Maintenance and Preservation of Fungal Cultures

Pure culture of edible fungi is prepared either by multispore culture or tissue culture; the former is suitable for obtaining fruiting cultures of A. bisporus but is not a suitable technique for heterothallic species. Tissue cultures derived from the stipe or pileus of the mushrooms, both homothallic as well as heterothallic species, can be used to raise fruiting cultures. For multispore culture, a healthy and mature fruitbody of the mushroom is first washed in sterile water, surface-sterilized with alcohol, and is placed on a spiral wire loop kept in sterile petriplate covered with a beaker. Mushroom sheds spores on petriplates from which a loopful of spores is transferred on suitable growth medium, generally malt extract agar in case of A. bisporus. Spores after germination give rise to multispore culture. In case of tissue culture, a piece from a suitable place of fruitbody is cut and after surface-sterilization, the piece is transferred onto sterile growth medium slants. Different mushrooms may require different growth medium and incubation temperatures; for example, A. bisporus grows best on malt extract agar medium between 24-28°C while Volvariella spp. are best maintained on potato dextrose agar medium at 35°C. It is desirable that the cultures are not maintained on the same type of medium in each subculturing for a long time. Degeneration of cultures or spawn, which refers to the loss of desired traits, survival, growth rate, and productivity, is not uncommon (Chang and Miles 1989; Stadelmann 1986) and has, of late, attracted the attention of the researchers to understand the reasons. Authentic pure cultures of mushrooms should preferably be obtained from the reputed mushroom germplasm banks. Now a days, mushroom strains of commercial importance are patented and thus free availability is restricted (Jong and Birmingham 1991).

Though the pure cultures of mushrooms, once raised or obtained as described above, are traditionally maintained by periodic subculturing and/or cold storing between 2-5°C, however, better long-term preservation methods of fungal cultures are advisable and practiced now, which are required for maintenance of vigor and genetic characteristics especially related with productivity and quality (Chang and Miles 1989). Frequent subculturing is not only time consuming but also costly and risky (Smith and Onions 1983). Other methods of preserving fungal cultures including mushrooms have been described (Jong 1989; Singh and Upadhyay 2002; Smith and Kolkowski 1996; Smith and Onions 1983), which include storage in mineral oil (paraffin wax), lyophilisation, cryopreservation at low temperatures (— 70°C), in liquid nitrogen or in mechanical freezers. The choice of method depends on many factors like requirement, resources, cost, etc. It is advisable that each mushroom strain should be maintained by at least two different methods; liquid nitrogen and mineral oil preservation have been found highly suitable and are popular for preservation of mushroom cultures. Mushroom culture repositories/banks play a vital role in the supply of pure and authentic cultures to the spawn production units. American Type Culture Collection in USA; International Mycological Institute in UK; Institute of Microbial Technology and National Research Centre for Mushroom in India are some of the reputed mushroom culture banks.

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