Conifer somatic embryogenesis (SE) is a process driven by exogenously supplied plant growth regulators (PGRs). Exogenous PGRs and endogenous phytohormones trigger particular ontogenetic events. Complex mechanisms involving a number of endogenous phytohormones control the differentiation of cells and tissues, as well as the establishment of structures and organs. Most of the mechanisms and hormonal functions in the SE of conifers have not yet been described. With the aim to better understand these mechanisms, we provided detailed analysis of the spectrum of endogenous phytohormones over the course of SE in Norway spruce (Picea abies). Concentrations of endogenous phytohormones including auxins, cytokinins (CKs), abscisic acid (ABA), jasmonates, and salicylic acid (SA) in somatic P. abies embryos were analyzed by HPLC-ESI-MS/MS. The results revealed that the concentrations of particular phytohormone classes varied substantially between proliferation, maturation, desiccation, and germination. Endogenous ABA showed a maximum concentration at the maturation stage, which reflected the presence of exogenous ABA in the medium and demonstrated its efficient perception by the embryos as a prerequisite for their further development. Auxins also had concentration maxima at the maturation stage, suggesting a role in embryo polarization. Endogenous jasmonates were detected in conifer somatic embryos for the first time, and reached maxima at germination. According to our knowledge, we have presented evidence for the involvement of the non-indole auxin phenylacetic acid, cis-zeatin- and dihydrozeatin-type CKs and SA in SE for the first time. The presented results represent the currently most comprehensive overview of plant hormone levels in embryos throughout the whole process of conifer SE. The differences in concentrations of various classes of phytohormones over the proliferation, maturation, desiccation, and germination in somatic P. abies embryos clearly indicate correlations between endogenous phytohormone profiles and particular developmental stages of the SE of conifers.

Laboratory of Biologically Active Compounds Institute of Experimental Botany of the Czech Academy of Sciences Prague Czechia

Laboratory of Hormonal Regulations in Plants Institute of Experimental Botany of the Czech Academy of Sciences Prague Czechia

Laboratory of Mass Spectrometry Institute of Experimental Botany of the Czech Academy of Sciences Prague Czechia

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An C. F., Mou Z. L. (2011). Salicylic acid and its function in plant immunity. J. Int. Plant Biol. 53 412–428. 10.1111/j.1744-7909.2011.01043.x PubMed DOI

Cairney J., Pullman G. S. (2007). The cellular and molecular biology of conifer embryogenesis. New Phytol. 176 511–536. 10.1111/j.1469-8137.2007.02239.x PubMed DOI

Chalupa V. (1985). Somatic embryogenesis and plantlet regeneration from cultured immature and mature embryos of Picea abies (L.) Karst. Com. Inst. Forest. Cech. 14 65–90.

Chiwocha S., von Aderkas P. (2002). Endogenous levels of free and conjugated forms of auxin, cytokinins and abscisic acid during seed development in Douglas fir. Plant Growth Regul. 36 191–200. 10.1023/A:1016522422983 DOI

Djilianov D. L., Dobrev P. I., Moyankova D. P., Vankova R., Georgieva D. T., Gajdosova S., et al. (2013). Dynamics of endogenous phytohormones during desiccation and recovery of the resurrection plant species Haberlea rhodopensis. J. Plant Growth Regul. 32 564–574. 10.1007/s00344-013-9323-y DOI

Dobrev P. I., Vankova R. (2012). Quantification of abscisic acid, cytokinin, and auxin content in salt-stressed plant tissues. Methods Mol. Biol. 913 251–261. 10.1007/978-1-61779-986-0_17 PubMed DOI

Filonova L. H., Bozhkov P. V., von Arnold S. (2000). Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking. J. Exp. Bot. 51 249–264. 10.1093/jexbot/51.343.249 PubMed DOI

Find J. I. (1997). Changes in endogenous ABA levels in developing somatic embryos of Norway spruce (Picea abies (L) Karst) in relation to maturation medium, desiccation and germination. Plant Sci. 128 75–83. 10.1016/S0168-9452(97)00141-6 DOI

