Somaclonal variability of conifers in culture in vitro

Cover Page
Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

Abstract


The use of somatic embryogenesis is one of the promising methods of conifer propagation on an industrial scale. However, this technology has a number of problems, which include the appearance of somaclonal variation in cell and tissue culture. The review considers the causes and methods for detecting somaclonal variability of conifer in culture in vitro. It is shown that it is necessary to use a complex of molecular, cytogenetic, morphological, physiological methods for the analysis of somaclonal changes in embryogenic plant cultures.


Full Text

Restricted Access

About the authors

Elena N. Gulyaeva

Karelian Research Centre of the Russian Academy of Sciences

Email: gln7408@gmail.com
SPIN-code: 4967-5430

Russian Federation, Petrozavodsk

Junior Research Associate, Laboratory for Plant Biotechnology, Department for Multidisciplinary Scientific Research

Roman V. Ignatenko

Karelian Research Centre of the Russian Academy of Sciences

Author for correspondence.
Email: ocean-9@mail.ru
SPIN-code: 8298-4063

Russian Federation, Petrozavodsk

PhD, Senior Researcher, Laboratory for Plant Biotechnology, Department for Multidisciplinary Scientific Research

Natalia A. Galibina

Karelian Research Centre of the Russian Academy of Sciences

Email: galibina@krc.karelia.ru
SPIN-code: 7262-5860

Russian Federation, Petrozavodsk

Doctor of Science, Main Researcher, Laboratory for Plant Biotechnology, Department for Multidisciplinary Scientific Research

