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Occurrence of Latent Bacteria During Cryopreservation of Long-term in Vitro Cultures of Coltsfoot, Tussilago Farfara

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BACKGROUND: Long-term in vitro cultures of Tussilago farfara (L.), a traditional medicinal plant in Austria, had been stored at 14 °C for over 20 years. The cultures were vigorous and showed no visual signs of bacterial presence. The transfer from growth regulator-free culture medium to medium containing kinetin and the increase of temperature from 14 °C to 25 °C for fast propagation led to the emergence of latent bacteria in all twelve accessions studied. OBJECTIVE: To investigate latent infections occurring during the development of a cryopreservation protocol of genetically interesting material using droplet-vitrification. MATERIALS AND METHODS: Two protocols for droplet-vitrification were tested using plant vitrification solutions (PVS) 2 and 3. The bacteria were isolated and identified using 16S rDNA analysis. Next, non-cryopreserved in vitro plantlets were acclimatized and transferred to the glasshouse. After 6 weeks, shoot tips were harvested from the pot plants, surface-sterilized and initiated into culture. Further, newly acquired achenes of Tussilago were surface-sterilized and germinated in vitro and seedlings checked for bacteria. RESULTS: The bacteria from the long-term cultures were isolated and identified as Luteibacter. Regeneration after cryopreservation using PVS3 was successful despite the continuing presence of Luteibacter. Luteibacter could no longer be detected in the newly-initiated in vitro material in subsequent tests and it was also not detected in the seedlings. CONCLUSION: Luteibacter withstood the cryopreservation procedure. Re-initiation of infected material may be an efficient alternative to antibiotic treatment to manage bacteria in micropropagation systems.
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Keywords: ANTIBIOTICS; CONTAMINATION; ENDOPHYTIC-FREE CULTURES; INFECTION; SEQUENCING

Document Type: Research Article

Publication date: November 1, 2019

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  • CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation

    The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.

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