Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

BioForA INRA ONF Orléans France

BIOGECO INRA University of Bordeaux Cestas France

Centre de Génomique Fonctionnelle Plateforme Protéome University of Bordeaux Bordeaux France

FCBA Pôle Biotechnologie et Sylviculture Avancée Cestas France

Institute of Experimental Botany of the Czech Academy of Sciences Prague Czechia

PEIRENE Sylva LIM Université de Limoges Limoges France

UCA INRA UMR PIAF Clermont Ferrand France

Zobrazit více v PubMed

Adrian A., Rahnenfuhrer J. (2016). topGO: Enrichment Analysis for Gene Ontology. R package version 2.30.1.

Amsterdam A., Pitzer F., Baumeister W. (1993). Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control. Proc. Natl. Acad. Sci. U.S.A. 90 99–103. PubMed PMC

Bastien J. C., Sanchez L., Michaud D. (2013). “Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco),” in Forest Tree Breeding in Europe: Current State-of-the-Art and Perspectives, Managing Forest Ecosystems, ed. Pâques L. (Berlin: Springer Science+Business Media; ), 325–369.

Benjamini Y., Hochberg Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57 289–300.

Bonga J. M., Klimaszewska K. K., von Aderkas P. (2010). Recalcitrance in clonal propagation, in particular of conifers. Plant Cell Tissue Organ Cult. 100 241–254. 10.1007/s11240-009-9647-2 DOI

Bonhomme M., Peuch M., Ameglio T., Rageau R., Guilliot A., Decourteix M., et al. (2010). Carbohydrate uptake from xylem vessels and its distribution among stem tissues and buds in walnut (Juglans regia L.). Tree Physiol. 30 89–102. 10.1093/treephys/tpp103 PubMed DOI

Brand U., Grunewald M., Hobe M., Simon R. (2002). Regulation of CLV3 expression by two homeobox genes in Arabidopsis. Plant Physiol. 129 565–575. 10.1104/pp.001867 PubMed DOI PMC

Bravo S., Bertín A., Turner A., Sepúlveda F., Jopia P., Parra M. J., et al. (2017). Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of Pinus radiata D. don. Plant Cell Tissue Organ Cult. 130 521–529. 10.1007/s11240-017-1242-3 DOI

Businge E., Brackmann K., Moritz T., Egertsdotter U. (2012). Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies). Tree Physiol. 32 232–244. 10.1093/treephys/tpr142 PubMed DOI

Carlson M. (2017). GO.db: A Set of Annotation Maps Describing the Entire Gene Ontology. R package version 3.5.0.

Chang S., Puryear J., Cairney J. (1993). A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. 11 113–116. PubMed

Chin C. F., Tan H. S. (2018). The use of proteomic tools to address challenges faced in clonal propagation of tropical crops through somatic embryogenesis. Proteomes 6:E21. 10.3390/proteomes6020021 PubMed DOI PMC

Correia S., Vinhas R., Manadas B., Lourenço A. S., Veríssimo P., Canhoto J. M. (2012). Comparative proteomic analysis of auxin-induced embryogenic and nonembryogenic tissues of the solanaceous tree Cyphomandra betacea (Tamarillo). J. Prot. Res. 11 1666–1675. 10.1021/pr200856w PubMed DOI

Crouzet J., Trombik T., Fraysse A. S., Boutry M. (2006). Organization and function of the plant pleiotropic drug resistance ABC transporter family. FEBS Lett. 580 1123–1130. 10.1016/j.febslet.2005.12.043 PubMed DOI

Cutler A. J., Krochko J. E. (1999). Formation and breakdown of ABA. Trends Plant Sci. 4 472–478. 10.1016/s1360-1385(99)01497-1 PubMed DOI

Dawson R. J. P., Locher K. P. (2006). Structure of a bacterial multidrug ABC transporter. Nature 443 180–185. 10.1038/nature05155 PubMed DOI

Dobrev P. I., Kamínek M. (2002). Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J. Chromatogr. A 950 21–29. 10.1016/S0021-9673(02)00024-9 PubMed DOI

Dronne S., Label P., Lelu M.-A. (1997). Desiccation decreases abscisic acid content in hybrid larch (Larix × leptoeuropaea) somatic embryos. Physiol. Plant. 99 433–438.

Durzan D., Gupta P. (1987). Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures. Plant Sci. 52 229–235. 10.1016/0168-9452(87)90056-2 DOI

Edgar R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy, and high throughput. Nucleic Acids Res. 32 1792–1797. 10.1093/nar/gkh340 PubMed DOI PMC

Edreva A. (2005). Pathogenesis-related proteins: research progress in the last 15 years. Gen. Appl. Plant Physiol. 31 105–124.

