Cryopreservation of the Norway spruce tissue culture line able to produce extracellular lignin
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A cryopreservation method was developed for a Norway spruce (Picea abies L. Karst.) cell line characterised by highly vacuolated cells and ability to produce natural-like extracellular lignin in a cell suspension culture. Spruce callus cultured in a photoperiod of 16 h light, 8 h dark contained two types of callus morphologies. Soft callus was composed of loosely bound cells that dispersed into single cells and small cell aggregates when transferred into liquid medium. The callus with hard morphology had also cells that were more tightly attached to each other; this callus formed bigger cell aggregates in liquid medium in addition to single cells and small cell aggregates. The hard callus contained higher concentration of intracellular phenolic compounds as compared to soft callus. For cryopreservation, a vitrification method with plant vitrification solution 2 (PVS2) was used. To reduce cellular water content, spruce calli were pre-cultured on a culture medium with increasing sucrose concentration (0.2 and 0.4 M; one day on each). The cryopreservation survival rate of callus with hard morphology was significantly higher than that with soft morphology (45 ± 8% and 5 ± 5%, respectively). Pre-culturing in continuous light for several weeks led exclusively to formation of a hard-type callus, which had a survival rate of 48 ± 16% in cryopreservation. Expression of candidate genes of the monolignol biosynthesis pathway, Fourier transform infrared spectra and pyrolysis breakdown products of extracellular lignin were similar in control cultures and those originating from cryopreserved cells suggesting that cryopreservation is a feasible method for long-term storage of the lignin-forming cell line.
KeywordsCryopreservation Lignin formation Norway spruce cell culture Picea abies Vitrification
Cinnamyl alcohol dehydrogenase
Fourier transform infrared spectroscopy
Milled wood lignin
Plant vitrification solution 2
Pyrolysis gas chromatography mass spectrometry
We thank Ms Kaija Porkka for the initial characterization of the hard and soft calli. We thank funding from the Academy of Finland (decisions #251390, #256174 and #283245 to A.K.) and the Natural History Society of Oulu (Grant to S.V.). Te. L. thanks the Finnish Cultural Foundation, Häme Regional Fund for funding. Green chemistry from forest—Natural Resources Institute Finland self-funded project is also acknowledged.
AK, HH, JE, SJ-L and PS conceived and designed the experiments. SV, ED, JT, JE, KN, TeL, TaL, and AK performed the research. RK performed pyrolysis and data analysis. SV, ED, SJ-L, TaL, RK, PS, AK, and HH wrote the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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