Integrated proteomics and histochemical analysis of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) in embryogenic suspension culture


  • Francis Pereira Dias Federal University of Santa Catarina
  • Neusa Steiner Federal University of Santa Catarina
  • Gabriela C. Cangahaula-Inocente Federal University of Santa Catarina
  • Ana Paula Lando Federal University of Santa Catarina
  • Marisa Santos Federal University of Santa Catarina
  • Miguel Pedro Guerra Federal University of Santa Catarina



Brazilian pine, cell suspension, proteomics, proembryogenic masses, somatic embryogenesis


Cell suspension culture is a useful in vitro model-system for both scaling up and conserving the Brazilian conifer Araucaria angustifolia. In the present work, cell suspension of Brazilian pine was subjected to proteomics, biochemical and histochemical analyses. The results revealed new insights underlying the molecular mechanism of proembryogenic masses transition in cell suspension. Embryogenic cell cultures were cultivated in a basal liquid medium modified in a Steward apparatus (orbital agitator). Cell growth dynamics was evaluated using cell volume after sedimentation, fresh weight, mitotic index, conductivity, pH, and the number of proembryogenic masses (PEMs: I, II, III). Histochemical parameters, cell viability, and cell death analyses were performed to pinpoint growth rates. Proteomics analysis was performed using two-dimensional electrophoresis, and protein identification was carried out by MALDI-TOF-TOF tandem mass spectrometry. Cell growth dynamics showed a predominance of PEM III. Maximum slope of the exponential phase growth in fresh weight occurred at exponential phase after 15 days (optimal cultivation time), after which cell viability and pH decreased, thereby allowing the identification of stress-related proteins. Several metabolism and growth proteins were abundant, such as: cytoskeletal, WOX1, cytokinin-related, and auxin-related proteins acting on cell wall modification, suspensor cell formation, and PEM I to PEM III transition.


Abrahamsson M., Valladares S., Larsson E., Clapham D., von Arnold S., 2012. Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death. Plant Cell Tissue, Organ and Culture 109: 391-400. DOI: 10.1007/s11240-011-0103-8.Balbuena T.S., 2009. Proteômica do desenvolvimento da semente de Araucaria angustifolia, Instituto de Biociências, Universidade de São Paulo, São Paulo, 102 p.Balbuena T.S., Jo L., Pieruzzi F.P., Dias L.L.C., Silveira V., Santa-Catarina C., Junqueira M., Thelen J.J., Schevchenki A., Floh E.S.I., 2011. Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia. Phytochemistry 72: 302-311. DOI: 10.1016/j.phytochem.2010.12.007.Balbuena T.S., SIlveira E.R., Junqueira M., Dias L.L.C., Santa-Catarina C., Shevchenko A., Floh E.S.I., 2009. Changes in the 2-DE protein profile during zygotic embryogenesis in the Brazilian pine (Araucaria angustifolia). Journal of Proteomics 72: 337-352. DOI: 10.1016/j.jprot.2009.01.011.Barkla B.J., Vera-Estrella R., Hernández-Coronado M., Pantoja O., 2009. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance. The Plant Cell 21: 4044-4058. DOI: 10.1105/tpc.109.069211.Benzanilla M., Gladfelter A.S., Kovar D.R., Lee W.