Root Organ Culture Technique

The ROC system is the most attractive cultivation methodology for research; it uses root-inducing transfer-DNA-transformed roots of a host plant to develop the symbiosis on a specific medium in vitro (Becard and Fortin 1988). The pathogenic condition known as "hairy root" occurs due to the transfer of root inducing Ri-plasmid from the bacterium Agrobacterium rhizogenes (Schenck 1992). These techniques, though challenging have proved useful additions to our knowledge for various aspects of the AM fungal-host symbiosis (Douds 1997). The problem of producing inoculum in bulk is addressed by the ROC of AM isolates, which provides pure, viable, contamination-free inoculum using less space and thus has an edge over the conventional mode of pot-culture multiplication. Pioneering work on in vitro cultures was initiated in the early 1960s (Chabot et al. 1992). The use of Ri T-DNA transformed roots of Daucus carrota as host by A. rhizogenes has permitted an increase in spore production of Glomus mosseae (Mugnier and Mosse 1987). Only few fungal species have being successfully grown using ROC mode such as Gigaspora margarita, Glomus fasciculatum, and G. macrocarpum, G. intraradices, Gigaspora gigantia, G. versiforme, G. caledonium, G. clarum, G. fistulosum, and G. etunicatum (Chabot et al. 1992; Declerck et al. 1996a,b; Declerck et al. 1998; Diop et al. 1992; Douds and Becard 1993; Gryndler et al. 1998; Karandashov et al. 2000; Pawlowska et al. 1999; Souza and Barbara 1999). Though process of bringing them is very difficult, but the list of the new species under in vitro is increasing every day. Mass production of spores is prerequisite and mathematical models may be useful as descriptive and predictive tools of sporulation dynamics (Declerck et al. 2001). This mode of culturing AM fungi also provides an opportunity for biochemical and molecular investigations of AM symbiosis, which are otherwise unclear.

Isolation and inoculum production of ectomycorrhiza (EM) and AM fungi present very different problems. Many EM fungi can be cultured on artificial media. Therefore, isolates of EM can be obtained by placing surface-disinfected portions of sporocarps or mycorrhizal short roots on growth medium. The resulting fungal biomass can be used directly as inoculum but, for ease of use, inoculum often consists of the fungal material mixed with a carrier or bulking material such as peat. They can now be produced in a fermentor and by entrapping the mycelium in alginate beads (Le Tacon et al. 1985; Mauperin et al. 1987). Obtaining isolates of AM fungi is more difficult because they will not grow apart from their hosts. Spores can be sieved from soil, surface disinfected, and used to initiate "pot cultures" on a susceptible host plant in sterile soil or an artificial plant-growth medium. Inoculum is typically produced in scaled-up pot cultures. Alternatively, hydroponic or aeroponic culture systems are possible; a benefit of these systems is that plants can be grown without a supporting substrate, allowing colonized roots to be sheared into an inoculum of high propagule number.

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