Lambardi M, Fabbri A, Caccavale A
Istituto sulla Propagazione delle Specie Legnose, Consiglio Nazionale delle Ricerche, via Ponte di Formicola 76, 50018 Scandicci (Florence), Italy e-mail:
Dipartimento di Biologia Evolutiva e Funzionale, Università degli Studi, viale delle Scienze, 43100 Parma, Italy, , , , , , IT.
Plant Cell Rep. 2000 Jan;19(3):213-218. doi: 10.1007/s002990050001.
Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at -196 °C by one-step vitrification. After preculturing at 5 °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 M glycerol and 0.4 M sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 M sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0 °C and, following cryopreservation, rewarmed at 40 °C and washed in 1.2 M sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μM N-benzyladenine plus 0.5 μM gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μM indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips.
通过一步玻璃化法，成功地将体外培养的、经过抗寒锻炼的白杨（Populus alba L.）砧木的茎尖在-196°C下冷冻保存。在含有不同蔗糖浓度的无激素MS培养基上于5°C预培养2天，并用2M甘油和0.4M蔗糖加载20分钟后，将茎尖用PVS2玻璃化溶液处理并直接投入液氮中。当茎尖在0.09M蔗糖的无激素MS培养基上预培养，在0°C下用PVS2溶液处理60分钟进行玻璃化，冷冻保存后在40°C复温并在1.2M蔗糖溶液中洗涤20分钟时，获得了最佳存活率（90%）。将茎尖接种在含有1.5μM N-苄基腺嘌呤加0.5μM赤霉素的凝胶化MS培养基上可促进再生，而在含有3μM吲哚-3-丁酸的MS培养基上可实现茎生根。按照此程序，从冷冻保存的茎尖中获得了近60%的生根芽。
