Meta-topolin applied in proliferation increases the yield of Norway spruce somatic embryos

Abstract

In four unrelated embryogenic lines of Norway spruce, the application of meta-topolin (mT) instead of the commonly used 6-benzyladenine (BAP) during proliferation led to a positive effect on the subsequent maturation phase, when cytokinin is no longer applied. Although, both, BAP and mT are aromatic cytokinins, and differ only in the presence of a hydroxyl group in mT, the application of mT resulted in an increased yield of good-quality mature embryos. Furthermore, in selected line C (derived in our laboratory) we have examined the consequences of BAP vs. mT use that underlay the modified culture behaviour, persisting until late maturation. Our results showed modifications of hormonal balance in BAP, resp. mT variants including not only members of the cytokinin group (including metabolites), but also other phytohormones (abscisic acid, indole acetic acid, salicylic acid) and compounds with prominent protective and signalling roles (ascorbate, glutathione, sugars). This indicates the creation of a memory imprint of mT in the proliferation phase that is preserved and passed onto subsequent phases, directing further development. We assume that the mT could allow for better realization of the embryonic potential of a given line, that the BAP is unable to accomplish.

Keywords

Ascorbate / Carbohydrates / Cytokinins / Glutathione / Picea abies / Redox status

References

[1] Abdalla N, Dobránszki J. Meta-topolin as an effective benzyladenine derivative to improve the multiplication rate and quality of in vitro axillary shoots of Húsvéti rozmaring apple scion. Plants (Basel), 2024, 13(11): 1568

[2] Ahmad N, Strnad M. Meta-topolin: a growth regulator for plant biotechnology and agriculture, 2021, Singapore, Springer

[3] Ahmad N, Fatima N, Faisal M, Alatar AA, Pathirana R. Photosynthetic parameters and oxidative stress during acclimation of crepe-myrtle (Lagerstroemia speciosa (L.) pers.) in a meta-topolin-based micropropagation system and genetic fidelity of regenerated plants. Plants (Basel), 2022, 11(9): 1163

[4] Amoo SO, Finnie JF, Van Staden J. The role of meta-topolins in alleviating micropropagation problems. Plant Growth Regul, 2011, 63(2): 197-206

[5] Aremu AO, Bairu MW, Doležal K, Finnie JF, Van Staden J. Topolins: a panacea to plant tissue culture challenges?. Plant Cell Tissue Organ Cult PCTOC, 2012, 108(1): 1-16

[6] Aremu AO, Plačková L, Bairu MW, Novák O, Plíhalová L, Doležal K, Finnie JF, Van Staden J. How does exogenously applied cytokinin type affect growth and endogenous cytokinins in micropropagated Merwilla plumbea?. Plant Cell Tissue Organ Cult, 2014, 118(2): 245-256

[7] Bairu MW, Stirk WA, Dolezal K, Van Staden J. Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin?. Plant Cell Tissue Organ Cult, 2007, 90(1): 15-23

[8] Bairu MW, Novák O, Doležal K, Van Staden J. Changes in endogenous cytokinin profiles in micropropagated Harpagophytum procumbens in relation to shoot-tip necrosis and cytokinin treatments. Plant Growth Regul, 2011, 63(2): 105-114

[9] Bajguz A, Piotrowska A. Conjugates of auxin and cytokinin. Phytochemistry, 2009, 70(8): 957-969

[10] Bartoli CG, Yu JP, Gómez F, Fernández L, McIntosh L, Foyer CH. Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves. J Exp Bot, 2006, 57(8): 1621-1631

[11] Bartoli CG, Tambussi EA, Diego F, Foyer CH. Control of ascorbic acid synthesis and accumulation and glutathione by the incident light red/far red ratio in Phaseolus vulgaris leaves. FEBS Lett, 2009, 583(1): 118-122

[12] Belmonte MF, Stasolla C. Altered HBK3 expression affects glutathione and ascorbate metabolism during the early phases of Norway spruce (Picea abies) somatic embryogenesis. Plant Physiol Biochem, 2009, 47(10): 904-911

[13] Belmonte MF, Macey J, Yeung EC, Stasolla C. The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development. Plant Physiol Biochem, 2005, 43(4): 337-346

[14] Belmonte MF, Tahir M, Schroeder D, Stasolla C. Overexpression of HBK3 a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos. J Exp Bot, 2007, 58(11): 2851-2861

[15] Bettinger P, Clutter M, Siry J, Kane M, Pait J. Broad implications of southern United States pine clonal forestry on planning and management of forests. Int for Rev, 2009, 11(3): 331-345

[16] Cairns NG, Pasternak M, Wachter A, Cobbett CS, Meyer AJ. Maturation of Arabidopsis seeds is dependent on glutathione biosynthesis within the embryo. Plant Physiol, 2006, 141(2): 446-455