-L., 2015. Cytoskeletal dynamics: a view from the membrane. The Jounal of Cell Biology 209: 329-337. DOI: 10.1083/jcb.201502062.Bevan M., Bancroft I., Bent E., Love K., Goodman H., Dean C., Bergkamp R., ... Chalwatzis N., 1998. Analysis of 1.9Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391: 485-488. DOI: 10.1038/35140.Bou Daher F., Braybrook S.A., 2015. How to let go: pectin and plant cell adhesion. Frontiers in Plant Science 6: 5-23. DOI: 10.3389/fpls.2015.00523.Bozhkov P.V., Filanova L.H., von Arnold S., 2001. A key development switch during Norway spruce somatic embryogenesis is induced by withdrawl of growth regulators and is associated with cell death and extracellular acidification. Biotechnology and bioengineering 77: 658-667. DOI: 10.1002/bit.10228.Bradford M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Analytical Biochemistry 71: 248-256. DOI: 10.1006/abio.1976.9999.Branco C.S., Doung A., Machado A.K., Scola G., Andreazza A.C., Salvador M., 2019. Modulation of mitochondrial and epigenetic targets by polyphenols-rich extract from Araucaria angustifolia in larynx carcinoma. Anti-cancer Agents in Medicinal Chemistry 19: 130-139. DOI: 10.2174/1871520618666180816142821.Buchanan B.B., Gruissem W., Jones R.L., 2000. Biochemistry and molecular biology of plants. American Society of Plant Physiology, Maryland, 1367 p.Cai G., 2010. Assembly and disassembly of plant microtubules: tubulin modifications and binding to MAPs. Journal of Experimental Botany 61: 623-626. DOI: 10.1093/jxb/erp395.Cangahuala-Inocente G.C., Amaral F.P.d., Faleiro A.C., Huergo L.F., Arisi A.C.M., 2013. Identification of six differentially accumulated proteins of Zea mays seedlings (DKB240 variety) inoculated with Azospirillum brasilense strain FP2. European Journal of Soil Biology 58: 45-50. DOI: 10.1016/j.ejsobi.2013.06.002 46.Carlsson L., Nyström L.-E., Sundkvist I., Markey F., Lindberg U., 1977. Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells. Journal of Molecular Biology 115: 465-483. DOI: 10.1016/0022-2836(77)90166-8.Chau N.-H., Ramachandran S., Molager H.E., Hedehusene C., 2002. Alteration of plant morphology by control of profilin expression. In: Patent US (ed). Institute of Molecular Agrobiology, US pp. 1-10.Chen S., Harmon A.C., 2006. Advances in plant proteomics. Proteomics 6: 5504-5516. DOI: 10.3389/fpls.2015.00209.De Oliveira L.F., Navarro B.V., Cerruti G., Elbl P., Minocha R., Minocha S.C., Dos Santos A.L.W., Floh E.S.I., 2018. Polyamines and amino acid related metabolism: the roles of argenine and ornithine are associated with embryogenic potential. Plant Cell Physiology 9(5): 1084–1098. DOI: 10.1093/pcp/pcy049.Dos Santos A.L.W., Elbl P., Navarro B.V., Oliveira L.F., Salvato F., Balbuena T.S., Floh E.S.I., 2016. Quantitative proteomic analysis of Araucaria angustifolia (Bertol.) Kuntze cell lines with contrasting embryogenic potential. Proteomics 130: 180-189. DOI: 10.1016/j.jprot.2015.09.027.Dos Santos A.L.W., Silveira V., Steiner N., Maraschin M., Guerra M.P., 2010. Biochemical and morphological changes during the growth kinectics of Araucaria angustifolia suspension cultures. Brazilian Archives of Biology and Technology 53: 497-504. DOI: 10.1590/S1516-89132010000300001.Elbl P., Lira B.S., Andrade S.C.S., Jo L., dos Santos A.L.W., Coutinho L.L., Floh E.S.I., Rossi M., 2015. Comparative transcriptome analysis of early somatic embryo formation and seed development in Brazilian pine, Araucaria angustifolia (Bertol.) Kuntze. Plant Cell Tissue, Organ and Culture 120: 903-915. DOI: 10.1016/j.jprot.2014.01.007.Farias-Soares F.L., Burrieza H.P., Steiner N., Maldonado S., Guerra M.P., 2013. Immunoanalysis of dehydrins in Araucaria angustifolia embryos. Protoplasma 250: 911-918. DOI: 10.1007/s00709-012-0474-7.Farias-Soares F.L., Steiner N., Schmidt E.C., Pereira M.L.T., Rogge-Renner G.D., Bouzon Z.L., Floh E.S.I., Guerra M.P., 2014. The transition of proembryogenic masses to somatic embryos in Araucaria angustifolia (Bertol.) Kuntze is related to the endogenous contents of IAA, ABA and polyamines. Acta Physiologiae Plantarum 36: 1853-1865. DOI: 10.1007/s11738-014-1560-6.Fehér A., Pasternak T.P., Dutis D., 2003. Transition of somatic plant cells to an embryogenic state. Plant Cell, Tissue Organ and Culture 74: 201-228. DOI: 10.1023/A:1024033216561.Fey S.J., Larsen P.M., 2001. 