Fischerova L., Fischer L., Vondrakova Z., Vagner M. (2008). Expression of the gene encoding transcription factor PaVP1 differs in Picea abies embryogenic lines depending on their ability to develop somatic embryos. Plant Cell Rep. 27 435–441. 10.1007/s00299-007-0469-6 PubMed DOI

Frebort I., Kowalska M., Hluska T., Frebortova J., Galuszka P. (2011). Evolution of cytokinin biosynthesis and degradation. J. Exp. Bot. 62 2431–2452. 10.1093/jxb/err004 PubMed DOI

Gemperlova L., Fischerova L., Cvikrova M., Mala J., Vondrakova Z., Martincova O., et al. (2009). Polyamine profiles and biosynthesis in somatic embryo development and comparison of germinating somatic and zygotic embryos of Norway spruce. Tree Physiol. 29 1287–1298. 10.1093/treephys/tpp063 PubMed DOI

Gomez-Cadenas A., Vives V., Zandalinas S. I., Manzi M., Sanchez-Perez A. M., Perez-Clemente R. M., et al. (2015). Abscisic acid: a versatile phytohormone in plant signaling and beyond. Cur. Protein Peptide Sci. 16 413–434. 10.2174/1389203716666150330130102 PubMed DOI

Grossmann K. (2010). Auxin herbicides: current status of mechanism and mode of action. Pest Manag. Sci. 66 113–120. 10.1002/ps.1860 PubMed DOI

Guevin T. G., Kirby E. G. (1997). Induction of embryogenesis in cultured mature zygotic embryos of Abies fraseri (Pursh) Poir. Plant Cell Tissue Organ Cult. 49 219–222. 10.1023/A:1005747026269 DOI

Gupta P. K., Durzan D. J. (1986). Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos of Picea abies (Norway Spruce). In vitro Cell. Dev. Biol. 22 685–688. 10.1007/BF02623484 DOI

Hakman I., von Arnold S., Eriksson T. (1985). Somatic embryogenesis in Norway Spruce. Physiol. Plant. 64:A12.

Jain S., Gupta P. K., Newton R. J. (1995). Somatic Embryogenesis in Woody Plants. Dordrecht: Kluwer Academic Publishers.

Jiskrova E., Novak O., Pospisilova H., Holubova K., Karady M., Galuszka P., et al. (2016). Extra- and intracellular distribution of cytokinins in the leaves of monocots and dicots. New Biotech. 33 735–742. 10.1016/j.nbt.2015.12.010 PubMed DOI

Jourdain I., Lelu M. A., Label P. (1997). Hormonal changes during growth of somatic embryogenic masses in hybrid larch. Plant Physiol. Biochem. 35 741–749.

Kaminek M., Brezinova A., Gaudinova A., Motyka V., Vankova R., Zazimalova E. (2000). Purine cytokinins: a proposal of abbreviations. Plant Growth Regul. 32 253–256. 10.1023/a:1010743522048 DOI

Liao Y. K., Liao C. K., Ho Y. L. (2008). Maturation of somatic embryos in two embryogenic cultures of Picea morrisonicola Hayata as affected by alternation of endogenous IAA content. Plant Cell Tissue Organ Cult. 93 257–268. 10.1007/s11240-008-9371-3 DOI

Linkies A., Leubner-Metzger G. (2012). Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination. Plant Cell Rep. 31 253–270. 10.1007/s00299-011-1180-1 PubMed DOI

Lorenzo O., Solano R. (2005). Molecular players regulating the jasmonate signalling network. Curr. Opin. Plant Biol. 8 532–540. 10.1016/j.pbi.2005.07.003 PubMed DOI

Montalbán I. A., Novak O., Rolcik J., Strnad M., Moncaleán P. (2013). Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees. Physiol. Plant 148 214–231. 10.1111/j.1399-3054.2012.01709.x PubMed DOI

Norgaard J. V., Krogstrup P. (1991). Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk. Plant Cell Rep. 9 509–513. 10.1007/BF00232107 PubMed DOI

