A simple cryopreservation protocol of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. embryogenic calli by encapsulation-vitrification |
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Authors: | Yin Ming-Hua Hong Sen-Rong |
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Institution: | (1) College of Life Sciences, Shangrao Normal University, 334001 Shangrao, Jiangxi, People’s Republic of China; |
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Abstract: | Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification 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 α-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 CaCl2 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 PVS2 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 μmol 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. |
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