References

  1. Aronen T, Pehkonen T, Ryynänen L. Enhancement of somatic embryogenesis from immature zygotic embryos of Pinus sylvestris. Scand J For Res. 2009;24(5):372-383. https://doi.org/10.1080/02827580903228862.
  2. Klimaszewska K, Cyr DR. Conifer somatic embryogenesis: I. Development. Dendrobiology. 2002;48:31-39.
  3. Третьякова И.Н., Барсукова А.С. Соматический эмбриогенез в культуре in vitro трех видов лиственницы // Онтогенез. – 2012. – Т. 43. – № 6. – С. 425. [Tretyakova IN, Barsukova AV. Somatic embryogenesis in in vitro culture of three larch species. Russian Journal of Developmental Biology. 2012;43(6):425. (In Russ.)]. https://doi.org/10.1134/s1062360412060082.
  4. Hakman I, Fowke LC, von Arnold S, et al. The development of somatic embryos in tissue cultures initiated from immature embryos of Pice aabies (Norway spruce). Plant Science. 1985;38(1):53-59. https://doi.org/10. 1016/0168-9452(85)90079-2.
  5. Nagmani R, Bonga JM. Embryogenesis in subcultured callus of Larix decidua. Can J For Res. 1985;15:1088-1091. https://doi.org/10.1139/x85-177.
  6. Celestino C, Carneros E, Ruiz-Galea M, et al. Cloning stone pine (Pinus pinea L.) by somatic embryogenesis. In: Mutke S, Piqué M and Calama R, eds. Options Méditerranéennes, A, no 105, Mediterranean Stone Pine for Agroforestry. International Centre for Advanced Mediterranean Agronomic Studies. Zaragoza Spain; 2013. Р. 89-96.
  7. Ворошилова Е.В. Индукция соматического эмбриогенеза в культуре in vitro у гибридных семян Pinus sibirica Du Tour: Дис. … канд. биол. наук. – Красноярск, 2013. – 138 с. [Voroshilova EV. Induktsiya somaticheskogo embriogeneza v kul’ture in vitro u gibridnykh semyan Pinus sibirica Du Tour. [dissertation] Krasnoyarsk; 2013. 138 p. (In Russ.)]
  8. Mahdavi-Darvari F, Noor NM, Ismanizan I. Epigenetic regulation and gene markers as signals of early somatic embryogenesis. Plant Cell Tissue Organ Cult. 2015;120:407-422. https://doi.org/10.1007/s11240-014-0615-0.
  9. Sarmast MK. Genetic transformation and somaclonal variation in conifers. Plant Biotechnol Rep. 2016;10(6): 309-325. https://doi.org/10.1007/s11816-016-0416-5.
  10. Isabel N, Bovin R, Levasseur C, et al. Occurrence of somaclonal variation among somatic embryoderived white spruce (Picea glauca, Pinacae). Amer J Bot. 1996;83(9):1121-1130. https://doi.org/10.1002/j.1537-2197.1996.tb13892.x.
  11. Fourré JL, Berger P, Niquet L, et al. Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches. Theor Appl Genet. 1997;94(2): 159-169. https://doi.org/10.1007/s001220050395.
  12. Tremblay L, Levasseur C, Tremblay FM. Frequency of somaclonal variation in plants of black spruce (Picea mariana, Pinaceae) and white spruce (P. glauca, Pinaceae) derived from somatic embryogenesis and identification of some factors involved in genetic instability. Am J Bot. 1999;86(10):1373-1381. https://doi.org/10.2307/2656920.
  13. Marum L, Rocheta M, Maroco J, et al. Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster). Plant Cell Rep. 2009;28(4):673-682. https://doi.org/10.1007/s00299-008-0668-9.
  14. Fourré JL. Somaclonal variation and genetic molecular markers in woody plants. Molecular biology of woody plants. 2000;1:425-449. https://doi.org/10.1007/978-94-017-2311-4_18.
  15. Bairu MW, Aremu AO, Staden JV. Somaclonal variation in plants: causes and detection methods. Plant Growth Regulation. 2011;63(2):147-173. https://doi.org/10.1007/s10725-010-9554-x.
  16. Hazubska-Przybyl T, Dering M. Somaclonal variation during Picea abies and P. omorika somatic embryiogenesis and cryopreservation. Acta Biologica Cracoviensia Botanica Acta. 2017;59(1):93-103. https://doi.org/10.1515/abcsb-2017-0003.
  17. Mishiba K, Nishihara M, Nakatsuka T, et al. Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J. 2005;44(4): 541-556. https://doi.org/10.1111/j.1365-313X. 2005.02556.x.