Ribeiro L. M., Garcia Q. S., Mueller M., Munne-Bosch S. (2015). Tissue-specific hormonal profiling during dormancy release in macaw palm seeds. Physiol. Plant. 153 627–642. 10.1111/ppl.12269 PubMed DOI

Roberts D. R., Flinn B. S., Webb D. T., Webster F. B., Sutton B. C. S. (1990). Abscisic-acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce. Physiol. Plant. 78 355–360. 10.1111/j.1399-3054.1990.tb09048.x DOI

Rodriguez-Gacio M. D. C., Matilla-Vazquez M. A., Matilla A. J. (2009). Seed dormancy and ABA signaling: the breakthrough goes on. Plant Signal. Behav. 4 1035–1049. 10.4161/psb.4.11.9902 PubMed DOI PMC

Sakakibara H. (2006). Cytokinins: activity, biosynthesis, and translocation. Ann. Rev. Plant Biol. 57 431–449. 10.1146/annurev.arplant.57.032905.105231 PubMed DOI

Sauer M., Robert S., Kleine-Vehn J. (2013). Auxin: simply complicated. J. Exp. Bot. 64 2565–2577. 10.1093/jxb/ert139 PubMed DOI

Schneider E. A., Kazakoff C. W., Wightman F. (1985). Gas chromatography-mass spectrometry evidence for several endogenous auxins in pea seedling organs. Planta 165 232–241. 10.1007/bf00395046 PubMed DOI

Schwarzerova K., Vondrakova Z., Fischer L., Borikova P., Bellinvia E., Eliasova K., et al. (2010). The role of actin isoforms in somatic embryogenesis in Norway spruce. BMC Plant Biol. 10:89 10.1186/1471-2229-10-89 PubMed DOI PMC

Senaratna T., Merritt D., Dixon K., Bunn E., Touchell D., Sivasithamparam K. (2003). Benzoic acid may act as the functional group in salicylic acid and derivatives in the induction of multiple stress tolerance in plants. Plant Growth Regul. 39 77–81. 10.1023/a:1021865029762 DOI

Simon S., Petrasek J. (2011). Why plants need more than one type of auxin. Plant Sci. 180 454–460. 10.1016/j.plantsci.2010.12.007 PubMed DOI

Smehilova M., Dobruskova J., Novak O., Takac T., Galuszka P. (2016). Cytokinin-specific glycosyltransferases possess different roles in cytokinin homeostasis maintenance. Front. Plant Sci. 7:1264 10.3389/fpls.2016.01264 PubMed DOI PMC

Song Y. (2014). Insight into the mode of action of 2,4-dichlorophenoxyacetic acid (2,4-D) as an herbicide. J. Int. Plant Biol. 56 106–113. 10.1111/jipb.12131 PubMed DOI

Stasolla C., Yeung E. C. (2003). Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult. 74 15–35. 10.1023/a:1023345803336 DOI

Su Y. H., Liu Y. B., Zhang X. S. (2011). Auxin-cytokinin interaction regulates meristem development. Mol. Plant 4 616–625. 10.1093/mp/ssr007 PubMed DOI PMC

Sugawara S., Mashiguchi K., Tanaka K., Hishiyama S., Sakai T., Hanada K., et al. (2015). Distinct characteristics of indole-3-acetic acid and phenylacetic acid, two common auxins in plants. Plant Cell Physiol. 56 1641–1654. 10.1093/pcp/pcv088 PubMed DOI PMC

Sun J., Xu Y., Ye S., Jiang H., Chen Q., Liu F., et al. (2009). Arabidopsis ASA1 is important for jasmonate-mediated regulation of auxin biosynthesis and transport during lateral root formation. Plant Cell 21 1495–1511. 10.1105/tpc.108.064303 PubMed DOI PMC

Svobodova H., Albrechtova J., Kumstyrova L., Lipavska H., Vagner M., Vondrakova Z. (1999). Somatic embryogenesis in Norway spruce: anatomical study of embryo development and influence of polyethylene glycol on maturation process. Plant Physiol. Biochem. 37 209–221. 10.1016/S0981-9428(99)80036-9 DOI

Vagner M., Vondrakova Z., Opatrna J., Kumstyrova L., Gosslova M., Svobodova H., et al. (1999a). Endogenous IAA and cytokinins during somatic and zygotic embryo development of Norway spruce. Biol. Plant. 42 S60–S60.

