Methods

Primary explants In most herbaceous plants, shoot tip explants may be derived from either apical or lateral buds of an intact plant, and consist of a meristematic stem apex with a subtended rudimentary stem bearing several leaf initials (Fig 2.3). In the axils of the more developed leaf primordia there will be axillary bud meristems. In some species (e.g. Eucalyptus) it is an advantage to commence shoot cultures with a piece of the stem of the mother plant bearing one or more buds (stem nodes). Shoot growth from the bud, and treatment of the culture, is thereafter the same as in conventional shoot tip culture. The use of nodal explants should not be confused with node culture in which a method of shoot multiplication is used that is different to that in shoot culture (see later).

Fig. 2.3 Shoot tip culture.

Separation of axillary shoots for rooting (or subculture) is easier in species which naturally produce long shoots.

Fig. 2.3 Shoot tip culture.

Separation of axillary shoots for rooting (or subculture) is easier in species which naturally produce long shoots.

Shoot tips from trees, or other woody perennials, Standardi and Catalano (1985) preferred to initiate can be difficult to decontaminate. Because of this, shoot cultures of Actinidia chinensis from meristem tips which could be sterilised more easily. Shoot tips of woody plants are more liable than those of herbaceous species to release undesirable phenolic substances when first placed onto a growth medium. Buds taken from mature parts of the shrub or tree can also be reluctant to grow in vitro and seasonal factors may reinforce natural dormancy in buds from any source, so that cultures can only be readily initiated at certain times of the year (see Chapter 11). Shoot tip or lateral bud explants are usually most readily induced into growth if taken from juvenile shoots (see Chapter 11) such as those of seedlings or young plants. The juvenile shoots which sometimes emerge from the base of mature plants or which arise form heavily pruned or coppiced bushes and trees, are alternative sources. However, developing techniques have made it possible to propagate some woody ornamentals, forest trees and fruit trees, using explants derived from mature shoots (see Chapter 11). De Fossard et al. (1977) could initiate cultures of Eucalyptus ficifolia with shoot tips from 36 year-old trees, but forest-gathered material was very difficult to decontaminate unless covered and protected for some period before excision (stage 0).

Secondary explants. Stage II subcultures are initiated from axillary shoots separated from primary shoot clusters. The place of the secondary explant within the primary shoot (cluster) can have a remarkable influence on the subsequent performance of the subcultures. A higher rate of shoot proliferation is often obtained from nodal explants or by subdivision of the basal shoot mass. Shoot tips were the best secondary explant for Rosa 'Fraser McClay', but with cherry ('F12/1') nodal explants gave more than twice as many shoots, and basal masses, three times as many as shoot tips (Hutchinson, 1985). In Sitka spruce, cultures that had been apices in the previous subculture were able to proliferate buds at higher rates than those that had been axillary buds (John and Murray, 1981). The origin of an explant can also have a tremendous influence on the subsequent behaviour of the plant when established under field conditions. This was illustrated by Marks and Meyers (1994) for Daphne odorata.

To minimise the risk of genetic change in ramets, explants for subculture and shoots to be transferred to Stage III, should, as far as possible, be chosen from new shoots of axillary origin. It may be advisable to adjust the growth regulator content of the medium so that adventitious shoots are not formed, even though the rate of overall shoot multiplication is thereby reduced. In some circumstances callus arising at the base of an explant may be semi-organised and therefore capable of producing genetically-stable plants (Vol. 2).

Stage II cultures are typically without roots, and shoots need to be detached and treated as miniature cuttings which, when rooted, will provide the new plants that are required. An alternative is to allow shoot clusters to elongate and to root singulated shoots under ex vitro conditions (Fig 2.2). Media and growth regulators Advice on the selection of appropriate media for shoot cultures is given in Volume 2. A notable feature of shoot cultures of most plant species is the need for high cytokinin levels at Stage II to promote the growth of multiple axillary shoots. A description of the compounds which can be employed and effective rates of treatment are given in Chapters 3-7.

Cytokinin growth regulators are usually extremely effective in removing the apical dominance of shoots. Their use can be combined with pinching the apex of shoots, or placing explants in an horizontal position (Chapter 6). A cytokinin treatment can not only promote the formation of multiple shoots (axillary and/or adventitious), but also (if the compound used is unsuitable, or the concentration used is too high), cause the shoots formed to be too short for rooting and transfer.

Because or their nature, or the absence of an adequate method of culture, plants of some kinds fail to produce multiple shoots at Stage II and retain their apical dominance. In shoot cultures of Gymnocladus dioicus, for example, despite the formation of several axillary shoots in the presence of BA cytokinin, one shoot nearly always became dominant over the others (Geneve et al., 1990). Most plants of this kind are best propagated by node culture (see below).

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