Unless we are dealing with a sudden mutation which occurs naturally and which can be vegetatively propagated then we will need to employ specialised selection techniques to develop our new variety. The hand pollination technique can be employed to cross-pollinate flowers of particular varieties to produce hybrid types. The flowers are covered at all times to avoid accidental pollination which would make our results unpredictable. The seeds are then grown and allowed to freely cross-pollinate. The next generation can then be selected and only the best plants grown on. It is sometimes necessary to self-pollinate the plant over several generations to ensure that the phenotype we are trying to stabilise becomes homozygous. This does lead to problems if we are dealing with an out-breeder type, since it will lead to inbreeding depression. To overcome this problem we would have to produce a number of pure lines and as they become inbred they can be crossed to restore their vigour by heterosis.

Clearly, there are many variations of this basic scheme and one particular variant gives rise to the commercially sold F₁ HYBRID seeds so common in garden centres.

The breeder maintains pure bred lines which are suffering from inbreeding depression and when these varieties are crossed they give rise to vigorous hybrids which are heterozygous for the particular phenotype required. When gardeners collect seed from these plants and grow them they will find that the plants which develop are poor specimens lacking the required phenotype, so they must return to the garden centre to by more seed next year if they want good looking plants.

A more complex crossing known as F₂ hybrids are also commercially available and are produced as follows:

When breeding programmes are used for evaluating plant crops it is often necessary to trial the varieties under a range of growth conditions. The plants will be evaluated for a range of agronomic features, eg germination rate, growth rate, flowering time, yield, degree of uniformity etc , and will be tested under a range of conditions of soil type, light level, temperature etc. The plants are grown in fields under a random block design so that the new variety is not subject to undue environmental influence relative to the varieties it is being compared with.

There are a number of problems to be overcome with any selective plant breeding program. Inbreeders may show cleistogamy, in which pollination and fertilisation occur before the flower has opened (eg lettuce). In the tomato the stamens form a covering over the stigma in the opened flower and this tends to restrict the entry of foreign pollen. These can be overcome by hand pollination at the bud stage. The biggest problem in outbreeders is caused by the presence of self-incompatibility systems in a number of commercially important crop plants. Self-incompatibility is the failure of pollen to either germinate when it reaches the stigma, or failure of the pollen tube to grow fast enough to allow fertilisation to be accomplished. The pollen is haploid and is growing through normal diploid maternal tissue. There is an interaction between the maternal and gametic tissue although no-one understands quite what it is. Generally, the pollen cannot function unless the stigma has a different phenotype to the pollen's. We use the symbol S (for sterility) with a number suffix to represent the self-incompatibility alleles. There are two major systems.

Gametophytic Self-incompatibility: There is no dominance between the S alleles thus if we have three alleles (the simplest arrangement) then we could have the following parent types:

S1/S2, S1/S3, S2/S3, note that S1/S1 or any similar combination is never possible with this type of self-incompatibility.

In the following diagram, the stamens of the parent plant have the genotype S1/S2 which gives pollen of types S1 and S2. These are transported to the stigma of a flower with the genotype S1/S3. Since the S1 type pollen is made incompatible due to the S1 allele in the stigma tissue it fails to germinate properly and cannot fertilise any of the ovules in the ovary. The S2 pollen has no such problems. Note that the phenotype of the pollen grain is the same as the single allele which it carries.

Sporophytic: Here the pollen producing tissue determines the phenotype of the pollen. If the plant producing the pollen has the alleles S1/S2 then S1 is dominant over S2 and the pollen will be of type S1 even though it ends up carrying the S2 allele. Remember in this type of self-incompatibility the pollen's phenotype is not always the same as its genotype. The alleles form a dominance series S1 > S2 > S3 >S4 etc.

Thus using the example above we get:

Since neither pollen type germinates we get no offspring from this cross! It is often possible to get around the problem of self-incompatibility by bud-pollination. The immature flower bud is split open and the pollen is transferred from the anthers onto the stigma. The self-incompatibility system does not appear to work in the early stages of flower development. When the self-incompatibility system fails to work under normal conditions the progeny produced are referred to as sibs and they are generally weak and poor yielding. Hybrid seed producers therefore try to build in the strongest possible self-incompatibility system to the parent lines to reduce the possibility of sib formation to a level of 5% or under which the seed users demand. The breeders check the degree of self-incompatibility by bud-pollination of immature flowers and then examination of the stigmas by fluorescent microscopy to establish the proportion of self-pollination that has occurred. Note that fruit trees produced asexually will have the same incompatibility alleles and an orchard needs two cross-compatible varieties.

Polymorphy: This is the existence of morphologically different types of an organism within the same population which are maintained in existence by a genetic system. It is also known as heteromorphy.

