Tissue culture cryopreservation

There has been a demand for the development of cryopreservation methods for plant cells to avoid the troublesome maintenance of tissue cultures and the danger of microbial contamination. The most successful method for cryopreservation of plant cells reported so far has been the freezing of callus cultures or shoot tips [36, 37]. As the system here enables us to obtain sufficient initial shoot materials, its potential practical application to cryopreservation is in progress. In addition, the system of adventitious shoot formation might be a promising tool to investigate relationship between morphogenesis (shoot formation) and alkaloid biosynthesis. 

Care must be taken not to overtrypsinize the cells when removing them from the tissue culture surface. The 2.2.15 cells are sticky and have a natural tendency to clump. Aggregated cells do not grow well and are difficult to count. The 2.2.15 cells also perform better in reseeding and cryopreservation procedures if they receive fresh medium 24 h before trypsinization. 

The cell and tissue culture of the major tropane alkaloid-producing species does not apparently offer any special problems. The regeneration of plantlets from callus and tissue cultures seems to be routine (286,307,309,323,325,332,350-352). Plants have also been regenerated from protoplasts of Atropa belladonna (353), Duboisia myoporoides (354), and Hyoscyamus muticus (355,356). Cryopreservation has been reported for Anisodus and Datura species (349,357). 

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