  18. DeVerno L, Park Y, Bonga J, et al. Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]. Plant Cell Reports. 1999;18(11):948-953. https://doi.org/10.1007/s002990050689.
  19. Klimaszewska K, Noceda C, Pelletier G, et al. Biological characterization of young andaged embryogenic cultures of Pinus pinaster (Ait). In Vitro Cell Dev Biol Plant. 2009;45(1):20-33. https://doi: 10.1007/s11627-008-9158-6.
  20. Hirochika H. Activation of tobacco retrotransposons during tissue-culture. EMBO J. 1993; 12(6):2521-2528. https://doi.org/10.1002/j.1460- 2075.1993.tb05907.x.
  21. Kaeppler SM, Kaeppler HF, Rhee Y. Epigenetic aspects of somaclonal variation in plants. Plant Gene Silencing. 2000;3:59-68. https://doi.org/10.1007/978-94-011-4183-3_4.
  22. Renau-Morata B, Nebauer SG, Sales E, et al. Genetic diversity and structure of natural and managed populations of Cedrus atlantica (Pinaceae) assessed using random amplified polymorphic DNA. Am J Bot. 2005;92(5):875-884. https://doi.org/10.3732/ajb.92.5.875.
  23. Burg K, Helmersson A, Bozhkov P, et al. Developmental and genetic variation in nuclear microsatellite stability during somatic embryogenesis in pine. J Exp Bot. 2007;58(3):687-698. https://doi.org/10.1093/jxb/erl241.
  24. Горячкина О.В., Пак М.Э., Третьякова И.Н. Цитогенетические особенности эмбриогенных клеточных линий Larix sibirica Ledeb. в культуре in vitro // Вестник Томского государственного университета. Биология. – 2017. – № 39. – С. 140–153. [Goryachkina OV, Park ME, Tretyakova NN. Cytogenetic peculiarities of Larix sibirica Ledeb. embryogenic cell lines in in vitro culture. Vestnik Tomskogo gosudarstvennogo universiteta. Biologiia. 2017;(39):140-153. (In Russ.)]. https://doi.org/10.17223/19988591/39/9.
  25. Karp A. Origins, causes and uses of variation in plant tissue cultures. Plant Cell and Tissue Culture. 1994;1:139-151. https://doi.org/10.1007/978-94-017-2681-8_6.
  26. Gupta P, Durzan D, Finkle B. Somatic polyembryogenesis in embryogenic cell masses of Picea abies (Norway spruce) and Pinus taeda (Loblolly pine) after thawing from liquid nitrogen. Can J For Res. 1987;17(9):1130-1134. https://doi.org/10.1139/x87-172.
  27. Roth R, Ebert I, Schmidt J. Trisomy associated with loss of maturation capacity in a long-term embryogenic culture of Abies alba. Theoretical and Applied Genetics. 1997;95(3):353-358. https://doi.org/10.1007/s001220050570.
  28. Nawrot-Chorabik K. Somaclonal variation in embryogenic cultures of silver fir (Abie salba Mill.). Plant Biosyst. 2009;143(2):377-385. https://doi.org/10.1080/11263500902722717.
  29. Egertsdotter U. Plant physiological and genetical aspects of the somatic embryogenesis process in conifers. Scand J For Res. 2019;34(5):360-369. https://doi.org/10.1080/02827581.2018.1441433.
  30. Lamhamedi MS, Chamberland H, Bernier PY, et al. Clonal variation in morphology, growth, physiology, anatomy and ultrastructure of container-grown white spruce somatic plants. Tree Physiology. 2000;20(13):869-880. https://doi.org/10.1093/treephys/20.13.869.
  31. Grossnickle SC, Major JE. Interior spruce seedlings compared with emblings produced from somatic embryogenesis. III. Physiological response and morphological development on a reforestation site. Can J For Res. 1994;24(7):1397-1407. https://doi.org/10.1139/x94-180.
  32. Nsangou M, Greenwood M. Physiological and morphological differences between somatic, in vitro germinated, and normal seedlings of red spruce (Picea rubens Sarg.). Can J For Res. 1998;28(7):1088-1092. https://doi.org/10.1139/cjfr-28-7-1088.
  33. Benowicz A, Grossnickle SC, El-Kassaby YA. Field assessment of Douglas-fir somatic and zygotic seedlings with respect to gas exchange, water relations, and frost hardiness. Can J For Res. 2002;32(10):1822-1828. https://doi.org/10.1139/x02-093.
