Stage II

Once a morphogenic callus has been isolated, propagation is carried out either by callus subdivision, or by the preparation of cell suspensions.

The success of each technique depends on the subcultured tissues or cells continuing to regenerate shoots.

Callus subdivision. Callus is cut into smaller pieces which increase in size when subcultured in a liquid or an agar-solidified medium. The callus can either be subdivided further, or shoot regeneration allowed to occur. This may take place on the same medium, or the callus may need to be transferred to another shoot-inducing medium. The organogenic capacity of callus is easily lost on repeated subculture. Use of high growth regulator levels can encourage the proliferation of non-regenerative callus which will displace tissues having the competence to form new shoots (e.g. in Pelargonium; Holdgate, 1977).

Fig. 2.5 Callus with different morphogenic potential is often isolated from a single explant.

The preparation of cell suspensions. Compared to the relatively rapid rates of propagation that are possible with shoot culture of some kinds of plants, propagation from morphogenically competent callus can be slow initially. Krikorian and Kann (1979) quoted a minimum of 135 days from the excision of daylily explants to the potting of plantlets. The rate at which propagation can proceed after that depends on the rate at which callus can be grown and subdivided. Providing a shoot-forming capacity is retained, a much faster rate of multiplication can be achieved by initiating a suspension culture from competent callus. After being increased by culture, the cells or cell aggregates can then be plated to produce new regenerative callus colonies. This is not an easy operation, as growth regulators favouring the formation of a dispersed cell suspension can cause the cells to lose their morphogenic capacity (see Chapter 10). There is also the problem that, by prolonging the period before shoot regeneration, genetic variability within the cell line will be increased.

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