Elhiti M., Stasolla C., Wang A. (2013). Molecular regulation of plant somatic embryogenesis. In Vitro Cell. Dev. Biol. Plant 49 631–642. 10.1007/s11627-013-9547-3 DOI

Etienne H., Sotta B., Montoro P., Miginiac E., Carron M. P. (1993). Relations between exogenous growth-regulators and endogenous indole-3-acetic-acid and abscisic acid in the expression of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.). Plant Sci. 88 91–96. 10.1016/0168-9452(93)90113-e DOI

Fehér A. (2015). Somatic embryogenesis - Stress-induced remodeling of plant cell fate. Biochim. Biophys. Acta 1849 385–402. 10.1016/j.bbagrm.2014.07.005 PubMed DOI

Friml J., Vieten A., Sauer M., Weijers D., Schwarz H., Hamann T., et al. (2003). Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426 147–153. 10.1038/nature02085 PubMed DOI

Fujita M., Fujita Y., Maruyama K., Seki M., Hiratsu K., Ohme-Takagi M., et al. (2004). A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J. 39 863–876. 10.1111/j.1365-313X.2004.02171.x PubMed DOI

Gälweiler L., Guan C. H., Muller A., Wisman E., Mendgen K., Yephremov A., et al. (1998). Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science 282 2226–2230. 10.1126/science.282.5397.2226 PubMed DOI

Gautier F., Eliášová K., Leplé J. C., Vondráková Z., Lomenech A. M., Le Metté C., et al. (2018). Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.]. BMC Plant Biol. 18:164. 10.1186/s12870-018-1337-y PubMed DOI PMC

Goncalves L. S. A., Rodrigues R., Diz M. S. S., Robaina R. R., do Amaral A. T., Carvalho A. O., et al. (2013). Peroxidase is involved in Pepper yellow mosaic virus resistance in Capsicum baccatum var. pendulum. Genet. Mol. Res. 12 1411–1420. 10.4238/2013.April.26.3 PubMed DOI

Götz S., García-Gómez J. M., Terol J., Williams T. D., Nagaraj S. H., Nueda M. J., et al. (2008). High-throughput functional annotation and data mining with the Blast2GO suite. Nucl. Acids Res. 36 3420–3435. 10.1093/nar/gkn176 PubMed DOI PMC

Gournas C., Papageorgiou I., Diallinas G. (2008). The nucleobase-ascorbate transporter (NAT) family: genomics, evolution, structure-function relationships and physiological role. Mol. Biosyst. 4 404–416. 10.1039/b719777b PubMed DOI

Haecker A., Gross-Hardt R., Geiges B., Sarkar A., Breuninger H., Herrmann M., et al. (2004). Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 131 657–668. 10.1242/dev.00963 PubMed DOI

Heim M. A., Jakoby M., Werber M., Martin C., Weisshaar B., Bailey P. C. (2003). The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol. Biol. Evol. 20 735–747. 10.1093/molbev/msg088 PubMed DOI

Helleboid S., Hendriks T., Bauw G., Inze D., Vasseur J., Hilbert J. L. (2000). Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins. J. Exp. Bot. 51 1189–1200. 10.1093/jexbot/51.348.1189 PubMed DOI

Heringer A. S., Santa-Catarina C., Silveira V. (2018). Insights from proteomic studies into plant somatic embryogenesis. Proteomics 18:1700265. 10.1002/pmic.201700265 PubMed DOI

Jourdain I., Lelu M. A., Label P. (1997). Hormonal changes during growth of somatic embryogenic masses in hybrid larch [Larix x leptoeuropaea]. 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

Klein M., Perfus-Barbeoch L., Frelet A., Gaedeke N., Reinhardt D., Mueller-Roeber B., et al. (2003). The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use. Plant J. 33 119–129. 10.1046/j.1365-313X.2003.016012.x PubMed DOI

Klimaszewska K., Hargreaves C., Lelu-Walter M.-A., Trontin J.-F. (2016). “Advances in conifer somatic embryogenesis since year 2000,” in In Vitro Embryogenesis in Higher Plants, Methods in Molecular Biology Vol. 1359 eds Germanà M., Lambardi M. (New York, NY: Humana Press; ), 131–166. PubMed

