Hong S.R. and Yin M.H. Plant Cell Tiss. Cult. 109: 287-296 (2012)
Embryogenic calli of Anemarrhena asphodeloides Bunge were successfully cryopreserved by vitrification. Excised embryogenic calli from in vitro grown tillers were precultured in liquid basal tissue culture medium from MS medium containing 2 mg L-1 Kinetin (Kn), 0.1 mg L-1a-naphthalene acetic acid (NAA) and 0.5 M sucrose for 2 days under continuous light at 25 ± 1C. Following preculture the calli were transferred to a 2 mL plastic cryotube. The calli was then loaded with a mixture of 2 M glycerol and 0.4 M sucrose for 30 min at 25 ± 1C and dehydrated with a highly concentrated vitrification solution (PVS2) for 40 min at 0C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen (LN). After rapid thawing in a water-bath at 35C for 5 min, the calli were then transferred onto solid MS medium supplemented with Kn 2 mg L-1, NAA 0.1 mg L-1, 3% (w/v) sucrose and 0.75% (w/v) agar. The cultures were kept in the dark for 5 days prior to exposure to the light (14 h light/dark cycle). After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L-1, NAA 0.1 mg L-1, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium) and developed normal shoots. The regrowth rate of vitrified embryogenic calli reached over 60%. Cryopreservation did not affect the plant regeneration potential of A. asphodeloides Bunge embryogenic calli through somatic embryogenesis. Morphologies of plants regenerated from cryopreserved calli were similar to those of the seedlings. This efficient cryopreservation protocol would be useful for the ex-situ conservation of A. asphodeloides Bunge germplasm in gene banks at very low temperatures
Developing a simple and reliable protocol for the cryopreservation of embryogenic calli of A. asphodeloides Bunge by vitrification.
Seeds of A. asphodeloides Bunge were used, which were obtained from Medicine professional cooperatives (Wutong Town, Jingning County, Zhejiang Province,China).
Strong and healthy seeds were chosen, soaked in saturated washing powder for about 20 min and were then thoroughly washed under running tap water for 30 min. Finally, seeds were surface-sterilized sequentially with 70% (v/v) alcohol for 30 s and 0.1% mercuric chloride for 15 min. The seeds were then thoroughly washed with sterile distilled water before culturing.
1/2MS-based solid medium with 30 g/L sucrose, 7.5 g/L agar was used in seed germination experiments). The pH of the medium was adjusted to 5.8–6.0 prior to autoclaving at 121℃ for 20 min. Cultures were maintained at 25 ± 1℃ with 75 ± 5% relative humidity under a 16-h photoperiod at a light intensity of 36 µmol m-2 s-1 for periods of 4 weeks. In such conditions, plantlets are developed.