2D or not 2D. Current opinion in Chemical Biology 5: 26-33. DOI: 10.1002/ar.23752.Fraga H.P.F., Vieira L.d.N., Heringer A.S., Puttkammer C.C., Silviera V., Guerra M.P., 2016. DNA methylation and proteome profiles of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures as affected by plant growth regulators supplementation. Plant Cell Tissue, Organ and Culture 125: 353–374. DOI: 10.1007/s11240-016-0956-y.Fraga H.P.F., Vieira L.d.N., Puttkammer C.C., Oliveira E.M., Guerra M.P., 2015. Time-lapse cell tracking reveals morphohistological features in somatic embryogenesis of Araucaria angustifolia (Bert) O. Kuntze. Trees 29: 1613-1623. DOI: 10.1007/s00468-015-1244-x.George E.F., Hall M.A., Klerk G.-J.d., 2008. Plant propagation by tissue culture. Springer, Dordrecht, 503 p.Guerra M.P., Steiner N., Farias-Soares F.L., Vieira L.d.N., Fraga H.P.F., Rogge-Renner G.D., Maldonado S., 2016. Somatic embryogenesis in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae). In: Germanà M.A., Lambardi M. (eds) In vitro embryogenesis in higher plants (Methods in molecular biology). Springer, New York, pp. 439-450.Gupta P.K., Pullman G.S., 1991. Method for reproducing coniferous plants by somatic embryogenesis using abscisic acid and osmotic potential variation. US patent 5: 36-37.Haecker A., Gross-Hardt R., Geiges B., Sarkar A., Breuninger H., Herrmann M., Laux T., 2004. Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 131: 656-668. DOI: 10.1242/ dev.00963.Hakman I., Fowke L.C., 1987. An embryogenic cell suspension culture of Picea glauca (white spruce). Plant Cell Reports 6: 20-22. DOI: 10.1007/BF00269730.Hedman H., Zhu T., von Arnold S., Sohlberg J.J., 2013. Analysis of the WUSCHEL-RELATED HOMEOBOX gene family in the conifer Picea abies reveals extensive conservation as well as dynamic patterns. BMC Plant Biology, 13(1): 89. DOI: 10.1186/1471-2229-13-89.Heldt H.-W., Piechulla B., Heldt F., 2011. Plant Biochemistry, Elsevier, London, p.Heng Y.-W., Koh C.-G., 2010. Actin cytoskeleton dynamic and the cell division cycle. The International Journal of Biochemistry and Cell Biology 42: 1622-1633. DOI: 10.1016/j.biocel.2010.04.007.Hussey P.J., Ketelaar T., Deeks M.J., 2006. Control of actin cytoskeleton in plant cell growth. Annuals Review Plant Biology 57: 109-125. DOI: 10.1146/annurev.arplant.57.032905.105206.Inza M.V., Aguirre N.C., Torales S.L., Pahr N.M., Fassola H.E., Fornes L.F., Zelener N., 2018. Genetic variability of Araucaria angustifolia in the Argentinean Parana Forest and implications for management and conservation. Trees 32(4): 1135-1146. DOI: 10.1007/s00468-018-1701-4.Issawi M., Muhieddine M., Girard C., Sol V., Rioul C., 2017. Unexpected features of exponentially growing Tobacco Bright yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition. Glycoconjugate Journal 34: 585-590. DOI: 10.1007/s10719-017-9782-7.IUCN. 2017. The IUCN Red List of Threatened Species. 