Exploring the role of endophytes for improving cryopreservation of potato

The potato ranks as the fourth most important food crop. Thus, it is essential to maintain the heterozygous genotypes in vitro to preserve its genetic diversity. In vitro culturing includes surface sterilization to remove external bacteria and fungi, followed by propagation under sterile, sugar-rich conditions. However, this approach is labor-intensive as the plants require periodic transfer to fresh growth medium. Therefore, cryopreservation approaches have been established and implemented in genebanks for long-term storage to preserve vegetatively propagated collections. For this, meristematic tissues, i.e. apical or lateral shoot tips, undergo tissue dissection, osmotic adaptation, cryoprotection, cryogenic treatment using liquid nitrogen to halt all biochemical activity and preserving genetic integrity, and controlled rewarming. However, rewarming can trigger the colonization of endophytes around the explant, which can compromise its ability to develop into a plant.
In this project, we analyzed 382 potato accessions for their regrowth potential post-cryopreservation. While 50% showed a good regrowth (≥60% of plants regrown), 39% recovered poorly (≤60% of plants regrown) and 11% failed to recover. To investigate microbial influences, an ITS and 16S rRNA gene amplicon survey was conducted on all these accessions. As anticipated, the microbial diversity was in general low due to sterile cultivation conditions. A total of 637 bacterial amplicon sequencing variants (ASVs) spanning 53 different orders were identified. While some ASVs might originate from exogenous sources, others belong to known plant-associated groups, including Bacillales. In addition, bacteria were isolated from 19 potato accessions resulting in 52 bacterial isolates, belonging to 19 different strains based on 16S rRNA gene sequence comparison. The isolates were characterized for their potential in producing IAA, siderophores and osmolytes, as well as in ACC deamination, and phosphate solubilization. This characterization guided the selection of strains isolated from both well and poorly recovering accessions to test their impact on in vitro plant performance and to analyze their genomes. In conclusion, this study provides first insights in the microbial communities associated with potato cryopreservation and their functional traits, representing a crucial step towards enhancing regrowth success and refining long-term preservation strategies in genebanks.