An optimal protocol for the cryopreservation of in vitro-grown mat rush (igusa) buds by vitrification has been successfully developed. Established multiple stemmed cultures, which were induced in liquid MS medium containing 8.9 M BA by roller culture, were cut into small clumps, plated on solid MS medium and cultured for three weeks at 25°C. Clumps that grew many buds were cold-hardened at 5°C, with an 8 h photoperiod, for more than 30 d. The basal stem bud (1 to 2 mm long) was dissected from the clumps and precultured at 5°C for 2 d on solid MS medium containing 0.3 M sucrose. The precultured buds were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml loading solution containing 2 M glycerol and 0.6 M sucrose for 30 min at 25°C. Then they were dehydrated in 1 ml PVS2 solution at 25°C for 30 min and immersed in liquid nitrogen. Using this protocol, the survival level of cryopreserved igusa 'NZ219' buds reached 87%. This protocol was successfully applied to 42 different lines from three Juncus species, which had relatively high survival levels ranging from 30% to 90% and an average of 63%.
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MULTIPLE STEMMED CULTURE;
Document Type: Research Article
Publication date: 01 May 2007
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CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation
The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.