Stage I

In most herbaceous broad leafed plants, it is possible to initiate morphogenically competent callus cultures from explants derived from many different tissues. Leaf, stem or root segments, pieces of storage tissue (e.g. tubers), seed embryos, shoot tips and seedling tissues have been used at various times. In monocotyledons there is a narrower range of suitable organs; embryos, very young leaf tissue, stem nodes and immature inflorescences being the most common sources. Initiation of callus cultures of many tree species, including gymnosperms, is frequently limited to explants derived from tissues near the vascular bundles or the cambium of stem or root sections. Explants containing actively dividing cells may be necessary if callus possessing a high level of morphogenic competence is to be isolated.

Callus growth is usually initiated by placing the chosen explant on a semi-solid medium into which auxin has been incorporated at a relatively high level, with or without a cytokinin. Details of the compounds used are given in Chapter 5. One or more transfers on the same medium may be necessary before the callus is separated from the parental tissue for subculture. Because more than one kind of callus may arise from a single explant, successful propagation can depend on being able to recognise and subculture only the type (or types) which will eventually be able to give rise to shoots or somatic embryos. In the absence of previous experience, samples of each type of callus may have to be carried forward for testing on inductive media. Translucent, watery callus is seldom morphogenic, whereas nodular callus frequently is.

Organised adventitious shoots are usually induced to form in callus or suspension cultures by reducing the auxin level in the medium and/or increasing the concentration of cytokinin. To grow callus-derived shoots into plantlets capable of survival in the soil, they must be rooted as micro-cuttings. Root production by callus is of little consequence for micropropagation purposes; even if roots are formed concurrently with adventitious shoots, the vascular connections between roots and shoots, through the callus tissue, are almost invariably insufficient for the development of a functional plantlet.

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