A system of incompatibility which includes heteromorphy is shown by the primrose, which has two different flower structures existing in each population.

There is a pair of self-incompatibility alleles, with S dominant over little s. The positions of the stigma and anthers are controlled by the following allele combinations:

The three genes A, G and S are so closely linked they are inherited as a unit with rarely any crossing over between the loci. This is an example of a supergene.

These flowers are pollinated by bees which have long tongue-like mouth parts to reach the nectar at the base of the petals. As they enter the pin eyed flowers the bees head will brush against the stigma and the tongue area will be covered by pollen. As this bee enters the thrum eyed flower the tongue will brush against the stigma while the head will be brushed by the stamens and covered in pollen. Thus the pollen is carried on different parts of the bee's head and the stigmas are designed only to touch those parts of the bee carrying pollen from the other type of flower. If any pollen is accidentally transferred to a flower of the same type, then the incompatibility allele prevents its germination.

Induced mutations: The plant breeder depends upon genetic variability to provide the new combinations which he can select for and develop into new types. The rate of natural mutations is relatively low (for each locus about 1 in 100,000 gametes). By using mutagens the rate of point mutation can be increased many times and the following have been used on crop plant; X-rays, alpha particles, neutrons, gamma-rays, ultra-violet light, and mustard gas. These may cause point mutations, or chromosomal changes such as deletions, inversions and translocations. These mutations must be produced in the gametic tissues to be passed to the next generation. The mutagen treated plants must be grown and crossed with non-treated plants and any dominant mutations will be detected in the F1 generation while recessives can be detected in the F2 generation. Several new varieties of barley, rice, mustard, bean and peanuts have been produced by induced mutation techniques. The technique has also been used to induce self-fertility in the sweet cherry by altering its self-incompatibility mechanism.

Anther culture: The anthers contain a great deal of haploid tissue and these can be induced to grow to form haploid plant material. The plants developed have only one set of chromosomes and therefore there is no masking by dominance. Once a suitable haploid line has been produced the chromosome number can be doubled using colchicine to yield fertile homozygous diploids. This technique is especially useful in the mutagenic studies mentioned above. It is also a fast route to homozygosity cutting out a great deal of time in the normal repeated cycles of inbreeding in the usual plant breeding program. The excised anthers can be cultured on solid or liquid media, with embryogenesis occurring within the anther; or pollen is removed and cultured in a liquid medium.

Haploid plants have been produced of over 121 species or hybrids from 20 families.

The stages in the culture technique are

1) Surface sterilisation of the flowers by immersion in hypochlorite solution.

2) Dissection of the anthers from flowers.

3) Culture of anthers on solid or liquid medium.

4) subculture of microspore callus tissue to induce embryos.

5) induction of roots and shoots by altering the levels of plant hormones present.

6) Determination of the chromosome number in plantlets using squash preparation in Feulgen stain technique.

Protoplast culture is used in many bioengineering schemes because the absence of the cell wall gives easier access to an manipulation of the genetic material of the cell. One important use of this technique is the fusion of plant cells to form hybrids. If the two cells fusing are of the same type then we have a homokaryocyte, whereas if the cells are different then we have a heterokaryocyte. If the nuclei can then be induced to fuse together a true hybrid protoplast is formed.

The technique is particularly useful where the plants to be crossed are sexually or physically incompatible.

Plasmid introduction into protoplast: The gene to be introduced is combined with the DNA of a vector and this is introduced into the protoplast. The plasmid can reproduce itself in the protoplast and in this way resistance genes have been introduced into plants from types which could never have transmitted the gene in the normal way. Human genes have been incorporated into plant cells in the hope of their being expressed as a usable product, eg insulin into potato. The procedure for protoplast culture is:

1) Surface sterilisation

2) Controlled plasmolysis in a solution of mannitol or sorbitol (13% w/v) to cause the protoplast to shrink away from the cell wall.

3) Enzyme treatment to degrade the cell wall using pectinase and cellulase, followed by agitation to release the protoplast.

4) Purification of isolated protoplasts by removal of cellular debris and enzymes.

5) Protoplast culture in a stationary liquid or on the surface of semi-solid agar. The protoplasts regenerate their cell wall and form a callus and are then sub-cultured and subjected to plant hormones to encourage differentiation of stems and roots.

6) If fusion of protoplasts is the aim, then the protoplasts are subjected to fusigenic agents after stage 4. These agents include high levels of calcium ions, polyethylene glycol, high pH or high temperature (but not too high!). The recognition of heteroplasts and their selection requires complex cytological techniques which leave the heteroplasts alive so that they remain useful.