In recent years, the application of micropropagation techniques as an alternative mean of asexual propagation of important plants has increased the interest of workers in various field. The micropropagation techniques are preferred over the conventional asexual propagation methods because of the following reasons : (a) in this method only a small amount of tissue is needed as the initial explant for regeneration of millions of clonal plants in a year, (b) this method provides a possible alternative method for developing resistance in many species; (c) it provides a mean for international exchange of plant materials, hence the problem for introduction of disease can be solved in quarantine; (d) in vitro stock can be quickly proliferated as it is not season dependent, and (e) valuable germplasm can be stored for a long time (Hu and Want, 1983; Mascarenhas and Muralidharan, 1989).
Regeneration of plantlets in cultured plant cell and tissues has been achieved in many trees of high economic value. Many of the studies are aimed at large scale micropropagation of important trees yielding fuel, pulp, timber, oils or fruits. Therefore, clonal forestry and horticulture are gaining an increasing recognition as an alternative for tree improvement. However, strategies for transferring cultured plants from in vitro to field conditions are based on relatively higher priced horticultural species rather than agricultural and forestry species (Fossard, 1987).
Regeneration of plantlets in cultured tissue has been described (Murashige, 1974) to be accomplished into 3 stages (see shoot culture)'. Fossard (1987) has given a detailed account of stages of micropropagation (see Shoot culture and micropropagation).
At planting out stage the plantlets fail to survive because of sudden change in the environment and invasion by soil microbes. Jagannathan (1987) has described that the regenerates should be transferred first to green house and then to field. The humidity should be controlled by covering the plants with transparent polyethylene sheets. This acclimatization requires several weeks which should be followed by potting into sterile peat or soil.
In recent years, the interest has, aroused in commercializing the in vitro propagation of forest trees. This will bring about refinement in the existing procedures to make micropropagation more cost effective. Mascarenhas, Muralidharan and coworkers are making efforts to commercialize this biotechnology with respect to forest trees. However, development of automated procedure, plant delivery systems using somatic embryos and artificial seeds are also in progress.
For betterment and improvement of tree plants of high economic value a break through in forestry research has come with production of artificial seeds in Eucalyptus (Muralidharan and Mascarenhas, 1989), and genetic transformation and in vitro regeneration in conifers (Gupta, 1989). Moreover, micropropagation has been successfully done in many trees (Gupta et al. 1980, 1981; Jaiswal and Pratap Narayan, 1985; Amin and Jaiswal, 1988; Mascarenhas and Muralidharan, 1989).
Mascarenhas and Muralidharan (1989) reviewed the tissue culture studies carried out on forest trees in India. Some of the important plants are : Acacia nilotica, Albizia lebbeck, A. procera, Azadirachta indica, Bauhinia purpurea, Butea monosperma, Dalbergia sp., Dendrocalmus strictus, Eucalyptus sp. Ficus religiosa, Morus sp., Populus sp., Shorea robusta, Tectona grandis (all angiosperms), Biota oriental's, Cedrus deodara, Cryptomena japonica, Picea smithiana, Pinus sp. (all gymnosperms).
In Vitro Establishment of Mycorrhiza
Mycorrhizal fungi show highest level of specialization of parasitism. But the major problems with them is their failure to grow on an artificial medium in laboratory. Therefore, establishment and multiplication of mycorrhizal fungi on cultured tissue of the same host plant, if successfully developed, may be a good tool for handling mycorrhizal fungi, production of high potential inoculum and their establishment in root systems of nursery plants in horticulture and forestry, and plantation of mycorrhiza-infested seedlings into field. Only one report is available on this work. Kiernan et al. (1984) successfully produced strawberry plants by tissue culture which was infected by a mycorrhizal fungus, Glomus sp.
Many attempts have been made to establish Vesicular Arbuscular Mycorrhizal (VAM) fungi in axenic culture but unfortunately none of them got success. It was assumed that self inhibition of hyphal growth occurs in the growing germ tubes and the self inhibition compounds were recovered by adding activated charcoal into an agar medium that absorbs inhibitory compounds produced by germ tubes into medium. Cultures of mycorrhizas synthesized aseptically are grouped into two : the whole plant cultures and excised root cultures. Both the types of cultures are known as genetobiotic or monogenic systems (Rhodes, 1983). Due to presence of two organisms, it is also known as two member culture.
Mosse (1962) for the first time, reported the establishment of two member cultures. Appressorium formation and root penetration were much more likely to occur if a Pseudomonas sp. was present in culture. It is, therefore, suggested that 3 organisms i.e. fungus-plant-bacterium might be necessary for the development of symbiosis.
Moreover, mycorrhizal fungi have been cultured only on cortical tissues of roots which were separated from the whole plant, as in root organ cultures where it acted as food base. VAM fungi have very high degree of specialization for food base on root cortex (Rhodes, 1983).