We assessed a new cryopreservation protocol that uses vermiculite as a culture substrate, called the vermiculite protocol (VP), by assessing the viability, recovery time of hyphae after revival, and colony diameter of cryosensitive ectomycorrhizal basidiomycete strains after storage for 2 weeks or 1 year in a vapour-phase liquid nitrogen tank. Twelve difficult-to-preserve strains of nine species (Amanita citrina, A. pantherina, A. rubescens, A. spissa, Kobayasia nipponica, Lactarius akahatsu, L. hatsudake, Sarcodon aspratus, and Tricholoma flavovirens) that did not achieve good revival after cryopreservation with our previous Homolka's perlite protocol and modified perlite protocol (MPP) experiments were used to assess the new methodology. Vermiculite and liquid medium were put into a cryotube and inoculated with an agar plug containing mycelia. The cryotube was cultured for various incubation times. After adequate mycelial growth, a mixture of cryoprotectants (5% dimethyl sulfoxide and 10% trehalose [5D10T] or 5% glycerol and 10% trehalose [5G10T]) was placed into the cryotube. The cryotube was frozen in a freezing container in a -80 °C freezer and then stored in vapour-phase liquid nitrogen. In the recovery test, 10 of 12 strains showed 100% revival after 2 weeks of storage in the 5G10T cryoprotectant, and all 12 strains showed 100% revival after 2 weeks of storage in the 5D10T cryoprotectant. Furthermore, all strains were viable after 1 year of storage in a vapour-phase liquid nitrogen tank. Thus, the VP is applicable to a wide range of ectomycorrhizal basidiomycete cultures, including highly cryosensitive strains.

中文翻译：

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蛭石作为培养基质大大提高了外生菌根担子菌冷冻培养物的生存能力

我们评估了一种使用蛭石作为培养基质的新冷冻保存方案，称为蛭石方案 (VP)，通过评估冷冻敏感外生菌根担子菌菌株在储存 2 周或 1 年后的活力、复苏后菌丝的恢复时间和菌落直径在气相液氮罐中。九个物种（鹅膏菌、A. pantherina、A. rubescens、A. spissa、Kobayasia nipponica、Lactarius akahatsu、L. hatsudake、Sarcodon aspratus和Tricholoma flavovirens）的12个难以保存的菌株使用我们之前的 Homolka 珍珠岩协议和改良珍珠岩协议 (MPP) 实验进行的冷冻保存来评估新方法。将蛭石和液体培养基放入冷冻管中并用含有菌丝体的琼脂塞接种。将冷冻管培养不同的孵育时间。在菌丝体充分生长后，将冷冻保护剂（5% 二​​甲亚砜和 10% 海藻糖 [5D10T] 或 5% 甘油和 10% 海藻糖 [5G10T]）的混合物放入冷冻管中。将冷冻管冷冻在-80°C 冰箱中的冷冻容器中，然后储存在气相液氮中。在恢复试验中，12 株中有 10 株在 5G10T 冷冻保护剂中储存 2 周后显示出 100% 恢复，所有 12 株在 5D10T 冷冻保护剂中储存 2 周后均显示出 100% 恢复。此外，所有菌株在汽相液氮罐中储存 1 年后仍可存活。因此，VP 适用于广泛的外生菌根担子菌培养物，