Minimizing the deleterious effects of endophytes in plant shoot tip cryopreservation

Authors: Henk, Adam; Bonnart, Remi; ARAUJO DE OLIVEIRA, AC - Universidade Federal De Sergipe; Volk, Gayle

Submitted to: Applications in Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2022
Publication Date: 8/26/2022
Citation: Henk, A.D., Bonnart, R.M., Araujo De Oliveira, A., Volk, G.M. 2022. Minimizing the deleterious effects of endophytes in plant shoot tip cryopreservation. Applications in Plant Sciences. Article e11489. https://doi.org/10.1002/aps3.11489.
DOI: https://doi.org/10.1002/aps3.11489

Interpretive Summary: Plant genebanks use cryopreservation technologies to preserve crop collections that can't be stored as seeds. Often, plant materials must be introduced from the field or greenhouse into tissue culture before they can be cryopreserved. This introduction is a critical and possibly challenging step. Microbial contamination can be minimized by effective surface sterilization procedures. In some cases, endophytes within plant tissues prevent successful tissue culture initiation and subsequent cryopreservation. This manuscript describes methods that have been used to control microbial contamination in plant tissue cultures, including a review of methods for the possible elimination of endophytes. In addition, the process of isolating and then sequencing two endophytes encountered during Citrus shoot tip cryopreservation is described.

Technical Abstract: Plant cryopreservation technologies are used within genebanks for the long-term preservation of vegetatively propagated fruit, vegetable, and nut collections. Surface sterilized plant tissues from the field or greenhouse/screenhouse or in vitro-grown plants are the source of shoot tips for vitrification-based cryopreservation methods. We describe methods 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 durations and during recovery after liquid nitrogen exposure. We describe two case studies whereby bacterial endophytes were observed in Citrus genebank accessions during recovery after cryopreservation. These were identified using the MinION Oxford Nanopore system and Kirby-Bauer disc diffusion assays revealed bacterial responses after antibiotic exposure. Although endophytes can hinder plant tissue culture and cryopreservation efforts, there may be future beneficial relationships that improve in vitro plant growth and development.