Elongation

The length of the axillary shoots produced in shoot cultures varies considerably from one kind of plant to another. Species which have an elongated shoot system in vivo will produce axillary shoots which can be easily separated as microcuttings and then individually rooted. Apically dominant shoots which have not branched can be treated in the same way.

At the other extreme are plants with a natural rosette habit of growth, which tend to produce shoot clusters in culture (Fig 2.3). When these are micropropagated, it is difficult to separate individual shoots for use as secondary explants. It may then only be practical to divide the shoot mass into pieces and re-culture the fragments. Such shoot clusters can be induced to form roots when plants with a bushy habit are required (e.g. many species sold in pots for their attractive foliage). Otherwise it is necessary to specially elongate shoots before they are rooted (Stage Ilia). Shoot clusters are treated in such a way that axillary shoot formation is reduced, and shoot growth promoted. Individual shoots are then more readily handled and can be rooted as microcuttings. Methods for elongating shoots are discussed in Volume 2.

Rooting and transfer The cytokinin growth regulators added to shoot culture media at Stage II to promote axillary shoot growth, usually inhibit root formation. Single shoots or shoot clusters must therefore be moved to a different medium for rooting in vitro before being transferred as plantlets to the external environment. An alternative strategy for some plants is to root the plant material ex vitro. The methods employed are described in Volume 2. Treatments need to be varied according to the type of growth; the nature of the shoot proliferation produced during Stage II culture; and the plant habit required by the customer.

Current applications Conventional shoot culture continues to be the most important method of micropropagation, although node culture is gaining in importance. It is very widely used by commercial tissue culture laboratories for the propagation of many herbaceous ornamentals and woody plants (see Volume 2 for further details). The large numbers of manipulations required do, however, make the cost of each plantlet produced by this method comparatively expensive. Some success has been achieved in automating some stages of the process, in applying techniques for large-scale multiplication and in the use of robotics for plant separation and planting (Vol. 2).

3.1.2. Shoot proliferation from meristem tips

Barlass and Skene (1978; 1980a,b; 1982a,b) have shown that new shoots can be formed adventitiously when shoot tips of grapevine or Citrus are cut into several pieces before culture. Tideman and Hawker (1982) also had success using fragmented apices with Asclepias rotundifolia but not with Euphorbia peplus. Usually leaf-like structures first develop from the individual fragments; these enlarge and shoots form from basal swellings. Axillary shoots often arise from the initial adventitious shoots.

Shoot cultures transferred to agitated liquid culture may form a proliferating mass of shoots. Although high rates of multiplication are possible, leafy shoots usually become hyperhydric (see

Volume 2). However, in some species at least, shoots can be reduced in size to little more than proliferating shoot initials (by adding plant growth retardants, Ziv et al,, 1994) which are then suitable for large-scale multiplication (Volume 2). A somewhat similar kind of culture consisting of superficial shoot meristems on a basal callus can sometimes be initiated from shoot tip explants or from the base of shoot cultures (see later in this chapter).

3.1.3. Single and multiple node culture (in vitro layering)

Single node culture is another in vitro technique which can be used for propagating some species of plants from axillary buds. As with shoot culture, the primary explant for single node culture is a shoot apex, a lateral bud or a piece of shoot bearing one or more buds (i.e. having one or more nodes). When shoot apices are used, it can be advantageous to initiate cultures with large explants (up to 20 mm), unless virus-tested cultures are required, and small meristem-tips will be employed. Unbranched shoots are grown at Stage I until they are 5-10 cm in length and have several discrete and separated nodes. An environment that promotes etiolated shoot growth may be an advantage. Then at Stage II, instead of inducing axillary shoot growth with growth regulators (as in shoot culture), one of two manipulative methods is used to overcome apical dominance and promote lateral bud break (Fig 2.2):

• intact individual shoots may be placed on a fresh medium in an horizontal position. This method has been used by Wang (1977) to propagate potatoes, and has been termed 'in vitro layering';

• each shoot may be cut into single-, or several-node pieces which are sub-cultured. Leaves are usually trimmed so that each second stage explant consists of a piece of stem bearing one or more lateral buds.

• each approach can be reiterated to propagate during stage II.

Unfortunately, in vitro layering seldom results in several axillary shoots of equal length, as shown in Fig 2.4; apical dominance usually causes the leading shoot, or shoots, to grow more rapidly than the rest. El Hasan and Debergh (1987) found that, even in potato, node culture was preferable. Node culture is therefore the simplest method of in vitro propagation, as it requires only that shoot growth should occur. Methods of rooting are the same as those employed for the microcuttings derived from shoot culture, except that prior elongation of shoots is unnecessary.

Note that "node culture" is distinct from shoot cultures started from the nodes of seedlings or mature plants.

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