The effect of dehydration, cryopreservation, and reculture conditions on growth recovery (%) of vanilla (Vanilla planifolia) shoot-tips was evaluated using a D-cryoplate procedure. Tissues were excised from in vitro grown plantlets, preconditioned on MS semisolid medium supplemented with 0.15 M trehalose for 1 d, loaded in a solution of 0.4 M sucrose or trehalose and 2 M glycerol for 30 min, and dehydrated within a laminar flow cabinet for various durations (30, 60, 90, 120, 150, and 180 min). The same preconditioning and loading treatments were compared using dehydration with vitrification solutions (PVS2 or PVS3) for 30 min at room temperature according to droplet-vitrification and V-cryoplate methods. The highest (33%) recovery of cryopreserved shoot-tips was achieved using the D-cryoplate method after 0.15 M trehalose preconditioning, loading with sucrose-glycerol solution and desiccation for 180 min. DSC analyses revealed that the osmotically active water (OAW) content of the shoot-tips was reduced from 77% (fresh weight basis) to 17% after the only effective drying duration (180 min). Melting endotherms indicated that crystallization events accompanied cryopreservation of the tissues. Proliferation of multiple shoots was obtained by indirect organogenesis. Histological analysis of the explants during post-cryopreservation recovery confirmed the organogenic nature of the callus formed after 3–4 mo of reculture in the dark on semisolid multiplication medium. This was followed by a secondary organogenesis on MS medium with kinetin (2 mg L⁻¹) and exposure to a photoperiod. At present, this is the most optimized cryopreservation protocol for shoot-tips of V. planifolia.

使用D型冷冻板法评估了脱水，冷冻保存和再培养条件对香草（Vanilla planifolia）梢的生长恢复（％）的影响。从体外切除组织生长的小植株，在补充有0.15 M海藻糖的MS半固体培养基上预处理1 d，装入0.4 M蔗糖或海藻糖和2 M甘油的溶液中30分钟，并在层流柜中脱水不同的时间（30、60， 90、120、150和180分钟）。根据液滴玻璃化法和V型冷冻板法，在室温下使用玻璃化溶液（PVS2或PVS3）脱水30分钟，比较了相同的预处理和负载处理。在0.15 M海藻糖预处理，用蔗糖甘油溶液上样并干燥180分钟后，使用D-cryoplate方法可实现冷冻保存的茎尖的最高回收率（33％）。DSC分析表明，仅在有效干燥时间（180分钟）后，枝梢的渗透活性水（OAW）含量从77％（以鲜重计）降低到17％。吸热融化表明结晶事件伴随组织的冷冻保存。通过间接器官发生获得多芽的增殖。在冷冻保存后的恢复过程中对外植体的组织学分析证实，在黑暗中半固态繁殖培养基上再培养3–4 mo后形成的愈伤组织的器官发生特性。随后在含激动素（2 mg L 在冷冻保存后的恢复过程中对外植体的组织学分析证实，在黑暗中半固态繁殖培养基上再培养3–4 mo后形成的愈伤组织的器官发生特性。随后在含激动素（2 mg L 在冷冻保存后的恢复过程中对外植体的组织学分析证实，在黑暗中半固态繁殖培养基上再培养3–4 mo后形成的愈伤组织的器官发生特性。随后在含激动素（2 mg L^-1）并暴露于光周期。目前，这是最优化的平叶葡萄球茎冷冻保存方案。