Although not given a special numerical stage by Murashige, the methods whereby plantlets are transferred from the in vitro to the ex vitro external environment are extremely important. If not carried out carefully, transfer can result in significant loss of propagated material. There are two main reasons: —Shoots developed in culture have often been produced in high humidity and a low light 'intensity'. This results in there being less leaf epicuticular wax or wax with an altered chemical composition, than on plants raised in growth chambers or greenhouses. In some plants, the stomata of leaves produced in vitro may also be atypical and incapable of complete closure under conditions of low relative humidity. Tissue cultured plants therefore lose water rapidly when moved to external conditions (Sutter and Langhans, 1979, 1980).
—When supplied with sucrose (or some other carbohydrate) and kept in low light conditions, micropropagated plantlets are not fully dependent on their own photosynthesis (they are mixotrophic -Chapter 12). A stimulus which is not provided in the closed in vitro environment seems to be needed for them to change to being fully capable of producing their own requirements of carbon and reduced nitrogen (i.e. before they become capable of feeding themselves - autotrophic) (Marin and Gella, 1987). The change only occurs after the plants have spent a period of several days ex vitro.
In practice, plantlets are removed from their Stage III containers, and if they have been grown on agar medium, the gel is carefully washed from the roots. The application of an anti-transpirant film to the leaves has been recommended at this stage, but in practice, seems to be seldom used. Plantlets are then transplanted into an adequate rooting medium (such as a peat:sand compost) and kept for several days in high humidity and reduced light intensity. A fog of water vapour is very effective for maintaining humidity. Alternatively, intermittent water misting may be applied automatically, or the plants placed inside a clear plastic enclosure and misted by hand. With some plants, an in vitro Stage III can be omitted; shoots from Stage II are rooted directly in high humidity, and, at the same time, gradually hardened to the exterior environment.
The rooting of shoots using these methods is discussed fully in Volume 2.
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