BACKGROUND: Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster's three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. CONCLUSIONS AND SIGNIFICANCE: This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function.

Central European Institute of Technology Brno University of Technology Technicka 3058 10 CZ 616 00 Brno Czech Republic European Union

CESAM Centre for Environmental and Marine Studies and Department of Chemistry University of Aveiro 3810 193 Aveiro Portugal European Union

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 CZ 613 00 Brno Czech Republic European Union

Department of Plant Biology Mendel University in Brno Zemedelska 1 CZ 613 00 Brno Czech Republic European Union

Institute of Scientific Instruments Academy of Sciences of the Czech Republic Kralovopolska 147 CZ 612 64 Brno Czech Republic European Union

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Zimmerman JL (1993) Somatic embryogenesis—A model for early development in higher-plants. Plant Cell 5: 1411–1423. PubMed PMC

Boo KH, Cao DV, Pamplona RS, Lee D, Riu KZ, Lee DS (2015) In vitro plant regeneration of Aster scaber via somatic embryogenesis. Biosci Biotechnol Biochem 79: 725–731. 10.1080/09168451.2014.996202 PubMed DOI

Kumar V, Moyo M, Van Staden J (2015) Somatic embryogenesis of Pelargonium sidoides DC. Plant Cell Tissue Organ Cult 121: 571–577.

Leljak-Levanic D, Mihaljevic S, Bauer N (2015) Somatic and zygotic embryos share common developmental features at the onset of plant embryogenesis. Acta Physiol Plant 37: 1–14.

Nic-Can GI, Galaz-Avalos RM, De-la-Pena C, Alcazar-Magana A, Wrobel K, Loyola-Vargas VM (2015) Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus. PLoS One 10: 1–21. PubMed PMC

Raju CS, Aslam A, Shajahan A (2015) High-efficiency direct somatic embryogenesis and plant regeneration from leaf base explants of turmeric (Curcuma longa L.). Plant Cell Tissue Organ Cult 122: 79–87.

Feher A (2015) Somatic embryogenesis—Stress-induced remodeling of plant cell fate. Biochim Biophys Acta-Gene Regul Mech 1849: 385–402. PubMed

Gomez-Garay A, Manzanera JA, Pintos B (2014) Embryogenesis in Oak species. A review. For Syst 23: 191–198.

Smertenko A, Bozhkov PV (2014) Somatic embryogenesis: life and death processes during apicalbasal patterning. J Exp Bot 65: 1343–1360. 10.1093/jxb/eru005 PubMed DOI

Chi C-M, Vits H, Staba EJ, Cooke TJ, Hu W-S (1994) Morphological kinetics and distribution in somatic embryo cultures. Biotechnol Bioenerg 44: 368–378. PubMed

Chi C-M, Zhang C, Steba EJ, Cooke TJ, Hu W-S (1996) An andvanced image analysis system for evaluation of somatic embryo development. Biotechnol Bioenerg 50: 65–72. PubMed

Olofsdotter M (1993) Image processing: a non-destructive method for measuring growth in cell and tissue culture. Plant Cell Rep 12: 216–219. 10.1007/BF00237057 PubMed DOI

Hamalainen JJ, Kurten U, Kauppinen V (1993) Classification of plant somatic embryos by computer vision. Biotechnol Bioenerg 41: 35–42. PubMed

Padmanabhan K, Cantliffe DJ, Harrell RC, Harrison J (1998) Computer vision analyses of somatic embryos of sweet potato [Ipomoea batatas (L.) Lam.] for assessing their ability to convert to plants. Plant Cell Rep 17: 681–684. PubMed

Honda H, Ito T, Yamada J, Hanai T, Matsuoka M, Kobayashi T (1999) Selection of embryogenic sugarcane callus by image analyses. J Biosci Bioeng 87: 700–702. PubMed

Schroder P, Fischer C, Debus R, Wenzel A (2003) Reaction of detoxification mechanisms in suspension cultured spruce cells (Picea abies L. Karst.) to heavy metals in pure mixture and in soil eluates. Environ Sci Pollut Res 10: 225–234. PubMed

Petrek J, Vitecek J, Vlasinova H, Kizek R, Kramer KJ, Adam V, et al. (2005) Application of computer imaging, stripping voltammetry and mass spectrometry for study of the effect of lead (Pb-EDTA) on growth and viability of early somatic embryos of Norway spruce (Picea abies /L./ Karst.). Anal Bioanal Chem 383: 576–586. PubMed

Garcia-Mendiguren O, Montalban IA, Stewart D, Moncalean P, Klimaszewska K, Rutledge RG (2015) Gene Expression Profiling of Shoot-Derived Calli from Adult Radiata Pine and Zygotic Embryo-Derived Embryonal Masses. PLoS One 10: 1–19. PubMed PMC

Duval I, Lachance D, Giguere I, Bomal C, Morency MJ, Pelletier G, et al. (2014) Large-scale screening of transcription factor-promoter interactions in spruce reveals a transcriptional network involved in vascular development. J Exp Bot 65: 2319–2333. 10.1093/jxb/eru116 PubMed DOI PMC

Vondrakova Z, Eliasova K, Vagner M, Martincova O, Cvikrova M (2015) Exogenous putrescine affects endogenous polyamine levels and the development of Picea abies somatic embryos. Plant Growth Regul 75: 405–414.