Klimaszewska K., Overton C., Stewart D., Rutledge R. (2011). Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process. Planta 233 635–647. 10.1007/s00425-010-1325-4 PubMed DOI

Klip A., Tsakiridis T., Marette A., Ortiz P. A. (1994). Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J. 8 43–53. PubMed

Klubicová K., Uvácková L., Danchenko M., Nemecek P., Skultéty L., Salaj J., et al. (2017). Insights into the early stage of Pinus nigra Arn. somatic embryogenesis using discovery proteomics. J. Proteomics 166 99–111. 10.1016/j.jprot.2017.05.013 PubMed DOI

Kumaravel M., Uma S., Backiyarani S., Saraswathi M. S., Vaganan M. M., Muthusamy M., et al. (2017). Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine. Plant Cell Rep. 36 163–178. 10.1007/s00299-016-2067-y PubMed DOI

Kurdyukov S., Faust A., Trenkamp S., Bar S., Franke R., Efremova N., et al. (2006). Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain alpha-,omega-dicarboxylic fatty acids and formation of extracellular matrix. Planta 224 315–329. 10.1007/s00425-005-0215-7 PubMed DOI

Laux T., Mayer K. F. X., Berger J., Jurgens G. (1996). The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development 122 87–96. PubMed

Lê S., Josse J., Husson F. (2008). FactoMineR: an R package for multivariate analysis. J. Stat. Softw. 25 1–18.

Lelu-Walter M.-A., Gautier F., Eliášová K., Sanchez L., Teyssier C., Lomenech A.-M., et al. (2018). High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in Pseudotsuga menziesii [Mirb.]. Plant Cell Tissue Organ Cult. 132 137–155. 10.1007/s11240-017-1318-0 DOI

Lelu-Walter M.-A., Thompson D., Harvengt L., Sanchez L., Toribio M., Pâques L. E. (2013). Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Gen. Genomes 9 883–899. 10.1007/s11295-013-0620-1 DOI

Léran S., Varala K., Boyer J. C., Chiurazzi M., Crawford N., Daniel-Vedele F., et al. (2014). A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci. 19 5–9. 10.1016/j.tplants.2013.08.008 PubMed DOI

Li H., Durbin R. (2009). Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 1754–1760. 10.1093/bioinformatics/btp324 PubMed DOI PMC

Li Q., Zhang S., Wang J. (2015). Transcriptomic and proteomic analyses of embryogenic tissues in Picea balfouriana treated with 6-benzylaminopurine. Physiol. Plant. 154 95–113. 10.1111/ppl.12276 PubMed DOI

Li Y. D., Zhu Y. X., Yao J., Zhang S. L., Wang L., Guo C. L., et al. (2017). Genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes. Sci. Rep. 7:12638. 10.1038/s41598-017-16207-6 PubMed DOI PMC

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

Lipavská H., Konrádová H. (2004). Somatic embryogenesis in conifers: the role of carbohydrate metabolism. In Vitro Cell. Dev. Biol. Plant 40 23–30.

Lippert D., Jun Z., Ralph S., Ellis D. E., Gilbert M., Olafson R., et al. (2005). Proteome analysis of early somatic embryogenesis in Picea glauca. Proteomics 5 461–473. 10.1002/pmic.200400986 PubMed DOI

Lombard V., Ramulu H. G., Drula E., Coutinho P. M., Henrissat B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42 D490–D495. 10.1093/nar/gkt1178 PubMed DOI PMC

Long T. A., Tsukagoshi H., Busch W., Lahner B., Salt D. E., Benfey P. N. (2010). The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots. Plant Cell 22 2219–2236. 10.1105/tpc.110.074096 PubMed DOI PMC

Love M. I., Huber W., Anders S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:550. 10.1186/s13059-014-0550-8 PubMed DOI PMC

Lyngved R., Renaut J., Hausman J.-F., Iversen T.-H., Hvoslef-Eide A. K. (2008). Embryo-specific Proteins in Cyclamen persicum analyzed with 2-D DIGE. J. Plant Growth Regul. 27:353 10.1007/s00344-008-9061-8 DOI

Maadon S. N., Rohani E. R., Ismail I., Baharum S. N., Normah M. N. (2016). Somatic embryogenesis and metabolic differences between embryogenic and non-embryogenic structures in mangosteen. Plant Cell Tissue Organ Cult. 127 443–459. 10.1007/s11240-016-1068-4 DOI

Mahdavi-Darvari F., Noor N., Ismanizan I. (2015). Epigenetic regulation and gene markers as signals of early somatic embryogenesis. Plant Cell Tissue Organ Cult. 120 407–422. 10.1007/s11240-014-0615-0 DOI

