BACKGROUND: To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g- 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g- 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels. RESULTS: Repetitive somatic embryogenesis dramatically improved the proliferating lines' cellular organization (genotype SD4's most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300-2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on "Proteolysis" and "Catabolic process" Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same "Plant development" and "Defense response" biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines. CONCLUSIONS: This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines' embryogenic status.

BioForA INRA ONF F 45075 Orléans France

BIOGECO INRA University Bordeaux F 33610 Cestas France

Institute of Experimental Botany of the Czech Academy of Sciences Rozvojová 263 165 02 Praha 6 Lysolaje Czech Republic

Plateforme Protéome Centre de Génomique Fonctionnelle University Bordeaux F 33000 Bordeaux France

Pôle Biotechnologie et Sylviculture Avancée FCBA Campus Forêt Bois de Pierroton F 33610 Cestas France

SylvaLIM University Limoges F 78060 Limoges France

University Clermont Auvergne INRA PIAF F 63000 Clermont Ferrand France

Zobrazit více v PubMed

Pijut PM, Lawson SS, Michler CH. Biotechnological efforts for preserving and enhancing temperate hardwood tree biodiversity, health, and productivity. In Vitro Cell Dev Biol Plant. 2011;47(1):123–147. doi: 10.1007/s11627-010-9332-5. DOI

Isah T. Induction of somatic embryogenesis in woody plants. Acta Physiol Plant. 2016;38(5):118. doi: 10.1007/s11738-016-2134-6. DOI

Klimaszewska K, Hargreaves CL, Lelu-Walter M-A, Trontin J-F. Advances in conifer somatic embryogenesis since year 2000. In: Germanà MA, Lambardi M, editors. In vitro embryogenesis in Higher plants. Springer science + business media, New York. 2016. pp. 131–162. PubMed

Durzan D, Gupta P. Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures. Plant Sci. 1987;52(3):229–235. doi: 10.1016/0168-9452(87)90056-2. DOI

Reeves C, Hargreaves C, Trontin J-F, Lelu-Walter M-A. Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir. Trees. 2017;32(1):175–190. doi: 10.1007/s00468-017-1622-7. DOI

Lelu-Walter M-A, Gautier F, Eliášová K, Sanchez L, Teyssier C, Lomenech A-M, Le Metté C, Hargreaves C, Trontin J-F, Reeves C. 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. 2017;132(1):137–155. doi: 10.1007/s11240-017-1318-0. DOI

Lelu-Walter M-A, Thompson D, Harvengt L, Sanchez L, Toribio M, Pâques LE. Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Gen Genomes. 2013;9(4):883–899. doi: 10.1007/s11295-013-0620-1. DOI

Lelu-Walter M-A, Klimaszewska K, Miguel C, Aronen T, Hargreaves C, Teyssier C, Trontin J-F. Somatic embryogenesis for more effective breeding and deployment of improved varieties in Pinus spp.: bottlenecks and recent advances. In: Loyola-Vargas VM, Ochoa-Alejo N, editors. Somatic embryogenesis: fundamental aspects and applications. Cham: Springer International Publishing; 2016. pp. 319–365.

Trontin J-F, Klimaszewska K, Morel A, Hargreaves CL, Lelu-Walter M-A. Molecular aspects of conifer zygotic and somatic embryo development: a review of genome-wide approaches and recent insights. In: Germana M, Lambardi M, editors. In Vitro Embryogenesis in Higher Plants Methods Mol Biol. New York: SpringerScience + BusinessMedia; 2016. pp. 131–166. PubMed

Williams EG, Maheswaran G. Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group. Ann Bot. 1986;57(4):443–462. doi: 10.1093/oxfordjournals.aob.a087127. DOI

von Aderkas P, Teyssier C, Charpentier J-P, Gutmann M, Pâques L, Le Metté C, Ader K, Label P, Kong L, Lelu-Walter M-A. Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii) Ann Bot. 2015;115(4):605–615. doi: 10.1093/aob/mcu254. PubMed DOI PMC

