Cryopreservation of horseradish hairy root cultures using encapsulation-dehydration method

Rapid progress in plant biotechnology and genetic engineering has led to the development of various new plants and cultured cell lines which are modified to take advantage of their useful properties. Important stock cultures for all of these applications can be maintained by serial subcultures on appropriate nutrient media. However, factor such as progressive changes in their characters, contamination with other organisms, and the labor and space required for maintenance create problems for the preservation of valuable plant materials by subculture. Cryopreservation has thus become important for long-term and stable preservation of such materials. In this study, I used the encapsulation-dehydration method for cryopreservation of plant roots which has not been successfully cryopreserved in many species. Horseradish (Armoracia rusticana) hairy root cultures well known as a typical internal tissue of "artificial seed" having high germination rate was used as materials for cryopreservation. Under light condition, the primordia were induced from branch portions in hairy roots by preculture on Murashige-Skoog medium with 0.2 mg/l naphthaleneacetic acid. They were excised and encapsulated in 2 percent Ca-alginate beads containing 0.5 M sucrose and 1 M glycerol and then precultured for one day on solid medium containing the same concentration of sucrose and glycerol. Then, they were gradually slowly dehydrated to 70 percent of the initial bead weight with dried silica gel for about 60 hours, and rapidly dehydrated to about 40 percent at a rate of 10 percent of water content loss per hour under reducing pressure before being immersed into liquid nitrogen. After warming in water bath at 40 deg C, the beads were directly cultivated on the medium containing 0.09 M (3 percent) sucrose and 1 mg/l benzyladenine under light condition. The average rate of plant regeneration was more than 90 percent, and the revived primordia produced shoots within 2 weeks after cultivation. A long-term preservation of shoot primordia was also achieved by the technique. No morphological abnormalities of regenerated plants were observed. The hairy roots were also easily developed from the primordia cryopreserved when they were cultivated on the hormone-free medium in the dark. In addition, root tips of horseradish hairy root cultures were also cryopreserved by the technique with small modifications, the survival of cryopreserved roots was about 60 percent. This encapsulation-dehydration method including slow dehydration step is promising as a practical procedure for cryopreserving meristems, somatic embryos, and hairy roots.