10.2305/IUCN.UK.2008.RLTS.Jamet E., Canut H., Boudart G., Pont-Lezica R.F., 2006. Cell wall proteins: a new insight through proteomics. Trends in Plant Science 38: 33-39. DOI: 10.1016/j.tplants.2005.11.006.Jensen W.A., 1962. Botanical histochemistry (principles and pratice). W. H. Freeman and Company, San Francisco, 408 p.Jo L., Dos Santos A.L.W., Bueno C.A., Barbosa H.R., Floh E.S.I., 2014. Proteomic analysis and polyamines, ethylene and reactive oxygen species levels of Araucaria angustifolia (Brazilian pine) embryogenic cultures with different embryogenic potential. Tree Physiology 34: 94-104. DOI: 10.1093/t ree phy s/tp t102.Johansen D.A., 1940. Plant microtechnique, Mc Graw Hill New York, 523 p.Knox J.P., 1992. Cell adhesion, cell separation and plant morphogenesis. The Plant Journal 2: 137-141. DOI: 10.1111/j.1365-313X.1992.00137.x.Kraus J.E., Arduin M., 1997. Manual básico de métodos em morfologia vegetal, Universidade Rural do Rio de Janeiro, Seropédica, 198 p.Kurakawa T., Ueda N., Maekawa M., Kobayashi K., Kojima M., Nagato Y., Sakakibara H., Kyozuka J., 2007. Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445: 652-655. DOI: 10.1038/nature05504.Kuroha T., Tokunaga H., Kojima M., Ueda N., Ishida T., Nagawa S., Fukuda H., Sugimoto K., Sakakibara H., 2009. Functional analyses of LONELY GUY cytokinin-activating enzymes reveal the importance of the direct activation pathway in Arabidopsis. The Plant Cell 21: 3152-3169. DOI: 10.1105/tpc.109.068676Larsson E., Sitbon F., Liung K., von Arnold S., 2008. Inhibited polar auxin transport results in aberrant embryo development in Norway spruce. New Phytologist 177: 365-366. DOI: 10.1111/j.1469-8137.2007.02289.x.Li X., Han J.-D., Fang S.-N., Bai S.-N., Rao G.-Y., 2017. Embryogenesis-associated genes during somatic embryogenesis of Adiantum capillus-veneris L. in vitro: new insights into the evolution of reproductive organs in land plants. Frontiers in Plant Science 8: 1-13. DOI: 10.3389/fpls.2017.00658.Lian G., Ding Z., Wang Q., Zhang D., Xu J., 2014. Origins and evolution of WUSCHEL-Related Homeobox protein family in plant kingdom. The Scientific World Journal 2014: 1-12. DOI: 10.1155/2014/534140.Llyod C., Chan J., 2008. The parallel lives of microtubules and cellulose microfibrils. Current opinion in Cell Biology 11: 641-646. DOI: 10.1016/j.pbi.2008.10.007.Lulsdorf M.M., Tautorus T.E., Kikcio S.I., Dunstan D.I., 1992. Growth parameters of embryogenic suspension cultures of interior spruce (Picea glauca-engelmannii) and black spruce (Picea mariana Mill.). Plant Science 82: 227-234. DOI: 10.1016/0168-9452(92)90224-A.Mathesius U., Keijzers G., Natera S.H.A., Weinman J.J., Djordjevic M.A., Rolfe B.G., 2001. Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting. Proteomics 1: 1424-1440. DOI: 10.1002/1615-9861.Maurer J.B.B., Bacic A., Pereira-Netto A.B., Donatti L., Zawadzki-Baggio S.F., Pettolino F.A., 2010. Arabinogalactan-proteins from cell suspension cultures of Araucaria angustifolia. Phytochemistry 71: 1400-1409. DOI: 10.1016/j.phytochem.2010.04.021.Mortier V., Wasson A., Jaworek P., Keyser A.d., Decroos M., Holster M., Trakowski P., Mathesius U., Goormachtig S., 2014. Role of LONELY GUY genes in indeterminate nodulation on Medicago truncatula. New Phytologist 202: 582-593. DOI: 10.1111/nph.12681.Moser J.R., Gonçalves Garcia M., Viana M., 2004. Establishment and growth of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Plant Cell Tissue, Organ and Culture 78: 37-42. DOI: 10.1023/B:TICU.0000020387.96568.25.Muschitz A., Riou C., Mollet J.