[17] Carrier DJ, Cunningham JE, Taylor DC, Dunstan DI. Sucrose requirements and lipid utilization during germination of interior spruce (Picea glauca engelmannii complex) somatic embryos. Plant Cell Rep, 1997, 16(8): 550-554

[18] Chen Z, Silva H, Klessig DF. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science, 1993, 262(5141): 1883-1886

[19] Chen XY, Zhang LY, Liu CB, Wang R, Dai JF, Kong LS, Zhang JF, Zhao J. The transcription factor LpWRKY65 enhances embryogenic capacity through reactive oxygen species scavenging during somatic embryogenesis of larch. Plant Physiol, 2025, 198(3): kiaf286

[20] Chen SG, Gao F, Cao X, Dong HY, Zhan YC, Zhu LH, Tao J. Roles of reactive oxygen species, storage compounds, and endogenous hormones in regulating somatic embryogenesis in blue spruce (Picea pungens Engelm.). Front Plant Sci, 2026, 16: 1646777

[21] Chiwocha S, von Aderkas P. Endogenous levels of free and conjugated forms of auxin, cytokinins and abscisic acid during seed development in Douglas fir. Plant Growth Regul, 2002, 36(3): 191-200

[22] De Diego N, Montalbán IA, Moncaleán P. In vitro regeneration of adult Pinus sylvestris L. trees. S Afr N J Bot, 2010, 76(1): 158-162

[23] do Nascimento Vieira L, Santa-Catarina C, de Freitas Fraga HP, dos Santos ALW, Steinmacher DA, Schlogl PS, Silveira V, Steiner N, Floh EIS, Guerra MP (2012) Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission. Plant Sci 195:80–87.

[24] Durner J, Klessig DF. Inhibition of ascorbate peroxidase by salicylic acid and 2, 6- dichloroisonicotinic acid, two inducers of plant defense responses. Proc Natl Acad Sci USA, 1995, 92(24): 11312-11316

[25] Egertsdotter U. Plant physiological and genetical aspects of the somatic embryogenesis process in conifers. Scand J Res, 2019, 34(5): 360-369

[26] Elhiti M, Yang CC, Belmonte MF, Gulden RH, Stasolla C. Transcriptional changes of antioxidant responses, hormone signalling and developmental processes evoked by the Brassica napus SHOOTMERISTEMLESS during in vitro embryogenesis. Plant Physiol Biochem, 2012, 58: 297-311

[27] Eliášová K, Konrádová H, Dobrev PI, Motyka V, Lomenech AM, Fischerová L, Lelu-Walter MA, Vondráková Z, Teyssier C. Desiccation as a post-maturation treatment helps complete maturation of Norway spruce somatic embryos: carbohydrates, phytohormones and proteomic status. Front Plant Sci, 2022, 13: 823617

[28] Filonova LH, Bozhkov PV, von Arnold S. Developmental pathway of somatic embryogenesis in Picea abies as revealed by time‐lapse tracking. J Exp Bot, 2000, 51(343): 249-264

[29] Find JI, Charity JA, Grace LJ, Kristensen MMMH, Krogstrup P, Walter C. Stable genetic transformation of embryogenic cultures of Abies nordmanniana (nordmann fir) and regeneration of transgenic plants. In Vitro Cell Dev Biol Plant, 2005, 41(6): 725-730

[30] Fischerová L, Gemperlová L, Cvikrová M, Matušíková I, Moravčíková J, Gerši Z, Malbeck J, Kuderna J, Pavlíčková J, Motyka V, Eliášová K, Vondráková Z. The humidity level matters during the desiccation of Norway spruce somatic embryos. Front Plant Sci, 2022, 13: 968982

[31] Galuszka P, Frebort I, Sebela M, Sauer P, Jacobsen S, Pec P. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals. Eur J Biochem, 2001, 268(2): 450-461

[32] Gautier F, Eliášová K, Leplé JC, Vondráková Z, Lomenech AM, Le Metté C, Label P, Costa G, Trontin JF, Teyssier C, Lelu-Walter MA. Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb. BMC Plant Biol, 2018, 18(1): 164

[33] Gemperlová L, Fischerová L, Cvikrová M, Malá J, Vondráková Z, Martincová O, Vágner M. Polyamine profiles and biosynthesis in somatic embryo development and comparison of germinating somatic and zygotic embryos of Norway spruce. Tree Physiol, 2009, 29(10): 1287-1298

[34] Gentile A, Jàquez Gutiérrez M, Martinez J, Frattarelli A, Nota P, Caboni E. Effect of meta-topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell Tissue Organ Cult PCTOC, 2014, 118(3): 373-381

[35] Gentile A, Frattarelli A, Nota P, Condello E, Caboni E. The aromatic cytokinin meta-topolin promotes in vitro propagation, shoot quality and micrografting in Corylus colurna L. Plant Cell Tissue Organ Cult PCTOC, 2017, 128(3): 693-703

