In the past, several ornamental plants [e.g. Freesia (Hussey and Hargreaves, 1974) and Pelargonium (Holdgate, 1977)] have been micropropagated from adventitious shoots produced indirectly from callus. It had been hoped to extend the technique to other species possessing a strong natural tendency towards diploidy (e.g. some forest trees) where plantlets produced in vitro might have a normal karyotype (Mott, 1981), but it is now realised that the genetic changes which are almost universally induced in the genotype of cells during callus and cell culture make cloning by this technique inadvisable except where new genotypes are required for selection or further plant breeding. Another possibility is that mutated somatic cells, already present in the mother plant, are given the opportunity to develop into a plantlet.
Indirect formation of somatic embryos (or adventitious somatic embryogenesis) from callus or suspension cultures is observed more frequently than direct embryogenesis. Frequently callus which is wholly or partly embryogenic can be induced during the initial culture of explants derived from young meristematic tissues (see below), but induction is less common in cultures which have been kept and transferred for some period without organogenesis.
There are important requirements for the successful induction of embryogenic callus and suspension cultures:
• The plant genotype must be capable of embryogenesis on the chosen system of induction (medium plus added growth regulators). In some genera most genotypes are competent, but in others there may be a wide variation in competence even between different varieties or cultivars within a species.
• In most practical situations, cultures should be grown in the presence of an auxin for the induction (and initiation) of embryogenesis (Stage I).
• The level of sugar (e.g. sucrose or glucose) in the medium may need to be within critical concentrations, and no embryos may be formed at all if the sugar concentration is too high (Lippman and Lippmann, 1984).
• After the beginning of embryogenesis, it is usually (but not invariably) necessary for Stage I tissues or cells to be subcultured to a medium containing a reduced auxin concentration, or containing no auxin at all (Stage II) (Chapter 9).
• There may be an optimum length of time during which the Stage I routine should be maintained. An extended period before subculture can result in the failure to obtain embryogenesis at Stage II (e.g. Dos Santos et al.1980). Maintenance of the cultures on high auxin usually causes embryo development to be arrested or a loss of embryogenic capability.
• A supply of reduced nitrogen is required. This may be supplied in the form of NH4+ ion and/or as an amino acid such as glutamine or alanine (see Chapter 3).
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