Mahmud I., Shrestha B., Boroujerdi A., Chowdhury K. (2015). NMR-based metabolomics profile comparisons to distinguish between embryogenic and non-embryogenic callus tissue of sugarcane at the biochemical level. In Vitro Cell. Dev. Biol. Plant 51 340–349. 10.1007/s11627-015-9687-8 DOI

Marsoni M., Bracale M., Espen L., Prinsi B., Negri A. S., Vannini C. (2008). Proteomic analysis of somatic embryogenesis in Vitis vinifera. Plant Cell Rep. 27 347–356. 10.1007/s00299-007-0438-0 PubMed DOI

Martin A. B., Cuadrado Y., Guerra H., Gallego P., Hita O., Martin L., et al. (2000). Differences in the contents of total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from Medicago arborea L. Plant Sci. 154 143–151. 10.1016/S0168-9452(99)00251-4 PubMed DOI

Martinoia E., Klein M., Geisler M., Bovet L., Forestier C., Kolukisaoglu U., et al. (2002). Multifunctionality of plant ABC transporters - more than just detoxifiers. Planta 214 345–355. 10.1007/s004250100661 PubMed DOI

Mayer K. F. X., Schoof H., Haecker A., Lenhard M., Jurgens G., Laux T. (1998). Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95 805–815. 10.1016/s0092-8674(00)81703-1 PubMed DOI

Mi H., Muruganujan A., Thomas P. (2013). PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res. 41 D377–D386. 10.1093/nar/gks1118 PubMed DOI PMC

Miguel C., Rupps A., Raschke J., Rodrigues A., Trontin J. (2016). “Impact of molecular studies on somatic embryogenesis development for implementation in conifer multi-varietal forestry,” in Vegetative Propagation of Forest Trees, eds Park Y., Bonga J., Moon H. (Seoul: Korea Forest Research Institute; ), 373–421.

Morel A., Teyssier C., Trontin J.-F., Eliášová K., Pešek B., Beaufour M., et al. (2014). Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses. Physiol. Plant. 152 184–201. 10.1111/ppl.12158 PubMed DOI

Nakano R. T., Matsushima R., Ueda H., Tamura K., Shimada T., Li L., et al. (2009). GNOM-LIKE1/ERMO1 and SEC24a/ERMO2 are required for maintenance of endoplasmic reticulum morphology in Arabidopsis thaliana. Plant Cell 21 3672–3685. 10.1105/tpc.109.068270 PubMed DOI PMC

Navarro B. V., Elbl P., De Souza A. P., Jardim V., de Oliveira L. F., Macedo A. F., et al. (2017). Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia. PLoS One 12:e0180051. 10.1371/journal.pone.0180051 PubMed DOI PMC

Neale D. B., McGuire P. E., Wheeler N. C., Stevens K. A., Crepeau M. W., Cardeno C., et al. (2017). The douglas-fir genome sequence reveals specialization of the photosynthetic apparatus in pinaceae. G3 7 3157–3167. 10.1534/g3.117.300078 PubMed DOI PMC

Nørgaard 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

Ohbayashi I., Sugiyama M. (2018). Plant nucleolar stress response, a new face in the NAC-dependent cellular stress responses. Front. Plant Sci. 8:2247. 10.3389/fpls.2017.02247 PubMed DOI PMC

Palovaara J., Hallberg H., Stasolla C., Hakman I. (2010). Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8/9 in developing seeds and somatic embryos of the gymnosperm Picea abies. New Phytol. 188 122–135. 10.1111/j.1469-8137.2010.03336.x PubMed DOI

Pascual M. B., Cánovas F. M., Ávila C. (2015). The NAC transcription factor family in maritime pine (Pinus pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol. 15:254. 10.1186/s12870-015-0640-0 PubMed DOI PMC

Pighin J. A., Zheng H. Q., Balakshin L. J., Goodman I. P., Western T. L., Jetter R., et al. (2004). Plant cuticular lipid export requires an ABC transporter. Science 306 702–704. 10.1126/science.1102331 PubMed DOI

Pollard M., Beisson F., Li Y. H., Ohlrogge J. B. (2008). Building lipid barriers: biosynthesis of cutin and suberin. Trends Plant Sci. 13 236–246. 10.1016/j.tplants.2008.03.003 PubMed DOI

Pullman G., Johnson S., Bucalo K. (2009). Douglas fir embryogenic tissue initiation. Plant Cell Tissue Organ Cult. 96 75–84. 10.1007/s11240-008-9462-1 DOI

Quesnelle P. E., Emery R. J. N. (2007). cis-Cytokinins that predominate in Pisum sativum during early embryogenesis will accelerate embryo growth in vitro. Can. J. Bot. 85 91–103. 10.1139/b06-149 DOI

R Development Core Team (2011). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing.