  34. Häggman H, Ryynänen L, Aronen T, et al. Cryopreservation of embryogenic cultures of Scots pine. Plant Cell Tissue Organ Cult. 1998;54(1):45-53. https://doi.org/10.1023/a:1006104325426.
  35. Aronen TS, Krajnakova J, Häggman HM, et al. Genetic fidelity of cryopreserved embryogenic cultures of open-pollinated Abies cephalonica. Plant Science. 1999;142(2):163-172. https://doi.org/10.1016/s0168-9452(98)00244-1.
  36. Marum L, Estêvão C, Oliveira MM, et al. Recovery of cryopreserved embryogenic cultures of maritime pine – effect of cryoprotectant and suspension density. Cryo Letters. 2004;25(5):363-374.
  37. Gale S, John A, Benson EE. Cryopreservation of Picea sitchensis (Sitka spruce) embryogenic suspensor masses. Cryo Letters. 2007;28:225-239.
  38. Salaj T, Matušiková I, Fráterová L, et al. Regrowth of embryogenic tissues of Pinus nigra following cryopreservation. Plant Cell Tiss Organ Cult. 2011;106(1):55-61. https://doi.org/10.1007/s11240-010-9893-3.
  39. Lineros Y, Balocchi C, Muñoz X, et al. Cryopreservation of Pinus radiate embryogenic tissue: effects of cryoprotective pretreatments on maturation ability. Plant Cell Tissue Organ Cult. 2018;135(2):357-366. https://https://doi.org/10.1007/s11240-018-1469-7.
  40. Latutrie M, Aronen T. Long-term cryopreservation of embryogenic Pinus sylvestris cultures. Scand J For Res. 2012;28(2):1-7. https://doi.org/10.1080/02827581.2012.701325.
  41. Nunes S, Marum L, Farinha N, et al. Somatic embryogenesis of hybrid Pinus elliottiivar. elliottii × P. caribaea var. hondurensis and ploidy assessment of somatic plants. Plant Cell Tiss Organ Cult. 2018;132(1):71-84. https://doi.org/10.1007/s11240-017-1311-7.
  42. Krajňáková J, Sutela S, Aronen T, et al. Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity. Cryobiology. 2011;63(1):17-25. https://doi.org/10.1016/j.cryobiol.2011.04.004.
  43. Kong L, von Aderkas P. Genotype effects on ABA consumption and somatic embryo maturation in interior spruce (Picea glauca × engelmanni). J Exp Bot. 2007;58(6):1525-1531. https://doi.org/10.1093/jxb/erm019.
  44. Salajova T, Salaj J. Somatic embryogenesis in European black pine (Pinus nigra Arn.). Biol Plant. 1992;34(3-4):213-218. https://doi.org/ 10.1007/bf02925871.
  45. O’Brien IE, Smith DR, Gardner RC, et al. Flow cytometric determination of genome size in Pinus. Plant Science. 1996;115(1):91-99. https://doi.org/10.1016/0168-9452(96)04356-7.
  46. Nkongolo KK, Klimaszewska K. Cytological and molecular relationships between Larix decidua, L. leptolepis and Larix × eurolepis: Identification of species-specific chromosomes and synchronization of mitotic cell. Theoretical and Applied Genetics. 1995;90(6):827-834. https://doi.org/10.1007/bf00222018.
  47. Krutovsky KV, Tretyakova IN, et al. Somaclonal variation of haploid in vitro tissue culture obtained from Siberian larch (Larix sibirica Ledeb.) megagametophytes for whole genome de novo sequencing. In Vitro Cell Dev Biol Plant. 2014;50(5):655-664. https://doi.org/10.1007/s11627-014-9619-z.
  48. Третьякова И.Н., Пак М.Э., Иваницкая А.С. Микроклональное размножение Larix sibirica и Larix sukaczewii с использованием биотехнологии соматического эмбриогенеза in vitro // Сохранение лесных генетических ресурсов Сибири: материалы 4-го международ. совещ., 24–29 августа 2015 г. – Барнаул, 2015. – С. 175. [Tretyakova IN, Park ME, Ivanitskaya AS. Microclonal propagation of Larix sibirica and Larix sukaczewii by somatic embryogenesis in vitro biotechnology. Conservation of Siberian Forest Genetic Resources: materials 4th international conferences. 24–29 August 2015. Barnaul; 2015. P. 175. (In Russ.)]
  49. Goryachkina OV, Park ME, Tretyakova IN, et al. Cytogenetic stability of young and long-term embryogenic cultures of Larix sibirica. Cytologia. 2018;83(3):323-329. https://doi.org/10.1508/cytologia.83.323.
  50. Lee M, Phillips RL. The chromosomal basis of somaclonal variation. Annu Rev Plant Physiol Plant Mol Biol. 1988;39(1):413-437. https://doi.org/10.1146/annurev.pp.39.060188.002213.
