Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Terezia SALAJ; Bart PANIS; Katarina KLUBICOVA; Jan SALAJ

doi:10.15835/nbha50312770

Authors

Terezia SALAJ Plant Science and Biodiversity Center SAS, Institute of Plant Genetics and Biotechnology, Akademicka 2, 950 07 Nitra (SK) https://orcid.org/0000-0002-8649-8246
Bart PANIS Alliance of Biodiversity International and International and CIAT, c/o KU Leuven, Willem de Croylaan 42, 3001 Leuven; Department of Biosystems, KU Leuven, Willem de Croylaan 42, 3001 Leuven (BE) https://orcid.org/0000-0001-6717-947X
Katarina KLUBICOVA Plant Science and Biodiversity Center SAS, Institute of Plant Genetics and Biotechnology, Akademicka 2, 950 07 Nitra (SK) https://orcid.org/0000-0003-0927-0253
Jan SALAJ Plant Science and Biodiversity Center SAS, Institute of Plant Genetics and Biotechnology, Akademicka 2, 950 07 Nitra (SK) https://orcid.org/0000-0003-0249-3657

DOI:

https://doi.org/10.15835/nbha50312770

Keywords:

conifers, cryo-tolerance, regeneration, silver fir, somatic embryogenesis

Abstract

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min^-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.

References

Ahn CH, Heo K, Partk HS, Choi YE (2019). In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis. In Vitro Cellular and Developmental Biology - Plant 55:605-614. https://doi.org/10.1007/s11627-019-09989-z

Alvarez JM, Cortizo M, Ordas RJ (2012). Cryopreservation of somatic embryogenic cultures of Pinus pinaster. CryoLetters 33(6):476-484.

Aronen TS, Krajnakova J, Häggman H, Ryynänen LA (1999). Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica. Plant Science 142:163-172. https://doi.org/10.1016/S0168-9452(98)00244-1

Bradai F, Sanches-Romero C (2021). Effect of cryopreservation on the ex vitro establishment of olive plants regenerated via somatic embryogenesis. Plants 10(2):396. https://doi.org/10.3390/plants 10020396

Carneros E, Hernandez I, Toribio M, Diaz-Sala C (2017). Effect of different cryoprotectant procedures on the recovery and saturation ability of cryopreserved Pinus pinea embryogenic cell lines. In Vitro Cellular and Developmental Biology - Plant 53:469-477. https://doi.org/10.1007/s11627-017-9833-6

Cyr DR, Lazaroff WR, Grimes MAS, Quan G, Bethune TD, Dunstan D, Roberts D (1994). Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures. Plant Cell Reports 13:574-577. https://doi.org/10.1007/BF00234514

Demarchi G, Stefenon VM, Steiner N, Vieira FN, Dal Vesco L, Guerra MP (2014). Ultra-low temperature conservation of Brazilian pine embryogenic cultures. Anais da Academia Brasileira de Ciencias 86:2057-2063. http://dx.doi.org/10.1590/0001-3765201420130405

Chalupa V (1985). Somatic embryogenesis and plant regeneration from cultured immature and mature embryos of Picea abies (L.) Karst. Communicationes Instituti Forestalis Czechosloveniae 14:57-63.

Engelmann F (2004). Plant cryopreservation: Progress and prospects. In Vitro Cellular and Developmental Biology - Plant 40:427-433. https://doi.org/10.1079/IVP2004541

Feher A (2015). Somatic embryogenesis - Stress-induced remodeling of plant cell fate. Biochimica et Biophysica Acta - Gene Regulatory Mechanism 1849(4):385-402. https://doi.org/10.1016/j.bbagrm.2014.07.005

Find JI, Floto F, Krogstrup P, Moller JD, Norgaard JV, Kristensen MH (1993). Cryopreservation of embryogenic suspension culture of Picea sitchensis and subsequent plant regeneration. Scandinavian Journal of Forest Research 8:156-162. https://doi.org/10.1080/02827589309382765

Find JI, Norgaard JV, Krogstrup P (1998). Growth parameters, nutrient uptake and maturation capacity of two cell lines of Norway spruce (Picea abies) in suspension culture. Journal of Plant Physiology 152:510-517. https://doi.org/10.1016/S0176-1617(98)80271-X