Reeves C., Hargreaves C., Trontin J.-F., Leu-Walter M.-A. (2018). Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir. Trees 32 175–190. 10.1007/s00468-017-1622-7 DOI

Rosenberg-Zand S. R., Jenkins D. J. A., Diamandis E. P. (2000). Steroid hormone activity of flavonoids and related compounds. Breast Cancer Res. Treat. 62 35–49. 10.1023/a:1006422302173 PubMed DOI

Rutledge R. G., Stewart D., Caron S., Overton C., Boyle B., MacKay J., et al. (2013). Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca). BMC Plant Biol. 13:116. 10.1186/1471-2229-13-116 PubMed DOI PMC

Rutledge R. G., Stewart D., Overton C., Klimaszewska K. (2017). Gene expression analysis of primordial shoot explants collected from mature white spruce (Picea glauca) trees that differ in their responsiveness to somatic embryogenesis induction. PLoS One 12:e0185015. 10.1371/journal.pone.0185015 PubMed DOI PMC

Sampedro J., Cosgrove D. J. (2005). The expansin superfamily. Genome Biol. 6:242. 10.1186/gb-2005-6-12-242 PubMed DOI PMC

Schmutz J., Cannon S. B., Schlueter J., Ma J. X., Mitros T., Nelson W., et al. (2010). Genome sequence of the palaeopolyploid soybean. Nature 463 178–183. 10.1038/nature08670 PubMed DOI

Sharifi G., Ebrahimzadeh H., Ghareyazie B., Gharechahi J., Vatankhah E. (2012). Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.). Proteome Sci. 10:3. 10.1186/1477-5956-10-3 PubMed DOI PMC

Shih M. D., Hsieh T. Y., Jian W. T., Wu M. T., Yang S. J., Hoekstra F. A., et al. (2012). Functional studies of soybean (Glycine max L.) seed LEA proteins GmPM6, GmPM11, and GmPM30 by CD and FTIR spectroscopy. Plant Sci. 196 152–159. 10.1016/j.plantsci.2012.07.012 PubMed DOI

Silva Rde C., Carmo L. S. T., Luis Z. G., Silva L. P., Scherwinski-Pereira J. E., Mehta A. (2014). Proteomic identification of differentially expressed proteins during the acquisition of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.). J. Proteomics 104 112–127. 10.1016/j.jprot.2014.03.013 PubMed DOI

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

Steinmann T., Geldner N., Grebe M., Mangold S., Jackson C. L., Paris S., et al. (1999). Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286 316–318. 10.1126/science.286.5438.316 PubMed DOI

Su Y. H., Su Y. X., Liu Y. G., Zhang X. S. (2013). Abscisic acid is required for somatic embryo initiation through mediating spatial auxin response in Arabidopsis. Plant Growth Regul. 69 167–176. 10.1007/s10725-012-9759-2 DOI

Teyssier C., Grondin C., Bonhomme L., Lomenech A.-M., Vallance M., Morabito D., et al. (2011). Increased gelling agent concentration promotes somatic embryo maturation in hybrid larch (Larix× eurolepsis): a 2-DE proteomic analysis. Physiol. Plant. 141 152–165. 10.1111/j.1399-3054.2010.01423.x PubMed DOI

Treutter D. (2006). Significance of flavonoids in plant resistance: a review. Environ. Chem. Lett. 4 147–157. 10.1007/s10311-006-0068-8 DOI

Trontin J. F., Aronen T., Hargreaves C., Montalbán I., Moncaleán P., Reeves C., et al. (2016a). “International effort to induce somatic embryogenesis in adult pine trees,” in Vegetative Propagation of Forest Trees, eds Park Y., Bonga J., Moon H. (Seoul: Korea Forest Research Institute; ), 211–260.

Trontin J.-F., Klimaszewska K., Morel A., Hargreaves C. L., Lelu-Walter M.-A. (2016b). “Molecular aspects of conifer zygotic and somatic embryo development: a review of genome-wide approaches and recent insights,” in In Vitro Embryogenesis in Higher Plants. Methods in Molecular Biology Vol. 1359 eds Germana M., Lambardi M. (Berlin: Springer Science+Business Media; ), 167–207. 10.1007/978-1-4939-3061-6_8 PubMed DOI

Vaňková 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; ), 67–78.

