Cryopreservation of highbush blueberry, strawberry, and saskatoon using V and D cryo-plate methods and monitoring of multiplication ability of regenerated shoots

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS ACS Applied Bio Materials Pub Date : 2024-01-16 DOI:10.1007/s11627-023-10399-5

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Abstract

The purpose of this work was to apply V and D cryo-plate methods for cryopreservation of Vaccinium corymbosum ʻToroʼ, Fragaria × ananassa ʻCleryʼ, and Amelanchier alnifolia (Nutt.) M. Roem. and to monitor the multiplication capacity of shoots regenerated from cryopreserved explants. Shoot tips pre-cultured for 1 d at 23°C in the dark on medium containing 0.3 M sucrose were used as explants. Loading was performed in a solution containing 2 M glycerol and 0.8 M sucrose (30 min at room temperature). In the V cryo-plate, dehydration was carried out at room temperature (20 to 50 min) using the following plant vitrification solutions: original PVS2, 90% PVS2 solution, and PVS3. Regarding the D cryo-plate, dehydration was performed in closed glass containers over silica gel for 2, 2.5, or 3 h. In both protocols, rewarming was carried out in a 1.0 M sucrose solution (15 min at 25°C). Regenerated shoots were multiplied and multiplication parameters were monitored after the second subculture. Using the V cryo-plate method, the highest regrowth in highbush blueberry was obtained following 50-min treatment with all three VSs (61.7 to 80.9%). The D cryo-plate method was even more suitable with maximum regrowth of 89.4% achieved after 2.5 h of desiccation. For strawberry, 62.5% was the highest regrowth recorded using PVS3-based V cryo-plate method while 83.3% of regrowth was observed using D cryo-plate protocol. Regrowth of saskatoon reached a maximum of 50% after 50-min treatment with PVS3 while it did not exceed 40% in other treatments. By the second subculture, shoots regenerated from cryopreserved explants regained and even exceeded the multiplication capacity of shoots regenerated from non-cryopreserved explants. This study is the first to present the successful application of the V cryo-plate method in highbush blueberry, as well as the utilization of both V and D cryo-plate methods in saskatoon.

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使用 V 和 D 低温板方法低温保存高丛蓝莓、草莓和沙棘果，并监测再生嫩枝的繁殖能力

摘要本研究的目的是应用V型和D型低温板法低温保存Vaccinium corymbosum ʻToroʼ、Fragaria × ananassa ʻCleryʼ和Amelanchier alnifolia (Nutt.) M. Roem.，并监测低温保存的外植体再生芽的繁殖能力。将在 23°C 黑暗条件下在含 0.3 M 蔗糖的培养基上预培养 1 d 的嫩枝尖端作为外植体。装载在含有 2 M 甘油和 0.8 M 蔗糖的溶液中进行（室温下 30 分钟）。在 V 型低温板中，使用以下植物玻璃化溶液在室温下进行脱水（20 至 50 分钟）：原始 PVS2、90% PVS2 溶液和 PVS3。对于 D 型低温板，脱水是在硅胶上的密闭玻璃容器中进行的，时间为 2、2.5 或 3 小时。在第二次亚培养后，对再生芽进行繁殖并监测繁殖参数。采用 V 型低温板法，三种 VS 处理 50 分钟后，高丛蓝莓的再生率最高（61.7%-80.9%）。D 型低温板法更为合适，在干燥 2.5 小时后，再生率最高，达到 89.4%。对于草莓，使用基于 PVS3 的 V 型低温板方法记录到的最高再生率为 62.5%，而使用 D 型低温板方案则观察到 83.3%的再生率。用 PVS3 处理 50 分钟后，沙棘的再生率最高达到 50%，而其他处理的再生率均不超过 40%。第二次亚培养时，从低温保存的外植体上再生的嫩枝恢复甚至超过了从非低温保存的外植体上再生的嫩枝的繁殖能力。本研究首次在高丛蓝莓中成功应用了 V 型冷冻板法，并在沙士基诺中同时应用了 V 型和 D 型冷冻板法。

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