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  • Title: Cryopreservation of embryogenic callus of Dioscorea bulbifera by vitrification.
    Author: Hong S, Yin M, Shao X, Wang A, Xu W.
    Journal: Cryo Letters; 2009; 30(1):64-75. PubMed ID: 19274313.
    Abstract:
    Cryopreservation of callus of Dioscorea bulbifera by vitrification was optimized. Calli of Dioscorea bulbifera were pretreated in liquid Murashige and Skoog (MS) medium supplemented with 2 mg L(-1) kinetin (KT), 0.5 mg L(-1) NAA, 0.5 mg L(-1) 2,4-D and 0.2 M sucrose for 5 d under continuous light (36 microM m(-2) s(-1)) at 25 + or - 1 degree C. The material was then loaded with 60 percent vitrification solution (PVS2) for 20 min at room temperature and dehydrated with 100 percent PVS2 for 30 min at 0 degree C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen and conserved for 1- 360 d. After rapid thawing in a water-bath at 35 degree C, the calli were washed three times with liquid MS medium supplemented with 2 mg L(-1) KT, 0.5 mg L(-1) NAA, 0.5 mg L(-1) 2, 4-D and 1.2 M sucrose and then transferred onto solid MS medium supplemented with KT 2 mg L(-1), NAA 0.5 mg per liter, 0.09 M sucrose and 0.75 percent agar. The cultures were kept in the dark for 2 days prior to exposure to the light (12 h light-dark cycle). The TTC test showed that 80-90 percent of the calli survived this cryoprocedure and there was a 60-70 percent regeneration of plantlets from the calli. The regenerated material did not exhibit any morphological variations.
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