Morel A, Teyssier C, Trontin J-F, Eliášová K, Pešek B, Beaufour M, Morabito D, Boizot N, Le Metté C, Belal-Bessai L, et al. Early molecular events involved in Pinus pinaster Ait. Somatic embryo development under reduced water availability: transcriptomic and proteomic analyses. Physiol Plant. 2014;152(1):184–201. doi: 10.1111/ppl.12158. PubMed DOI

Merkle S. Strategies for dealing with limitations of somatic embryogenesis in hardwood trees. Plant Tiss Cult & Biotech. 1995;1:112–121.

Ballester A, Corredoira E, Vieitez A. Limitations of somatic embryogenesis in hardwood trees. In: Park Y, Bonga J, Moon H, editors. Vegetative Propagation of Forest Trees. Seoul, Korea: Korea Forest Research Institute; 2016. pp. 56–74.

von Aderkas P, Bonga J, Klimaszewska K, Owens J. Comparison of larch embryogeny in vivo and in vitro. In: Ahuja M, editor. Woody Plant biotechnology. Boston, MA: Springer; 1991. pp. 139–155.

Gautier F, Eliášová K, Reeves C, Sanchez L, Teyssier C, Trontin J-F, Le Metté C, Vágner M, Costa G, Hargreaves C, et al. What is the best way to maintain embryogenic capacity of embryogenic lines initiated from Douglas-fir immature embryos ? In: Bonga J, Park Y, Trontin J, et al., editors. Proceedings 4th International Conference of the IUFRO Unit 20902 on “Development and application of vegetative propagation technologies in plantation forestry to cope with a changing climate and environment”. 2017. pp. 283–286.

Hernández I, Cuenca B, Carneros E, Alonso-Blázquez N, Ruiz M, Celestino C, Ocaña L, Alegre J, Toribio M: Application of plant regeneration of selected cork oak trees by somatic embryogenesis to implement multivarietal forestry for cork production. In: Tree For Sci Biotechnol. Vol. 5; 2011: 19–26.

Lee NN, Moon HK, Lee J-W, Choi YE, Park S-Y. Somatic embryogenesis and plant regeneration from a 700-year-old Kalopanax septemlobus tree. Trees. 2017;31(5):1439–1451. doi: 10.1007/s00468-017-1560-4. DOI

Pinto G, Correia S, Corredoira E, Ballester A, Correia B, Neves L, Canhoto J. In: In vitro culture of Eucalyptus: where do we stand? In: Vegetative Propagation of Forest Trees. Park Y, Bonga J, Moon H, editors. Seoul, Korea: Korea Forest Research Institute; 2016. pp. 441–462.

Bonga JM, Klimaszewska KK, von Aderkas P. Recalcitrance in clonal propagation, in particular of conifers. Plant Cell Tiss Org. 2010;100(3):241–254. doi: 10.1007/s11240-009-9647-2. DOI

Trontin J-F, Aronen T, Hargreaves C, Montalbán I, Moncaleán P, Reeves C, Quoniou S, Lelu-Walter M-A, Klimaszewska K: International effort to induce somatic embryogenesis in adult pine Trees In: Vegetative Propagation of Forest Trees. Edited by Park Y, Bonga J, Moon H, Korea Forest Research Institute edn. Seoul, Korea: Korea Forest Research Institute; 2016: 211–260.