Capataz-Tafur J, Hernandez-Sanchez AM, Rodriguez-Monroy M, Trejo-Tapia G, Sepulveda-Jimenez G (2010) Sucrose induces arabinogalactan protein secretion by Beta vulgaris L. cell suspension cultures. Acta Physiol Plant 32: 757–764.

Letarte J, Simion E, Miner M, Kasha KJ (2006) Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture. Plant Cell Reports 24: 691–698. PubMed

Huska D, Zitka O, Krystofova O, Adam V, Babula P, Zehnalek J, et al. (2010) Effects of cadmium(II) ions on early somatic embryos of Norway spruce studied by using electrochemical techniques and nuclear magnetic resonance. Int J Electrochem Sci 5: 1535–1549.

Karcz W, Kurtyka R (2007) Effect of cadmium on growth, proton extrusion and membrane potential in maize coleoptile segments. Biol Plant 51: 713–719.

Nada E, Ferjani BA, Ali R, Bechir BR, Imed M, Makki B (2007) Cadmium-induced growth inhibition and alteration of biochemical parameters in almond seedlings grown in solution culture. Acta Physiol Plant 29: 57–62.

Tukaj Z, Bascik-Remisiewicz A, Skowronski T, Tukaj C (2007) Cadmium effect on the growth, photosynthesis, ultrastructure and phytochelatin content of green microalga Scenedesmus armatus: A study at low and elevated CO2 concentration. Environ Exp Bot 60: 291–299.

Wu Q, Su NN, Chen Q, Shen WB, Shen ZG, Xia Y, et al. (2015) Cadmium-Induced Hydrogen Accumulation Is Involved in Cadmium Tolerance in Brassica campestris by Reestablishment of Reduced Glutathione Homeostasis. PLoS One 10: 1–6. PubMed PMC

Elloumi N, Zouari M, Chaari L, Ben Abdallah F, Woodward S, Kallel M (2015) Effect of phosphogypsum on growth, physiology, and the antioxidative defense system in sunflower seedlings. Environ Sci Pollut Res 22: 14829–14840. PubMed

Hazubska-Przybyl T, Wawrzyniak M, Obarska A, Bojarczuk K (2015) Effect of partial drying and desiccation on somatic seedling quality in Norway and Serbian spruce. Acta Physiol Plant 37: 1–6.

Rejon JD, Delalande F, Schaeffer-Reiss C, Carapito C, Zienkiewicz K, Alche JD, et al. (2013) Proteomics profiling reveals novel proteins and functions of the plant stigma exudate. J Exp Bot 64: 5695–5705. 10.1093/jxb/ert345 PubMed DOI PMC

Sala K, Potocka I, Kurczynska E (2013) Spatio-temporal distribution and methyl-esterification of pectic epitopes provide evidence of developmental regulation of pectins during somatic embryogenesis in Arabidopsis thaliana. Biol Plant 57: 410–416.

Vatankhah E, Niknam V, Ebrahimzadeh H (2014) Histological and biochemical parameters of Crocus sativus during in vitro root and shoot organogenesis. Biol Plant 58: 201–208.

Tillman-Sutela E, Kauppi A (2014) Maturity of surface structures in northern Pinus sylvestris L. seeds: A key to improved prediction of germination potential. Flora 209: 45–53.

Vitecek J, Petrlova J, Adam V, Havel L, Kramer KJ, Babula P, et al. (2007) A fluorimetric sensor for detection of one living cell. Sensors 7: 222–238.

Blazquez S, Olmos E, Hernandez JA, Fernandez-Garcia N, Fernandez JA, Piqueras A (2009) Somatic embryogenesis in saffron (Crocus sativus L.). Histological differentiation and implication of some components of the antioxidant enzymatic system. Plant Cell Tissue Organ Cult 97: 49–57.

Durzan DJ, Jokinen K, Guerra MP, Santerre A, Chalupa V, Havel L (1994) Latent diploid parthenogenesis and parthenote cleavage in egg-equivalents of Norway spruce. Int J Plant Sci 155: 677–688.

VonArnold S (1987) Improved efficiency of somatic embryogenesis in mature embryos of Picea-abies (L) Karst. J Plant Physiol 128: 233–244.

Havel L, Durzan DJ (1996) Apoptosis during diploid parthenogenesis and early somatic embryogenesis of Norway spruce. Int J Plant Sci 157: 8–16.

Havel L, Durzan DJ (1996) Apoptosis in plants. Bot Acta 109: 268–277.