A simple cryopreservation protocol of Dioscorea bulbifera L. embryogenic calli by encapsulation-vitrification

Abstract

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulationvitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L -1 Kinetin (Kn), 0.5 mg L -1 a-naphthalene acetic acid (NAA) and 0.5 mg L -1 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl 2 solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS 2 solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L -1 , 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 lmol m -2 s -1 provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. 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 0.5 mg L -1 , 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.

Cryopreservation of white mulberry (Morus alba L.) by encapsulation-dehydration and vitrification

“…Recovery in the control explants was not significantly affected till 3 days-treatment (Table 1). Our findings showed that the application of a correct exposure time and concentration of sucrose is critical to insure re-growth in Morus alba as previously shown in cryopreservation of shoot apices of temperate and tropical species and embryogenic tissue of Dioscorea bulbifera and Pinus nigra (Engelmann et al 2008 and references within reported;Ming-Hua and Sen-Rong 2010;Salaj et al 2011).…”

Cryopreservation of wild Shih (Artemisia herba-alba Asso.) shoot-tips by encapsulation-dehydration and encapsulation-vitrification

“…Tissues conserved in vitro at low temperature might show some physiological disorders during conservation followed by a dramatic decrease in survival and regrowth ability (Tahtamouni and Shibli 1999). Moreover, maximum successes of Dioscorea bulbifera L. calli were observed by using of 0.75 M sucrose at 4°C for 7 days (Ming-Hua and Sen-Rong 2010).…”

Direct somatic embryogenesis of Malaxis densiflora (A. Rich.) Kuntze

“…Tissue culture provides an alternate method for large-scale propagation of threatened and endangered plants, including orchid micropropagation using various explants. Somatic embryogenesis is one of the most promising approaches for plant propagation due to the production of large numbers of plantlets [10], the possibility of producing synthetic seeds [11], [12], the ability to store and rapidly mobilize germplasm for cryopreservation [13], the opportunity for genetic manipulation [14] and production of bioactive compounds within a short period of time [15], [16]. It is necessary to develop a method for mass clonal propagation and conservation to satisfy the pharmaceutical demand of this high value medicinal plant.…”