Occurrence of Latent Bacteria during Cryopreservation of Long-Term In Vitro Cultures of Coltsfoot, Tussilago farfara.

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.

Micropropagation and Cryoconservation of the Endangered Plant Species Artemisia laciniata (Asteraceae).

BACKGROUND: Artemisia laciniata, mainly distributed in Siberia and Central Asia, is classified as critically endangered in Europe. OBJECTIVES: This study developed a protocol for its micropropagation and cryopreservation. MATERIALS AND METHODS: In vitro cultures from fresh seed and in vivo shoots were initiated. Micropropagation and cryopreservation protocols were developed. Bacteria detected after cryopreservation were investigated using 16S rRNA analysis. Genome size measurements of regenerated plants after cryopreservation using flow cytometry and carbon isotope measurements to evaluate stress status were also carried out. RESULTS: A. laciniata from both starting materials could be successfully propagated on MS medium with 0.5 uM BAP. Material initiated from in vivo shoots yielded lower regeneration percentages (16%) after cryopreservation than material generated from seed (57 and 63%) using the droplet-vitrification method and PVS3. Bacteria occurring after cryopreservation belonged to the genera Sphingomonas, Staphylococcus, Curtobacterium and Gordonia. There was no significant difference in the genome size and stress status between non-cryopreserved and cryopreserved plants. CONCLUSION: A. laciniata could be readily micropropagated and cryopreserved. No negative effects of cryopreservation on plant water use efficiency or on genetic stability were found.

An Improved Cryopreservation Protocol for Mentha spp. Based on Pvs3 as the Cryoprotectant.

BACKGROUND: Vitrification approaches are widely used to cryopreserve Mentha spp. genetic resources. OBJECTIVE: Here, we compared the response of 20 different Mentha species and hybrids during cryopreservation and elucidated the efficacy of two cryoprotectants. MATERIALS AND METHODS: One hundred and fifty three Mentha spp. accessions were cryopreserved using in vitro plants maintained under slow-growth storage and PVS2 or PVS3 as cryoprotectants. RESULTS: The cryoprotectant PVS2 was effective for all species, except M. requienii and M. villosanervata. The use of PVS3 increased the proportion of explants able to regrow after rewarming. The outbreak of endophytes upon rewarming was both less frequent and less severe when PVS3 replaced PVS2. CONCLUSION: Both PVS2 and PVS3 can be used as cryoprotectant for all the species and accessions of Mentha spp. surveyed. Since higher regenerations were achieved using PVS3, and since the risk of an endophyte outbreak was reduced, this cryoprotectant should be preferred in future for cryopreserving Mentha spp.