Shoots or plantlets derived from Stage II are small, and not yet capable of self-supporting growth in soil or compost. At Stage III, steps are taken to grow individual or clusters of plantlets, capable of carrying out photosynthesis, and survival without an artificial supply of carbohydrate. Some plantlets need to be specially treated at this stage so that they do not become stunted or dormant when taken out of the cultural environment. As originally proposed by Murashige, Stage III includes the in vitro rooting of shoots prior to their transfer to soil.
Rooting shoots is a very important part of any in vitro propagation scheme. A few species form adventitious roots on shoots during the course of Stage III culture, but usually it is necessary to adopt a separate rooting procedure using special media, or methods, to induce roots to form. Sometimes shoots may need to be specially elongated before rooting is attempted. To reduce the costs of micropropagation, many laboratories now remove unrooted shoots from the in vitro environment and root them outside the culture vessel (Fig 2.2). Therefore, in cultures where micropropagation relies on adventitious or axillary shoots, Stage III is often conveniently divided, as Debergh and Maene (1981) suggested, into:
• Stage IIIa, the elongation of buds or shoots formed during Stage II, to provide shoots of a suitable size for Stage IIIb;
• Stage IIIb, the rooting of Stage IIIa shoots in vitro or extra vitrum.
Procedures used to induce rooting are discussed in later chapters.
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