エゾワサビ培養体茎頂のガラス化法による超低温保存

概要

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The objective of this study is to establish a cryopreservation protocol for shoot apices of Cardamine yezoensis. Cryopreservation was carried out using a vitrification method on shoot apices excised from in vitro cultures. Plant Vitrification Solution 2 (PVS2) was demonstrated to be an optimal vitrification solution for shoot apices in terms of its higher recovery after cryopreservation compared with PVS1 and PVS3. Smaller excised apices (1mm x 1mm square in size) showed higher regrowth after cryopreservation using PVS2 compared with larger apices (3mm x 3mm). The vitrification protocol leading to the optimal regrowth was as follows: Excised shoot tips were pretreated for 24hr at 25℃ on hormone-free basal medium with 0.4mol/L sucrose, then precultured in liquid basal medium supplemented with 0.4mol/L sucrose and 2.0mol/L glycerol (a loading solution) for 30min at 25℃. Precultured specimens were soaked in PVS2 for 60min at 0℃ in cryotubes before cryostorage for 1hr. We also examined the effects of various nutrient media on regrowth of cryopreserved apices. It was demonstrated that 4-times dilution of inorganic salts of Murashige and Skoog's medium (1/4MS) or Woody Plant medium (WPM) as basal medium resulted in higher regrowth percentages (both 66.7%) than other six media. We succeeded in improving regrowth after vitrification-based cryopreservation up to 96.7% by exchanging PVS2 twice during 60min of PVS2 loading.
2010-10-15