Ford CS, Jones NB, van Staden J (2000). Cryopreservation and plant regeneration from somatic embryos of Pinus patula. Plant Cell Reports 19:610-615. https://doi.org/10.1007/s002990050781

Fraga HPF, Vieira LN, Puttkammer C, da Silva JM, dos Anjos KG, Oliveira EM, Guerra MP (2016). High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features. Plant Cell, Tissue and Organ Culture 124:307-318. https://doi.org/10.1007/s11240-015-0895-z

Gale S, John A, Benson E (2007). Cryopreservation of Picea sitchensis (Sitka spruce) embryogenic suspensor masses. CryoLetters 28:225-239.

Gupta PK, Durzan DJ (1985). Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Reports 4:177-179. https://doi.org/10.1007/BF00269282

Gupta PK, Durzan DJ, Finkle BJ (1987). Somatic polyembryogenesis in embryogeneic cell masses of Picea abies (Norway spruce) and Pinus taeda (loblolly pine) after thawing from liquid nitrogen. Canadian Journal of Forest Research 17:1130-1133. https://doi.org/10.1139/x87-172

Guzman Garcia E, Bradai F, Sanchez-Romero C (2013). Cryopreservation of avocado embryogenic cultures using the droplet vitrification. Acta Physiologiae Plantarum 35:183-193. https://doi.org/10.1007/s11738-012-1062-3

Hakman I, Fowke LC, von Arnold S, Eriksson T (1985). The development of somatic embryos in tissue cultures initiated from immature embryos of Picea abies (Norway spruce). Plant Science 38:53-59. https://doi.org/10.1016/0168-9452(85)90079-2

Hargreaves CI, Grace LJ, Holden DG (2002). Nurse culture for efficient recovery of cryopreserved Pinus radiata D. Don. embryogenic cell lines. Plant Cell Reports 21:40-45. https://doi.org/10.1007/s00299-002-0478-4

Hazubska-Przybyl T, Chmielarz P, Michalak M, Dering M, Bojarczuk K (2013). Survival and genetic stability of Picea abies embryogenic cultures after cryopreservation using pregrowth-dehydration method. Plant Cell, Tissue and Organ Culture 113:303-313. https://doi.org/10.1007/s11240-012-0270-2

Häggman H, Ryynänen LA, Aronen TS, Krajnakova J (1998). Cryopreservation of embryogenic cultures of Scots pine. Plant Cell, Tissue and Organ Culture 54:45-53. https://doi.org/10.1023/A:1006104325426

Klimaszewska K (1995). Somatic embryogenesis in Picea mariana (Mill.). In: Jain SM, Gupta PK, Newton RJ (Eds). Somatic Embryogenesis in Woody Plants. Gymnosperms Vol. 3. Kluwer Academic Publishers, Dordrecht pp 67-79.

Klimaszewska K, Hargreaves C, Lelu-Walter MA, Trontin JF (2016). Advances in conifer somatic embryogenesis since year 2000. In: Germana MA, Lambardi M (Eds). In vitro Embryogenesis in Higher Plants. Methods in Molecular Biology 1359. Springer Science+Business Media, New York pp 131-166.

Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-Walter MA (2009). Biological characterisation of young and aged embryogenic cultures of Pinus pinaster (Ait). In Vitro Cellular and Developmental Biology - Plant 45:20-33.

Krajňáková J, Sutela S, Aronen T, Gömöry D, Vianello A, Häggman H (2011). Long-term cryopreservation of Greek fir embryogenic cell lines: Recovery, maturation and genetic fidelity. Cryobiology 63:17-25. https://doi.org/10.1016/j.cryobiol.2011.04.004

Krajňáková J, Bertolini A, Gömöry D, Vianello A, Häggman H (2013). Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell line. In Vitro Cellular and Developmental Biology - Plant 49:560-571. https://www.jstor.org/stable/42568815

Laine E, Bade P, David A (1992). Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea. Plant Cell Reports 11:295-298. https://doi.org/10.1007/BF00235085

Lambardi M, De Carlo A, Capuana M (2005). Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification or one-step freezing. CryoLetters 26:185-192.