Ruaud J-N, Bercetche J, Pâques M. First evidence of somatic embryogenesis from needles of 1-year-old Picea abies plants. Plant Cell Rep. 1992;11(11):563–566. doi: 10.1007/BF00233093. PubMed DOI

Klimaszewska K, Overton C, Stewart D, Rutledge R. 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. 2011;233(3):635–647. doi: 10.1007/s00425-010-1325-4. PubMed DOI

Harvengt L, Trontin J-F, Reymond I, Canlet F, Paques M. Molecular evidence of true-to-type propagation of a 3-year-old Norway spruce through somatic embryogenesis. Planta. 2001;213(5):828–832. doi: 10.1007/s004250100628. PubMed DOI

Uddenberg D, Valladares S, Abrahamsson M, Sundström JF, Sundås-Larsson A, von Arnold S. Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor. Planta. 2011;234(3):527–539. doi: 10.1007/s00425-011-1418-8. PubMed DOI PMC

Charest PJ, Devantier Y, Lachance D. Stable genetic transformation of Picea mariana (black spruce) via particle bombardment. In Vitro Plant. 1996;32(2):91–99. doi: 10.1007/BF02823137. DOI

Lelu MA, Klimaszewska K, Charest P. Somatic embryogenesis from immature and mature zygotic embryos and from cotyledons and needles of somatic plantlets of Larix. Can J For Res. 1994;24(1):100–106. doi: 10.1139/x94-015. DOI

Saly S, Joseph C, Corbineau F, Lelu MA, Côme D. Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene. Plant Growth Regul. 2002;37(3):287–294. doi: 10.1023/A:1020856112765. DOI

Vooková B, Matúšová R, Kormuťák A. Secondary somatic embryogenesis in Abies numidica. Biol Plant. 2003;46(4):513–517. doi: 10.1023/A:1024899124774. DOI

Vooková B, Kormuťák A. Comparison of induction frequency, maturation capacity and germination of Abies numidica during secondary somatic embryogenesis. Biol Plant. 2006;50(4):785–788. doi: 10.1007/s10535-006-0132-z. DOI

Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-Walter M-A. Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.) In Vitro Cell Dev Biol Plant. 2009;45(1):20–33. doi: 10.1007/s11627-008-9158-6. DOI

von Aderkas P, Kong L, Prior N. In vitro techniques for conifer embryogenesis. In: Park Y, Bonga J, Moon H, editors. Vegetative Propagation of Forest Trees. Seoul, Korea: Korea Forest Research Institute; 2016. pp. 335–350.

Egertsdotter U, Von Arnold S. Classification of embryogenic cell-lines of Picea abies as regards protoplast isolation and culture. J Plant Physiol. 1993;141(2):222–229. doi: 10.1016/S0176-1617(11)80764-9. DOI

Breton D, Harvengt L, Trontin J-F, Bouvet A, Favre J-M. Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine. Plant Cell Tissue Organ Cult. 2006;87(1):95–108. doi: 10.1007/s11240-006-9144-9. DOI

Breton D, Harvengt L, Trontin J-F, Bouvet A, Favre J-M. High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine. In Vitro Cell Dev Biol Plant. 2005;41(4):494. doi: 10.1079/IVP2005671. DOI

Elhiti M, Stasolla C, Wang A. Molecular regulation of plant somatic embryogenesis. In Vitro Cell Dev Biol Plant. 2013;49(6):631–642. doi: 10.1007/s11627-013-9547-3. DOI

Rocha D, Dornelas M. Molecular overview on plant somatic embryogenesis. CAB Review. 2013;8:1–17. doi: 10.1079/PAVSNNR20138022. DOI

Lippert D, Jun Z, Ralph S, Ellis DE, Gilbert M, Olafson R, Ritland K, Ellis B, Douglas CJ, Bohlmann J. Proteome analysis of early somatic embryogenesis in Picea glauca. PROTEOMICS. 2005;5(2):461–473. doi: 10.1002/pmic.200400986. PubMed DOI

Businge E, Bygdell J, Wingsle G, Moritz T, Egertsdotter U. The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos. Physiol Plant. 2013;149(2):273–285. doi: 10.1111/ppl.12039. PubMed DOI

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

Teyssier C, Maury S, Beaufour M, Grondin C, Delaunay A, Le Metté C, Ader K, Cadene M, Label P, Lelu-Walter M-A. 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. doi: 10.1111/ppl.12081. PubMed DOI

