A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant Anemarrhena asphodeloides Bunge by vitrification

Abstract

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 Murashige and Skoog (MS medium) containing 2 mg L−1 Kinetin (Kn), 0.1 mg L−1α-naphthalene acetic acid (NAA) and 0.5 M sucrose for 2 days under continuous light at 25 ± 1°C. 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 ± 1°C and dehydrated with a highly concentrated vitrification solution (PVS2) for 40 min at 0°C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen (LN). After rapid thawing in a water-bath at 35°C 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.