Latutrie M, Aronen T (2013). Long-term cyropreservation of embryogenic Pinus sylvestris cultures. Scandinavian Journal of Forest Research 28:103-109. http://dx.doi.org/10.1080/02827581.2012.701325

Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2006). Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster. Plant Cell Reports 25:767-776. https://doi.org/10.1007/s00299-006-0115-8

Lelu-Walter MA, Thompson D, Harvengt L, Sanchez L, Toribio M, Paques L (2013). Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genetics and Genomes 9:883-899. https://doi.org/10.1007/s11295-013-0620-1

Li WF, Zhang SH, Han SY, Wu T, Zhang JH, Qi LW (2013). Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr. Plant Cell, Tissue and Organ Culture 113:131-136. https://doi.org/10.1007/s11240-012-0233-7

Lineros Y, Balocchi C, Munoz X, Sanchez C, Rios D (2018). Cryopreservation of Pinus radiata embryogenic tissue: effects of cryoprotective pretreatments on maturation ability. Plant Cell, Tissue and Organ Culture 135:357-366. https://doi.org/10.1007/s11240-018-1469-7

Ma X, Bucalo K, Determann RO, Cruse-Sanders JM, Pullman GS (2012). Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species. In Vitro Cellular and Developmental Biology - Plant 48:324-334. https://doi.org/10.1007/s11627-012-9433-4

Martin C, Kremer C, Gonzalez I, Gonzalez-Benito ME (2015). Influence of the cryopreservation technique, recovery medium and genotype on genetic stability of mint cryopreserved shoot tips. Plant Cell, Tissue and Organ Culture 122:185-195. https://doi.org/10.1007/s11240-015-0760-0

Marum L, Estevao C, Oliveira M, Amancio S, Rodrigues L, Miguel C (2004). Recovery of cryopreserved embryogenic cultures of maritime pine – effect of cryoprotectant and suspension density. CryoLetters 25:363-374.

Mathur G, Alkutkar VA, Nadgauda RS (2003). Cryopreservation of embryogenic culture of Pinus roxburghii. Biologia Plantarum 46:205-210. https://doi.org/10.1023/A:1022894409408

Merkle SA, Montello PM, Reece HM, Kong LS (2014). Somatic embryogenesis and cryostorage of eastern hemlok and Carolina hemlok for conservation and restoration. Trees - Structure and Function 28:1767-1776. https://doi.org/10.1007/s00468-014-1084-0

Misson JP, Druart P, Panis B, Watillon B (2006). Contribution to the study of the maintenance of somatic embryos of Abies nordmanniana LK: culture media and cryopreservation method. Propagation of Ornamental Plants 6:17-23.

Nörgaard JV, Baldursson S, Krogstrup P (1993a). Genotypic differences in ability of embryogenic Abies nordmanniana cultures to survive cryopreservation. Silvae Genetica 42:93-97.

Nörgaard JV, Duran V, Johnsen O, Krogstrup P, Baldursson S, von Arnold S (1993b). Variation in cryotolerance of embryogenic Picea abies cell lines and the association to genetic, morphological, and physiological factors. Canadian Journal of Forest Research 23:2560-2567. https://doi.org/10.1139/x93-317

Nunez S, Marum L, Farinha N, Tolentino Pereira V, Almeida T, Celeste Diaz M, Santos C (2017). Plant regeneration from ploidy-stable cryopreserved embryogenic lines of the hybrid Pinus elliottii x P. caribaea. Industrial Crops and Products 105:215-224. https://doi.org/10.1016/j.indcrop.2017.05.015

O'Brien C, Hiti-Bandaralage J, Folgado R, Hayward A, Lahmeyer S, Folsom J, Mitter N (2021). Cryopreservation of woody crops: the avocado case. Plants 10(5):934. https://doi.org/10.3390/plants10050934

Panis B, Lambardi M (2005). Status of cryopreservation technologies in plants (Crops and Forest Trees). In: International Workshop on: "The role of biotechnology for the characterisation and conservation of crop, forestry, animal and fishery genetic resources." 2005, Turin, Italy pp 43-53.

Pence VC, Chaiken MF (2021). Shoot tip cryopreservation as a conservation tool for species of Quercus: effects of species and environment on recovery. CryoLetters 42:159-167.