Zhao J, Li H, Fu S, Chen B, Sun W, Zhang J, Zhang J. An iTRAQ-based proteomics approach to clarify the molecular physiology of somatic embryo development in prince Rupprecht's larch (Larix principis-rupprechtii Mayr) PLoS One. 2015;10(3):e0119987. doi: 10.1371/journal.pone.0119987. PubMed DOI PMC

Zhen Y, Zhao Z-Z, Zheng R-H, Shi J. Proteomic analysis of early seed development in Pinus massoniana L. Plant Physiol Biochem. 2012;54(0):97–104. doi: 10.1016/j.plaphy.2012.02.009. PubMed DOI

Morel A, Trontin J-F, Corbineau F, Lomenech A-M, Beaufour M, Reymond I, Le Metté C, Ader K, Harvengt L, Cadene M, et al. Cotyledonary somatic embryos of Pinus pinaster Ait. Most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses. Planta. 2014;240(5):1075–1095. doi: 10.1007/s00425-014-2125-z. PubMed DOI

Hargreaves CL, Reeves CB, Find JI, Gough K, Josekutty P, Skudder DB, van der Maas SA, Sigley MR, Menzies MI, Low CB, et al. Improving initiation, genotype capture, and family representation in somatic embryogenesis of Pinus radiata by a combination of zygotic embryo maturity, media, and explant preparation. Can J For Res. 2009;39(8):1566–1574. doi: 10.1139/X09-082. DOI

Hargreaves C, Reeves C, Find K, Gough K, Menzies M, Low C, Mullin T. Overcoming the challenges of family and genotype representation and early cell line proliferation in somatic embryogenesis from control-pollinated seeds of Pinus radiata. 2011. p. 41.

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

Crouzet M, Claverol S, Lomenech A-M, Le Sénéchal C, Costaglioli P, Barthe C, Garbay B, Bonneu M, Vilain S. Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation. PLoS One. 2017;12(7):e0180341. doi: 10.1371/journal.pone.0180341. PubMed DOI PMC

Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Meth. 2007;4(11):923–925. doi: 10.1038/nmeth1113. PubMed DOI

Vizcaíno JA, Csordas A, del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez-Riverol Y, Reisinger F, Ternent T, et al. 2016 update of the PRIDE database and its related tools. Nucl Acids Res. 2016;44(D1):D447–D456. doi: 10.1093/nar/gkv1145. PubMed DOI PMC

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

Alexa A, Rahnenführer J, Lengauer T. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics. 2006;22(13):1600–1607. doi: 10.1093/bioinformatics/btl140. PubMed DOI

Gupta P, Timmis R, Timmis K, Carlson W, Welty E. Somatic embryogenesis in Douglas-fir (Pseudotsuga menziesii) In: Jain S, Gupta P, Newton R, editors. Somatic embryogenesis in woody plants. vol. 3. The Netherlands: Kluwer Academic Publishers, Dordrecht; 1995. pp. 303–313.

Pilarska M, Malec P, Salaj J, Bartnicki F, Konieczny R. High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens. Protoplasma. 2016;253(2):345–355. doi: 10.1007/s00709-015-0814-5. PubMed DOI PMC

Rocha DI, Monte-Bello CC, Aizza LCB, Dornelas MC. A passion fruit putative ortholog of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 gene is expressed throughout the in vitro de novo shoot organogenesis developmental program. Plant Cell Tissue Organ Cult. 2016;125(1):107–117. doi: 10.1007/s11240-015-0933-x. DOI

Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Pâques M. Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos. Plant Sci. 2001;160(3):473–479. doi: 10.1016/S0168-9452(00)00410-6. PubMed DOI

Winkelmann T. International Society for Horticultural Science (ISHS) Belgium: Leuven; 2013. Recent advances in propagation of woody plants; pp. 375–381.