  51. Von Aderkas P, Pattanavibool R, Hristoforoglu K, Ma Y. Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch. Plant Cell Tissue Organ Cult. 2003;75(1):27-34. https://doi.org/10.1023/a:1024614209524.
  52. Doležel J, Vrana J, Šafař J, et al. Chromosomes in the flow to simplify genome analysis. Functional Integrative Genomics. 2012;12(3):397-416. https://doi.org/10.1007/s10142-012-0293-0.
  53. Lopez CM, Wetten AC, Wilkinson MJ. Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.). Theoretical Applied Genetics. 2004;110(1):157-166. https://doi.org/10.1007/s00122-004-1823-5.
  54. Nkongolo KK, Klimaszewska K. Karyotype analysis and optimization of mitotic index in Picea mariana (black spruce) preparations from seedling root tips and embryogenic cultures. Heredity. 1994;73(1):11-17. https://doi.org/10.1038/hdy.1994.93.
  55. Gajdošová A, Vookova B, Kormutak A, et al. Induction, protein composition and DNA ploidy level of embryogenic calli of silver fir and its hybrids. Biologia Plantarum. 1995;37(2):169-176. https://doi.org/10.1007/bf02913205.
  56. Fallour D, Fady B, Lefevre F. Study on isozyme variation in Pinus pinea L.: evidence for low polymorphism. Silvae Genet. 1997;46:201-207.
  57. Gad MA, Mohamed SY. Phylogenetic evaluation of some Pinus species from different genetic resources using protein, isoenzymes, RAPD, ISSR analysis. J Am Sci. 2012;8(3):311-321.
  58. Зацепина К.Г. Дифференциация популяций и клонов сосны обыкновенной (Pinus sylvestris L.) в южной части азиатского ареала: Дис. … канд. биол. наук. – Красноярск, 2014. – 115 с. [Zatsepina KG. Differentsiatsiya populyatsiy i klonov sosny obyknovennoy (Pinus sylvestris L.) v yuzhnoy chasti aziatskogo areala. [dissertation] Krasnoyarsk; 2014. 115 p. (In Russ.)]
  59. Eastman PA, Webster FB, Pitel JA, et al. Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmanii complex) using culture morphology and isozyme analysis. Plant Cell Rep. 1991;10(8):425-430. https://doi.org/10.1007/bf00232617.
  60. Noh EW, Minocha SC, Riemenschneider DE. Adventitious shoot formation from embryonic explants of red pine (Pinus resinosa). Physiol Plant. 1988;74(1):119-124. https://doi.org/10.1111/j.1399-3054.1988.tb04951.x.
  61. Richard S, Gauthier S, Laliberté S. Isozyme assessment of the genetic stability of micropropagated hybrid larch (Larix × eurolepis Henry). Can J ForRes. 1995;25(7):1103-1112. https://doi.org/10.1139/x95-122.
  62. Сулимова Г.Е. ДНК-маркеры в генетических исследованиях: типы маркеров, их свойства и области применения // Успехи современной биологии. – 2004. – Т. 124. – № 3. – С. 260–271. [Sulimova GE. DNK-markers in genetic studies: types of markers, their characteristics and application. Uspekhi sovremennoy biologii. 2004;124(3):260-271. (In Russ.)]
  63. Noh EW, Minocha SC. Pigment and isozyme variation in aspen shoots regenerated from callus culture. Plant Cell, Tissue and Organ Culture. 1990;23(1):39-44. https://doi.org/10.1007/bf00116087.
  64. Goto S, Thakur RC, Ishii K. Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers. Plant Cell Rep. 1998;18(3-4):193-197. https://doi.org/10.1007/s002990050555.
  65. Cuesta C, Ordás RJ, Fernández B, et al. Clonal micropropagation of six selected half-sibling families of Pinus pinea and somaclonal variation analysis. Plant Cell Tissue Organ Cult. 2008;95(1):125-30. https://doi.org/10.1007/s11240-008-9412-y.
  66. Isabel N, Tremblay L, Michaud M, et al. RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis derived populations of Picea mariana (Mill.) B.S.P. Theor Appl Genet. 1993;86(1):81-87. https://doi.org/10.1007/bf00223811.
  67. Heinz DJ, Mee GW. Morphologic, сytogenetic, and enzymatic variation in Saccharum species hybrid clones derived from callus tissue. American Journal Botany. 1971;58(3):257-262. https://doi.org/10.1002/j.1537-2197.1971.tb09971.x.