Vagner M., Vondrakova Z., Spackova J., Cvikrova M., Eder J., Lipavska H., et al. (1999b). “Norway spruce somatic embryogenesis: Endogenous levels of phytohormones during somatic embryo development,” in Plant Biotechnology and in vitro Biology in the 21st Century, eds Altman A., Ziv M., Izhar S. (Dordrecht: Kluwer Academic Publisher; ), 93–96.

Vagner M., Vondrakova Z., Strnadova Z., Eder J., Machackova I. (1998). Endogenous levels of plant growth hormones during early stages of somatic embryogenesis of Picea abies. Adv. Hort. Sci. 12 11–18.

Vankova R. (1999). “Cytokinin glycoconjugates - distribution, metabolism and function,” in Advances in Regulation of Plant Growth and Development, eds Strnad M., Pec P., Beck E. (Prague: Peres publishers; ), 67–78.

von Aderkas P., Bonga J. M. (2000). Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol. 20 921–928. 10.1093/treephys/20.14.921 PubMed DOI

von Aderkas P., Lelu M. A., Label P. (2001). Plant growth regulator levels during maturation of larch somatic embryos. Plant Physiol. Biochem. 39 495–502. 10.1016/s0981-9428(01)01271-2 DOI

von Arnold S., Clapham D., Egertsdotter U., Mo L. H. (1996). Somatic embryogenesis in conifers - a case study of induction and development of somatic embryos in Picea abies. Plant Growth Regul. 20 3–9. 10.1007/BF00024050 DOI

von Arnold S., Sabala I., Bozhkov P., Dyachok J., Filonova L. (2002). Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult. 69 233–249. 10.1023/a:1015673200621 DOI

Vondrakova Z., Eliasova K., Fischerova L., Vagner M. (2011). The role of auxins in somatic embryogenesis of Abies alba. Centr. Europ. J. Biol. 6 587–596. 10.2478/s11535-011-0035-7 DOI

Vondrakova Z., Krajnakova J., Fischerova L., Vagner M., Eliasova K. (2016). “Physiology and role of plant growth regulators in somatic embryogenesis,” in Vegetative Propagation of Forest Trees, eds Park Z. S., Bonga J., Moon H. K. (Seoul: National Institute of Forest Science; ), 123–169.

Wang J., Ma X. M., Kojima M., Sakakibara H., Hou B. K. (2011). N-Glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response in Arabidopsis thaliana. Plant Cell Physiol. 52 2200–2213. 10.1093/pcp/pcr152 PubMed DOI

Wang J., Ma X. M., Kojima M., Sakakibara H., Hou B. K. (2013). Glucosyltransferase UGT76C1 finely modulates cytokinin responses via cytokinin N-glucosylation in Arabidopsis thaliana. Plant Physiol. Biochem. 65 9–16. 10.1016/j.plaphy.2013.01.012 PubMed DOI

Yasin M., Andreasen C. (2015). Breaking seed dormancy of Alliaria petiolata with phytohormones. Plant Growth Regul. 77 307–315. 10.1007/s10725-015-0065-7 DOI

Zdarska M., Dobisova T., Gelova Z., Pernisova M., Dabravolski S., Hejatko J. (2015). Illuminating light, cytokinin, and ethylene signalling crosstalk in plant development. J. Exp. Bot. 66 4913–4931. 10.1093/jxb/erv261 PubMed DOI

Zhang H. B., Horgan K. J., Reynolds P. H. S., Jameson P. (2003). Cytokinins and bud morphology in Pinus radiata. Physiol. Plant. 117 264–269. 10.1034/j.1399-3054.2003.00026.x DOI

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