title: High-efficiency encapsulation-vitrification protocols for cryopreservation of embryogenic calli of the oriental medicinal plant Anemarrhena asphodeloides Bunge.
abstract: Embryogenic calli from in vitro grown tillers of Anemarrhena asphodeloides Bunge were successfully cryopreserved by the encapsulation-vitrification technique. Excised embryogenic calli were precultured for 4 days in liquid MS medium supplemented with 2 mg per liter kinetin (KIN), 0.1 mg per liter α-naphthalene acetic acid (NAA) and 0.75 M sucrose, then encapsulated in calcium alginate beads and loaded with a mixture of 2 M glycerol + 0.4 M sucrose for 60 min at 25 +/- 1 degree C. Calli were then dehydrated with the PVS2 solution for 80 min at 0 degree C. After changing the solution with fresh PVS2, calli were directly immersed in liquid nitrogen (LN). After rapid rewarming in a water-bath at 35 degree C for 5 min, calli were washed three times with liquid MS medium supplemented with 2 mg L-1 KIN, 0.1 mg per liter NAA and 1.2 M sucrose, then transferred on solid MS medium supplemented with 2 mg per liter KIN, 0.1 mg per liter NAA, 3 % (w/v) sucrose and 0.75 % (w/v) agar. Cryopreserved cultures were kept in the dark for 5 days prior to exposure to a 14h light/10h dark photoperiod with a light intensity of 36 μmol per square meter per sec provided by white cool fluorescent tubes at 25 +/- 1 degree C. Survival of cryopreserved embryogenic calli reached 80 percent, including after storage for c. 1 year. No significant difference was observed in the morphological development of plants coming from control and cryopreserved embryogenic calli. This encapsulation-vitrification method appears promising for the cryopreservation of A. asphodeloides Bunge germplasm.
authors: College of Life Sciences, Shangrao Normal University, Shangrao, China.
authors: S-R
authors: SR
authors: Hong
authors: M-H
authors: MH
authors: Yin
mesh_terms: D000464:Alginates / Q000737:chemistry; D031403:Anemarrhena / Q000196:embryology*; D018929:Cell Culture Techniques; D018914:Cells, Immobilized / Q000166:cytology / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D020723:Glucuronic Acid / Q000737:chemistry; D005990:Glycerol / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry; D007701:Kinetin / Q000378:metabolism; D010946:Plants, Medicinal / Q000196:embryology*; D012639:Seeds / Q000166:cytology* / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D058989:Vitrification
chemical_list: D000464:Alginates; D003470:Culture Media; D006603:Hexuronic Acids; D013395:Sucrose; D020723:Glucuronic Acid; D007701:Kinetin; D005990:Glycerol