cryopreservation of basal stem buds of in vitro-grown mat rush (Juncus spp.) By vitrification

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%.