Laukkanen H, Rautiainen L, Taulavuori E, Hohtola A. Changes in cellular structures and enzymatic activities during browning of scots pine callus derived from mature buds. Tree Physiol. 2000;20(7):467–475. doi: 10.1093/treephys/20.7.467. PubMed DOI

Baba AI, Nogueira FCS, Pinheiro CB, Brasil JN, Jereissati ES, Jucá TL, Soares AA, Santos MF, Domont GB, Campos FA. Proteome analysis of secondary somatic embryogenesis in cassava (Manihot esculenta) Plant Sci. 2008;175(5):717–723. doi: 10.1016/j.plantsci.2008.07.014. DOI

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(2):407–422. doi: 10.1007/s11240-014-0615-0. DOI

van Zyl L, Bozhkov PV, Clapham DH, Sederoff RR, von Arnold S. Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis. Gene Exp Pat. 2003;3(1):83–91. doi: 10.1016/S1567-133X(02)00068-6. PubMed DOI

Stasolla C, Bozhkov PV, Chu T-M, van Zyl L, Egertsdotter U, Suarez MF, Craig D, Wolfinger RD, Von Arnold S, Sederoff RR. Variation in transcript abundance during somatic embryogenesis in gymnosperms. Tree Physiol. 2004;24(10):1073–1085. doi: 10.1093/treephys/24.10.1073. PubMed DOI

Jo L, Dos Santos ALW, Bueno CA, Barbosa HR, Floh EIS. Proteomic analysis and polyamines, ethylene and reactive oxygen species levels of Araucaria angustifolia (Brazilian pine) embryogenic cultures with different embryogenic potential. Tree Physiol. 2014;34(1):94–104. doi: 10.1093/treephys/tpt102. PubMed DOI

Filonova LH, Bozhkov PV, Brukhin VB, Daniel G, Zhivotovsky B, von Arnold S. Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm Norway spruce. J Cell Sci. 2000;113:4399–4411. PubMed

Bozhkov PV, Filonova LH, Suarez MF: 4 - programmed cell death in plant embryogenesis. In: Current Topics in Developmental Biology. Edited by Schatten GP, vol. 67: academic press; 2005: 135–179. PubMed

Levine A, Tenhaken R, Dixon R, Lamb C. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell. 1994;79(4):583–593. doi: 10.1016/0092-8674(94)90544-4. PubMed DOI

dos Santos ALW, Elbl P, Navarro BV, de Oliveira LF, Salvato F, Balbuena TS, Floh EIS. Quantitative proteomic analysis of Araucaria angustifolia (Bertol.) Kuntze cell lines with contrasting embryogenic potential. J Proteome. 2016;130(Supplement C):180–189. doi: 10.1016/j.jprot.2015.09.027. PubMed DOI

Zhang Y, Zhang S, Han S, Li X, Qi L. Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis) Plant Cell Rep. 2012;31(9):1637–1657. doi: 10.1007/s00299-012-1277-1. PubMed DOI

Wiweger M, Farbos I, Ingouff M, Lagercrantz U, Von Arnold S. Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases. J Exp Bot. 2003;54(393):2691–2699. doi: 10.1093/jxb/erg299. PubMed DOI

Ge Y, Cai YM, Bonneau L, Rotari V, Danon A, McKenzie EA, McLellan H, Mach L, Gallois P. Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis. Cell Death Differ. 2016;23:1493. doi: 10.1038/cdd.2016.34. PubMed DOI PMC

Zhen Y, Chen J, Chen Q, Shi J. Elemental analyses of calli and developing somatic embryo of hybrid liriodendron. Pakistan J Bot. 2015;47(1):189–196.

