animal is born. The embryo is a source of stem cells, and the reason for making it by nuclear transfer is that the cells carry the patient's own nuclear DNA and so the patient's own antigens, which removes the rejection problem that limits every transplant. The cells obtained are pluripotent, so the range of tissues they could be directed into is much wider than that available from an adult source, which is the difference between the levels of potency.

There are two objections, of different kinds. The first is the ethical objection set out under stem cells. A human embryo is created and then destroyed, and the positions people hold on that do not change because the embryo was made by nuclear transfer, not by fertilisation. The second is practical, and it applies to the eggs. Human egg cells are needed in quantity, and collecting them requires hormone treatment and a procedure that carries risk for the women who donate.

A further argument is that the technique has been superseded. Induced pluripotent cells give a patient-matched pluripotent cell from a skin sample, with no egg and no embryo, and most laboratories that previously pursued therapeutic cloning now use them. State that development in an evaluation, since it changes the conclusion available.