Abnormal embryos and plantlets

Unfortunately embryogenesis in both callus and suspension cultures is seldom synchronous so that embryoids at different stages of development are usually present in a Stage II culture from the onset. This presents a major drawback for plant propagation which could otherwise be very rapid, especially from suspensions. A proportion of the seedlings developing from somatic embryos can also be atypical: abnormalities include the possession of multiple or malformed cotyledons, more than one shoot or root axis, and the presence of secondary adventive embryos. Embryos with three cotyledons have been observed to give rise to well-formed plantlets (Smith and Krikorian, 1990). Abnormal somatic embryos do however produce secondary embryos, which are usually of normal morphology.

Pretova and Williams (1986) suggested that embryo proliferation, or 'cleavage' (see earlier in chapter), and the formation of accessory cotyledons and root poles, to be homologous with the production of discrete complete embryoids. They suggested that production of accessory cotyledons and somatic embryos on the hypocotyl, and additional root poles near the base of existing embryos, may represent either a gradient along these organs in the early stages of determination, or, be caused by a factor affecting cell to cell co-ordination.

Differential filtering and sedimentation to separate embryos at different stages of development (Giuliano et al., 1983) can improve the uniformity of embryo populations in suspension cultures. More recently image analysis has been used to select embryos in specific developmental stages (Kurata, 1995). In addition cultures can be maintained on media containing high levels of sucrose (Ammirato and Steward, 1971), and/or low levels of abscisic acid (Ammirato, 1973; 1974). Both approaches, as well as the addition of imazalil to the culture medium (Werbrouck et al., 2000), limit the number of abnormalities and give a higher degree of synchronisation. High levels of sucrose and abscisic acid induce reversible dormancy in somatic embryos and thus might be used to temporarily suspend growth should this be advantageous in a planned micropropagation programme.

Dormancy is however not always reversible. Indeed somatic embryos can remain dormant, and conversion to plantlets can be problematic. Different types of approaches can be used to overcome this problem, i.e. desiccation, supplementing the medium with osmotic agents [e.g. polyethylene glycol (PEG), mannitol] (Capuana and Debergh, 1997).

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