  68. Беседина Е.Н., Агасьева И.С., Падалка С.Д., Исмаилов В.Я. Высокоспецифические RAPD-праймеры для ПЦР-анализа популяций энтомофага Habrobracon hebetor Say. // Международный научно-исследовательский журнал. – 2018. – № 12–2. – С. 14–17. [Besedina EN, Agasyeva IS, Padalka SD, Ismailov VYa. High-specific RAPD primers for PCR analysis of Habrobracon hebetor Say. entomophage populations. International Research Journal. 2018;(12-2):14-17. (In Russ.)]. https://doi.org/10.23670/IRJ.2018.78.12.038.
  69. Матвеева Т.В., Павлова О.А., Богомаз Д.И., и др. Молекулярные маркеры для видоидентификации и филогенетики растений // Экологическая генетика. – 2011. – Т. 9. – № 1. – С. 32–43. [Matveeva TV, Pavlova OA, Bogomaz DI, et al. Molecular markers for plant species identification and phylogenetics. Ecological genetics. 2011;9(1):32-43. (In Russ.)]. https://doi.org/10.17816/ecogen9132-43.
  70. Sun GL, Salomon B, von Bothmer R. Microsatellite polymorphism and genetic differentiation in three Norwegian populations of Elymusalaskanus (Poaceae). Plant Syst Evol. 2002;234(1):101-110. https://doi.org/10.1007/s00606-002-0211-3.
  71. Sun G, Salomon B. Microsatellite variability and heterozygote deficiency in the arctical pine Alaskan wheatgrass (Elymus alaskanus) complex. Genome. 2003;46(5):729-737. https://doi.org/10.1139/g03-052.
  72. Helmersson A, Jansson G, Bozhkov PV, von Arnold S. Genetic variation in microsatellite stability of somatic embryoplants of Picea abies: a case study using six unrelated full-sibfamilies. Scand J Forest Res. 2008;23(1):2-11. https://doi.org/10.1080/02827580701820043.
  73. Rogers SO, Bendich AJ. Heritability and variability in ribosomal RNA genes of Vicia faba. Genetics. 1987;117:285-295.
  74. Fraga M, Esteller M. DNA methylation: a profile of methods and applications. BioTechniques. 2002;33(3):632-649. https://doi.org/10. 2144/02333rv01.
  75. Fraga MF, Rodríguez R, Cañal MJ. Phase-change related epigenetic and physiological changes in Pinus radiata D. Don. Planta. 2002;215:672-678. https://doi.org/10.1007/s00425-002-0795-4.
  76. Valledor L, Hasbun R, Meijón M, et al. Involvement of DNA methylation in tree development and micropropagation. Plant Cell Tiss. Org. Cult. 2007;91(2):75-86. https://doi.org/10.1007/s11240-007-9262-z.
  77. Noceda C, Salaj T, Pérez M, et al. DNA demethylation and decrease on free polyamines is associated with the embryogenic capacity of Pinus nigra Arn. cell culture. Trees. 2009;23(6): 1285-1293. https://doi.org/10.1007/s00468-009- 0370-8.
  78. Teyssier C, Maury S, Beaufour M, et al. In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses. Physiol Plant. 2014;150(2):271-291. https://doi.org/10.1111/ppl.12081.
  79. Leljak-Levanić D, Mihaljević S, Jelaska S. Variations in DNA methylation in Picea omorika embryogenic tissue and the ability for embryo maturation. Propag Ornam Plants. 2009;9(1):3-9.
  80. Yakovlev IA, Fossdal CG, Johnsen Ø. MicroRNAs, the epigenetic memory and climatic adaptation in Norway spruce. New Phytologist. 2010;187(4):1154-1169. https://doi.org/10.1111/ j.1469-8137.2010.03341.x.
  81. Bräutigam K, Vining KJ, Lafon‐Placette C, et al. Epigenetic regulation of adaptive responses of forest tree species to the environment. Ecol Evol. 2013;3(2):399-415. https://doi.org/10.1002/ece3.461.
  82. Rehfeldt GE, Tchebakova NM, Parfenova YI, et al. Intraspecific responses to climate in Pinus sylvestris. Glob Change Biol. 2002;8:912-929. https://doi.org/10.1046/j.1365-2486.2002.00516.x.

Supplementary files

There are no supplementary files to display.

Statistics

Views

Abstract - 67

PDF (Russian) - 0

Cited-By


Article Metrics

Metrics Loading ...

PlumX

Dimensions


Copyright (c) 2020 Gulyaeva E.N., Ignatenko R.V., Galibina N.A.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies