Volk Gayle M, Bonnart Remi, de Oliveira Annie Carolina Araújo, Henk Adam D
USDA-ARS National Laboratory for Genetic Resources Preservation 1111 S. Mason St. Fort Collins Colorado 80521 USA.
Federal University of Sergipe Marechal Rondon Ave. São Cristóvão Sergipe 49100-000 Brazil.
Appl Plant Sci. 2022 Aug 26;10(5):e11489. doi: 10.1002/aps3.11489. eCollection 2022 Sep-Oct.
Plant cryopreservation technologies are used within gene banks for the long-term preservation of vegetatively propagated collections. Surface-sterilized plant tissues grown in the field, greenhouse/screenhouse, growth chamber, or in vitro are the source of shoot tips subjected to vitrification-based cryopreservation methods. Here, we describe the methods used to minimize microbial contamination during the tissue culture initiation process. We also discuss the occurrence and possible elimination of endophytes after extended in vitro culture and during recovery after liquid nitrogen exposure. We describe two case studies in which bacterial endophytes were observed in gene bank accessions during recovery after cryopreservation. These were identified using the MinION Oxford Nanopore system and Kirby-Bauer disc diffusion assays to examine the bacterial responses to antibiotic exposure. The methods used in this case study could be applied to identify endophytes to better target antimicrobial treatments of plant tissue collections.
植物冷冻保存技术用于基因库中营养繁殖种质资源的长期保存。在田间、温室/防虫网室、生长室或体外培养的经表面灭菌的植物组织是基于玻璃化冷冻保存方法处理的茎尖的来源。在这里，我们描述了在组织培养起始过程中用于最小化微生物污染的方法。我们还讨论了在体外长期培养后以及液氮暴露后的恢复过程中内生菌的出现及可能的消除方法。我们描述了两个案例研究，其中在冷冻保存后的恢复过程中，在基因库种质中观察到了细菌内生菌。使用MinION牛津纳米孔系统和 Kirby-Bauer 纸片扩散试验对这些细菌进行了鉴定，以检测细菌对抗生素暴露的反应。本案例研究中使用的方法可应用于鉴定内生菌，以便更好地针对植物组织种质进行抗菌处理。