Perez RM, Navarro L, Duran-Vila N (1997). Cryopreservation and storage of embryogenic callus cultures of several citrus species and cultivars. Plant Cell Reports 17:44-49. https://doi.org/10.1007/s002990050349

Pullman GS, Olson K, Fischer T, Egertsdotter U, Frampton J, Bucalo K (2016). Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation. New Forests 47:453–480. https://doi.org/10.1007/s11056-016-9525-9

Reinhoud PJ, van Iren F, Kijne JW (2000). Cryopreservation of undifferentiated plant cells. In: Engelmann F, Takagi H (Eds). Cryopreservation of Tropical Plant Germplasm: Current Research Progress and Application. IPGRI, Roma, Italy pp 91-102.

Salaj T, Matusova R, Salaj J (2015). Conifer somatic embryogenesis - an efficient plant regeneration system for theoretical studies and mass propagation. Dendrobiology 74:67-74. http://dx.doi.org/10.12657/denbio.074.007

Salaj T, Matusikova I, Panis B, Swennen R, Salaj J (2010). Recovery and characterisation of hybrid firs (Abies alba x A. cephalonica, Abies alba x A. numidica) embryogenic tissues after cryopreservation. CryoLetters 31:206-217.

Salaj T, Matusikova I, Swennen R, Panis B, Salaj J (2012). Long-term maintenance of Pinus nigra embryogenic cultures through cryopreservation. Acta Physiologiae Plantarum 34:227-233. https://doi.org/10.1007/s11738-011-0821-x

Salaj T, Klubicová K, Panis B, Swennen R, Salaj J (2020). Physiological and structural aspects of in vitro somatic embryogenesis in Abies alba Mill. Forests 11:1-15. https://doi.org/10.3390/f11111210

Salaj T, Matusova R, Panis B, Swennen R, Salaj J (2016). Tissue regeneration of Abies embryogenic cell lines after 1 year storage in liquid nitrogen. Biologia 71:93-99. https://doi.org/10.1515/biolog-2016-0004

Salaj T, Panis B, Swennen R, Salaj J (2007). Cryopreservation of embryogenic tissues of Pinus nigra Arn. by a slow freezing method. CryoLetters 28:69-76.

San Jose CdM, Corredoira E, Oliveira H, Santos C (2015). Cryopreservation of somatic embryos of Alnus glutinosa (L.) Gaertn. and confirmation of ploidy stability by flow cytometry. Plant Cell, Tissue and Organ Culture 123:489-499. https://doi.org/10.1007/s11240-015-0853-9

Sant R, Panis B, Taylor M, Tyagi A (2008). Cryopreservation of shoot tips by droplet vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions. Plant Cell, Tissue and Organ Culture 92:107-111. https://doi.org/10.1007/s11240-007-9302-8

Škrlep K, Bergant M, de Winter GM, Bohanec B, Žel J, Verpoorte R, van Iren F, Camloh M (2008). Cryopreservation of cell suspension cultures of Taxus x media and Taxus floridana. Biologia Plantarum 52:329-333. https://doi.org/10.1007/s10535-008-0067-7

Touchell DH, Chiang VL, Tsai CJ (2002). Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification. Plant Cell Reports 21:118-124. https://doi.org/10.1007/s00299-002-0490-8

von Arnold S, Hakman I (1988). Regulation of somatic embryo development in Picea abies by abscisic acid. Journal of Plant Physiology 132:164-169. https://doi.org/10.1016/S0176-1617(88)80155-X

Turner S, Senaratna T, Touchell D, Bunn E, Dixon K, Tan B (2001). Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Science 160:489-497. https://doi.org/10.1016/s0168-9452(00)00420-9

Varis S, Ahola S, Jaakola L, Aronen T (2017). Reliable and practical methods for cryopreservation of embryogenic cultures and cold storage of somatic embryos of Norway spruce. Cryobiology 76:8-17. https://doi.org/10.1016/j.cryobiol.2017.05.004

Volk GM, Caspersen AM (2007). Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha piperita. Protoplasma 231:215-226. https://doi.org/10.1007/s00709-007-0251-1

Vondrakova Z, Cvikrova M, Eliasova K, Martincova O, Vagner M (2010). Cryotolerance in Norway spruce and its association with growth rates, anatomical features and polyamines of embryogenic cultures. Tree Physiology 30:1335-1348. https://doi.org/10.1093/treephys/tpq074