Varhanikova M., Uvackova L., Skultety L., Pretova A., Obert B., Hajduch M. (2014). Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency. J. Proteomics 104 57–65. 10.1016/j.jprot.2014.02.003 PubMed DOI

Vestman D., Larsson E., Uddenberg D., Cairney J., Clapham D., Sundberg E., et al. (2011). Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression. Tree Gen. Genomes 7 347–362. 10.1007/s11295-010-0336-4 DOI

Vizcaíno J. A., Csordas A., del-Toro N., Dianes J. A., Griss J., Lavidas I., et al. (2016). 2016 update of the PRIDE database and related tools. Nucleic Acids Res. 44 D447–D456. 10.1093/nar/gkv1145 PubMed DOI PMC

von Aderkas P., Klimaszewska K., Bonga J. M. (1990). Diploid and haploid embryogenesis in Larix leptolepis, L. decidua, and their reciprocal hybrids. Can. J. Bot. 20 9–14. 10.1139/x90-002 DOI

Vondráková Z., Dobrev P., Pešek B., Fischerová L., Vágner M., Motyka V. (2018). Profiles of endogenous phytohormones over the course of Norway spruce somatic embryogenesis. Front. Plant Sci. 9:1283. 10.3389/fpls.2018.01283 PubMed DOI PMC

Vondráková Z., Eliášová K., Fischerová L., Vágner M. (2011). The role of auxins in somatic embryogenesis of Abies alba. Cent. Eur. J. Biol. 6 587–596. 10.2478/s11535-011-0035-7 DOI

Vondráková Z., Eliášová K., Vágner M. (2014). The anti-actin drugs latrunculin and cytochalasin affect the maturation of spruce somatic embryos in different ways. Plant Sci. 221–222, 90–99. 10.1016/j.plantsci.2014.02.006 PubMed DOI

Vondráková Z., Krajňáková J., Fischerová L., Vágner M., Eliášová K. (2016). “Physiology and role of plant growth regulators in somatic embryogenesis,” in Vegetative Propagation of Forest Trees, eds Park Y., Bonga J., Moon H. (Seoul: National Institute of Forest Science; ), 123–169.

Weber H., Borisjuk L., Heim U., Sauer N., Wobus U. (1997). A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds. Plant Cell 9 895–908. 10.1105/tpc.9.6.895 PubMed DOI PMC

Wickham H. (2009). ggplot2: Elegant Graphics for Data Analysis. New York, NY: Springer-Verlag.

Wickham H. (2017). tidyverse: Easily Install and Load the ‘Tidyverse’. R package version 1.2.1.

Williams L. E., Lemoine R., Sauer N. (2000). Sugar transporters in higher plants – a diversity of roles and complex regulation. Trends Plant Sci. 5 283–290. 10.1016/S1360-1385(00)01681-2 PubMed DOI

Willigen C. V., Verdoucq L., Boursiac Y., Maurel C. (2004). “Aquaporins in Plants,” in Membrane Transport in Plants Vol. 15 ed. Blatt M. R. (Hoboken, NJ: Blackwell Publishing Ltd; ), 247–281.

Withers S. G. (2001). Mechanisms of glycosyl transferases and hydrolases. Carbohydr. Polym. 44 325–337. 10.1016/s0144-8617(00)00249-6 DOI

Yu T., Li G., Dong S., Liu P., Zhang J., Zhao B. (2016). Proteomic analysis of maize grain development using iTRAQ reveals temporal programs of diverse metabolic processes. BMC Plant Biol. 16:241. 10.1186/s12870-016-0878-1 PubMed DOI PMC

Zhang J., Ma H., Chen S., Ji M., Perl A., Kovacs L., et al. (2009). Stress response proteins’ differential expression in embryogenic and non-embryogenic callus of Vitis vinifera L. cv. Cabernet Sauvignon - A proteomic approach. Plant Sci. 177 103–113. 10.1016/j.plantsci.2009.04.003 DOI

Zhao J., Wang B., Wang X., Zhang Y., Dong M., Zhang J. (2015). iTRAQ-based comparative proteomic analysis of embryogenic and non-embryogenic tissues of Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr). Plant Cell Tissue Organ Cult. 120 655–669. 10.1007/s11240-014-0633-y DOI