Li Z, Tang L, Qiu J, Zhang W, Wang Y, Tong X, Wei X, Hou Y, Zhang J. Serine carboxypeptidase 46 regulates grain filling and seed germination in Rice (Oryza sativa L.) PLoS One. 2016;11(7):e0159737. doi: 10.1371/journal.pone.0159737. PubMed DOI PMC

Nakano M, Kigoshi K, Shimizu T, Endo T, Shimada T, Fujii H, Omura M. Characterization of genes associated with polyembryony and in vitro somatic embryogenesis in Citrus. Tree Gen Genomes. 2013;9(3):795–803. doi: 10.1007/s11295-013-0598-8. DOI

Liu H-H, Tian X, Li Y-J, Wu C-A, Zheng C-C. Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA. 2008;14(5):836–843. doi: 10.1261/rna.895308. PubMed DOI PMC

Ahmadi B, Shariatpanahi ME, Teixeira da Silva JA. Efficient induction of microspore embryogenesis using abscisic acid, jasmonic acid and salicylic acid in Brassica napus L. Plant Cell Tissue Organ Cult. 2014;116(3):343–351. doi: 10.1007/s11240-013-0408-x. DOI

Linkies A, Leubner-Metzger G. Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination. Plant Cell Rep. 2012;31(2):253–270. doi: 10.1007/s00299-011-1180-1. PubMed DOI

Pourcel L, Irani NG, Koo AJK, Bohorquez-Restrepo A, Howe GA, Grotewold E. A chemical complementation approach reveals genes and interactions of flavonoids with other pathways. Plant J. 2013;74(3):383–397. doi: 10.1111/tpj.12129. PubMed DOI

Zhao J, Wang B, Wang X, Zhang Y, Dong M, Zhang J. 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. 2015;120(2):655–669. doi: 10.1007/s11240-014-0633-y. DOI

Agati G, Azzarello E, Pollastri S, Tattini M. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012;196(Supplement C):67–76. doi: 10.1016/j.plantsci.2012.07.014. PubMed DOI

Luo P, Shen Y, Jin S, Huang S, Cheng X, Wang Z, Li P, Zhao J, Bao M, Ning G. Overexpression of Rosa rugosa anthocyanidin reductase enhances tobacco tolerance to abiotic stress through increased ROS scavenging and modulation of ABA signaling. Plant Sci. 2016;245(Supplement C):35–49. doi: 10.1016/j.plantsci.2016.01.007. PubMed DOI

Zhang S-G, Han S-Y, Yang W-H, Wei H-L, Zhang M, Qi L-W. Changes in H2O2 content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis. Plant Cell Tissue Organ Cult. 2010;100(1):21–29. doi: 10.1007/s11240-009-9612-0. DOI

Kairong C, Ji L, Gengmei X, Jianlong L, Lihong W, Yafu W. Effect of hydrogen peroxide on synthesis of proteins during somatic embryogenesis in Lycium barbarum. Plant Cell Tissue Organ Cult. 2002;68(2):187–193. doi: 10.1023/A:1013871500575. DOI

Parent C, Capelli N, Dat J. Formes réactives de l'oxygène, stress et mort cellulaire chez les plantes. Comptes Rendus Biologies. 2008;331(4):255–261. doi: 10.1016/j.crvi.2008.02.001. PubMed DOI

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

Khan MIR, Fatma M, Per TS, Anjum NA, Khan NA. Salicylic acid-induced abiotic stress tolerance and underlying mechanisms in plants. Front Plant Sci. 2015;6:462. PubMed PMC

Gondor OK, Janda T, Soós V, Pál M, Majláth I, Adak MK, Balázs E, Szalai G. Salicylic acid induction of flavonoid biosynthesis pathways in wheat varies by treatment. Front Plant Sci. 2016;7:1447. doi: 10.3389/fpls.2016.01447. PubMed DOI PMC

Imin N, Goffard N, Nizamidin M, Rolfe BG. Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures. BMC Plant Biol. 2008;8(1):110. doi: 10.1186/1471-2229-8-110. PubMed DOI PMC

Ng TLM, Karim R, Tan YS, Teh HF, Danial AD, Ho LS, Khalid N, Appleton DR, Harikrishna JA. Amino acid and secondary metabolite production in embryogenic and non-embryogenic callus of Fingerroot ginger (Boesenbergia rotunda) PLoS One. 2016;11(6):e0156714. doi: 10.1371/journal.pone.0156714. PubMed DOI PMC

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.])