The use of cryopreservation techniques for plant species confined to wet habitats is difficult to standardize due to low tolerance for dehydration of plant tissue. The study aimed to develop a procedure for the long-term storage in liquid nitrogen (LN) of Viola stagnina, a rare and endangered species of moist habitats whose European range is constantly decreasing. Adventitious shoot apices of different sizes (small, medium and large) were cryopreserved using encapsulation/dehydration, encapsulation/vitrification (in PVS2 or PVS3 solutions) and droplet/vitrification methods. Encapsulation/dehydration was the most efficient technique, and 47.7 % regrowth was obtained with an average rate of shoot regeneration of 2.4 %. Less than 27.6 % of cryopreserved shoot apices were able to produce shoots with an efficiency of 0.35 or less based on vitrification procedures. In post-thaw recovery culture, adventitious shoots were produced indirectly (via callus). The genetic variation among recovered shoots after different cryopreservation methods was low (Dice coefficient not exceeded 0.09). Some clones were genetically uniform with the mother plant estimated by ISSR molecular markers. The genome size of all post-thaw recovered regenerants, except one individual with a multiplicated genome, was uniform with the initial plant.

This paper presents the first report on cryopreservation of V. stagnina with a developed protocol that can be used in ex situ conservation of the species.

由于对植物组织脱水的耐受性低，因此很难将冷冻保存技术用于局限在潮湿生境中的植物物种。该研究旨在开发一种用于中提琴中毒液氮（LN）的长期保存程序。是一种稀有且濒临灭绝的潮湿栖息地，其欧洲范围不断缩小。使用封装/脱水，封装/玻璃化（在PVS2或PVS3溶液中）和液滴/玻璃化方法将不同大小（小，中和大）的不定芽茎尖冷冻保存。包封/脱水是最有效的技术，再生长为47.7％，平均芽再生率为2.4％。根据玻璃化程序，少于27.6％的低温保存的芽尖能够产生0.35或更小的效率的芽。在解冻后的恢复文化中，不定芽是间接产生的（通过愈伤组织）。冷冻保存方法不同，恢复后的新芽之间的遗传变异较低（骰子系数不超过0.09）。通过ISSR分子标记估计，某些克隆与母本在遗传上是一致的。除一个具有多重基因组的个体外，所有解冻后回收的再生子的基因组大小与初始植物一致。

本文介绍了关于V. stagnina冷冻保存的第一份报告，该报告采用了可用于异地保存该物种的开发方案。