-C., GLoaguen V., Faugeron C., 2015. Modifications of cell wall pectin in tomato cell suspension in response to cadmium and zinc. Acta Physiologiae Plantarum 37: 245. DOI: 10.1007/s11738-015-2000-y.Mustafa N., Winter W., Iren F., Verpoorte R., 2011. Initiation, growth and crypreservation of plant cell suspensions cultures. Nature Protocols 6: 715-742. DOI: 10.1038/nprot.2010.144.Oppenheimer D.G., Haas N., Silflow C.D., Snustad D.P., 1988. The beta tubulin gene family of Arubidopsis thaliana: preferential accumulation of the beta1 transcript in roots. Gene 63: 87-102. DOI: 10.1016/0378-1119(88)90548-3.Palovaara J., Hakman I., 2008. Conifer WOX-related homeodomain transcription factors, developmental consideration and expression dynamic of WOX2 during Picea abies somatic embryogenesis. Plant Molecular Biology 66: 533-549. DOI: 10.1007/s11103-008-9289-5.Pieruzzi F.P., Dias L.L.C., Balbuena T.S., Santa-Catarina C., Dos Santos A.L.W., Floh E.S.I., 2011. Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm). Annals of Botany 108: 337-345. DOI: 10.1093/aob/mcr133.Pozo J.C.d., Manzano C., 2014. Auxin and the ubiquitin pathway. Two players–one target: the cell cycle in action. Journal of Experimental Botany 65: 2617-2632. DOI: 10.1093/jxb/ert363.Rabilloud T., Chevallet M., Luche S., Lelong C., 2010. Two-dimensional gel electrophoresis in proteomics: past, present and future. Journal of Proteomics 73: 2064-2077. DOI: 10.1016/j.jprot. 2010.05.016.Radauer C., Hoffmann-Sommergruber K., 2007. Profilins. In: Mills ENC, Shewry PR (eds) Plant food allergens. Blackwell Publishing, Oxford, pp. 105-124.Ramagli L.S., Rodriguez L.V., 1985. Quantitation of microgram amounts of protein in twodimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 1085: 559-563. DOI: 10.1002/elps.1150061109.Reis M.S.d., Montagna T., Mattos A.G., Filippon S., Ladio A.H., Marques A.d.C., Zechini A.A., Peroni N., Mantovani A., 2018. Domesticated landscapes in Araucaria forests, Southern Brazil: A multispecies local conservation-by-use system. Frontiers in Ecology and Evolution 6: 11. DOI: 10.3389/ fevo.2018.00011.Rockenbach M.F., Boneti J.I., Cangahuala-Inocente G.C., Gavioli-Nascimento M.C.A., Guerra M.P., 2015. Histological and proteomics analysis of apple defense responses to the development of Colletotrichum gloeosporioides on leaves. Physiological and Molecular Plant Pathology 89: 97-107. DOI: 10.1016/j.pmpp. 2015.01.003.Rogowska-Wrzesinska A., Bihan M.-C.L., Thaysen-Andersen M., Roepstorff P., 2013. 2D gels still have a niche in proteomics. Journal of Plant Proteomics 88: 4-13. DOI: 10.1016/j.jprot.2013.01.010.Salmen Espindola L., Noin M., Corbineau F., Côme D., 1994. Cellular and metabolic damage induced by desiccation in recalcitrant Araucaria angustifolia embryos. Seed Science Research 4: 1993-1201. DOI: 10.1017/S096025850000218X.Santer A., Estelle M., 2009. Recent advances and emerging trends in plant hormone signalling. Nature 459: 1071-1078. DOI: 10.1038/ nature08122.Sass J.E., 1951. Botanical microtechnique. Iowa State College, Ames, 228 p.Shi H.-Y., Zhang Y.-X., Chen L., 2013. Two pear auxin-repressed protein genes, PpARP1 and PpARP2, are predominantly expressed in fruit and involved in response to salicylic acid signaling. Plant Cell Tissue, Organ and Culture 114: 279-286. DOI: 10.1007/ s11240-013-0321-3.Silveira V., Balbuena T.S., Santa-Catarina C., Floh E.S.I., Guerra M.P., Handro W., 2004. Biochemical changes during seed development in Pinus taeda L. Plant Growth Regulation 44: 147-156. DOI: 10.1007/s10725-004-2601-8.Silveira V., Steiner N., Dos Santos A.L.W., Nodari R.O., Guerra M.P., 2002. Biotechnology tolls in Araucaria angustifolia conservation and improvement: inductive factors affecting somatic embryogenesis. Crop Breeding and Applied Biotechnology 2: 463-470. DOI: 10.12702/1984-7033.v02n03a18Somers D.E., Fujiwara S., 2009. Thinking outside the F-box: novel ligands for novel receptors. Trends in Plant Science 14: 206-213. DOI: 10.1016/j.tplants.2009.01.003.Stasolla C., Yeung E.C., 2003. Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell, Tissue Organ and Culture 73: 15-35. DOI: 10.1023/A: 1023345803336.Stefenon V.M., Gailing O., Finkeldey R., 2007. Genetic structure of Araucaria angustifolia (Araucariaceae) populations in Brazil: implications for the in situ conservation of genetic resources. Plant Biology 9: 516-525. DOI: 10.1055/s-2007-964974.Steiner N., Farias-Soares F.L., Schmidt E.C., Pereira M.L.T., Scheid B., Rogge-Renner G.D., Bouzon Z.L., Schmidt D., Maldonado S., Guerra M.P., 2015. Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze. Protoplasma 253(2), 487-501. DOI: 10.1007/ s00709-015-0827-0.Steiner N., Santa-Catarina C., Andrade J.B.R., Balbuena T.S., Guerra M.P., Handro W., Floh E.S.I., Silveira V., 2008. Araucaria angustifolia biotechnology. Functional Plant Science and Biotechnology 2: 20-28. DOI: 10.1590/S1516-89132002000100015Steiner N., Santa-Catarina C., Guerra M.P., Cutri L., Dornelas M., Floh E.S.I., 2012. A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis. Plant Cell Tissue, Organ and Culture 109: 41-50. DOI: 10.1007/s11240-011-0071-z.Steiner N., Santa-Catarina C., Silveira V., Floh E.S.I., Guerra M.P., 2007. Polyamine effects on growth and endogenous hormones levels in Araucaria angustifolia embryogenic cultures. Plant Cell Tissue, Organ and Culture 89: 55-62.Steiner N., Vieira F.d.N., Maldonado S., Guerra M.P., 2005. Effect of carbon source on morphology and histodifferentiation of Araucaria angustifolia embryogenic cultures. Brazilian Archives of Biology and Technology 48: 895-903. DOI: 10.1590/S1516-89132005000800005.Steward F.C., Mapes M.O., Mears K., 1958. Growth and organized development of cultured cells II. Organization in cultures grown from freely suspended cells. American Journal of Botany 45: 705-708. DOI: 10.1002/j.1537-2197.1958.tb10599.x.Straeten D.V.D., Pousada-Rodrigues R.A., Goodman H.M., Montagu M.V., 1991. Plant enolase: gene structure, expression and evolution. The Plant Cell 3: 719-735. DOI: 10.1105/tpc.3.7.719.Szabados L., Mroginski L.A., Roca W.M., 1993. Suspensiones celulares: descripción, manipulación y aplicaciones. In: Roca W.M., Mroginski L.A. (eds) Cultivo de tejidos en la agricultura: fundamentos y aplicaciones. CIAT, Cali, pp. 174-210.van der Graaff E., Laux T., Rensing S.A., 2009. The WUS homeobox-containing (WOX) protein family. Genome Biology 10(12): 248. DOI: 10.1186/gb-2009-10-12-248.Vogel G., 2005. How does a single somatic cell become a whole plant? Science 309: 86. DOI: 10.1126/science.309.5731.86.Von Arnold S., 2008. Somatic embryogenesis. In: George E.F., Hall M.A., Klerk G.-Jd. (eds) Plant propagation by tissue culture, 3 rd. edn. Springer, Dordrecht, pp. 335-358.Von Arnold S., Egertsdotter U., Ekberg I., Gupta P.K., Newton R.J., 1995. Somatic embryogenesis in Norway spruce (Picea abies). In: Jain S.M., Gupta P.K., Newton R.J. (eds) Somatic embryogenesis in woody plants. Springer Netherlands, Dordrchet, pp. 44-46.Von Arnold S., Sabala I., Bozhkov P.V., Dyachok J., Filanova L.H., 2002. Developmental pathways of somatic embryogenesis. Plant Cell, Tissue Organ and Culture 69: 233-249. DOI: 10.1023/A: 1015673200621.Webster J.M., Oxley D., Pettolino F.A., Bacic A., 2008. Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis. Phytochemistry 69: 873-881. DOI: 10.1016/j.phytochem.2007.10.009.Wink M., 1994. The cell culture medium - a functional extracellular compartment of suspension-cultured cells. Plant Cell Tissue, Organ and Culture 38: 307-319. DOI: 10.1007/BF00033891.






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