[36] Gibson SI. Control of plant development and gene expression by sugar signaling. Curr Opin Plant Biol, 2005, 8(1): 93-102

[37] Gillespie KM, Ainsworth EA. Measurement of reduced, oxidized and total ascorbate content in plants. Nat Protoc, 2007, 2(4): 871-874

[38] Grira M, Prinsen E, Werbrouck SPO. The effect of topophysis on the in vitro development of Handroanthus guayacan and on its metabolism of meta-topolin riboside. Plants, 2023, 12(20): 3577

[39] Grira M, Prinsen E, Werbrouck S. New understanding of meta-topolin riboside metabolism in micropropagated woody plants. Plants, 2024, 13(9): 1281

[40] Gupta PK, Durzan DJ. Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos of Picea abies (Norway spruce). Vitro Cell Dev Biol, 1986, 22(11): 685-688

[41] Habeeb J, Muralikrishna KS, Fathimath Zaeema MP, Nagaraja NR, Paulraj S, Nirmal Kumar BJ, Pandian RTP, Chandran KP, Rajesh MK. Aromatic cytokinin meta-topolin revitalizes indirect somatic embryogenesis and regeneration in immature inflorescence culture of dwarf arecanut hybrids. Plant Physiol Rep, 2026, 31(1): 19-32

[42] Hakman I, Von Arnold S. Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce). J Plant Physiol, 1985, 121(2): 149-158

[43] Hamet J, Konrádová H, Lipavská H. Drought-induced metabolic adjustments in woodland strawberry leaves: the role of soluble carbohydrates and starch. Plant Soil Environ, 2026, 72(2): 87-101

[44] Hartig K, Beck E. Crosstalk between auxin, cytokinins, and sugars in the plant cell cycle. Plant Biol, 2006, 8(3): 389-396

[45] Hazubska-Przybył T, Obarska A, Konecka A, Kijowska-Oberc J, Wawrzyniak MK, Piotrowska-Niczyporuk A, Staszak AM, Ratajczak E (2024) Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation. Front Plant Sci 15: 1372764. https://doi.org/10.3389/fpls.2024.1372764

[46] Hazubska-Przybył T, Wawrzyniak MK, Kijowska-Oberc J, Staszak AM, Ratajczak E. Somatic embryogenesis of Norway spruce and Scots pine: possibility of application in modern forestry. Forests, 2022, 13(2): 155

[47] Holub J, Hanuš J, Hanke DE, Strnad M. Biological activity of cytokinins derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul, 1998, 26(2): 109-115

[48] Hoyerová K, Hošek P. New insights into the metabolism and role of cytokinin N-glucosides in plants. Front Plant Sci, 2020, 11: 741

[49] Huaman E, Muñoz C, Prat L, Meléndez-Mori JB, Vargas R, Vigo CN, Tejada-Alvarado JJ, Huaman-Pilco AF, Oliva-Cruz M. Micropropagation of Vaccinium meridionale Sw.: interaction between basal media and cytokinins, physiological quality of shoots, and ex vitro rooting. J Genet Eng Biotechnol, 2026, 24(1): 100633

[50] Hudec L, Konrádová H, Hašková A, Lipavská H. Norway spruce embryogenesis: changes in carbohydrate profile, structural development and response to polyethylene glycol. Tree Physiol, 2016, 36(5): 548-561

[51] Iraqi D, Tremblay FM. Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development. J Exp Bot, 2001, 52(365): 2301-2311

[52] Jayakannan M, Bose J, Babourina O, Shabala S, Massart A, Poschenrieder C, Rengel Z. The NPR1-dependent salicylic acid signalling pathway is pivotal for enhanced salt and oxidative stress tolerance in Arabidopsis. J Exp Bot, 2015, 66(7): 1865-1875

[53] Jiang ZR, Wang M, Gouaille L, Barbier F, Nicolas M, Wang YH, Pérez-Garcia MD, Ogé L, Ledroit L, Cukier C, Lalande J, Hamama L, Crespel L, Ding YF, Lothier J, Le Gourrierec J, Bertheloot J, Li GH, Sakr S. Cytokinin-induced bud outgrowth depends on sugar metabolism and signalling. J Exp Bot, 2025, 76(18): 5351-5366

[54] Keunen EL, Peshev D, Vangronsveld J, Van Den Ende WI, Cuypers AN. Plant sugars are crucial players in the oxidative challenge during abiotic stress: extending the traditional concept. Plant Cell Environ, 2013, 36(7): 1242-1255

[55] Kiba T, Takei K, Kojima M, Sakakibara H. Side-chain modification of cytokinins controls shoot growth in Arabidopsis. Dev Cell, 2013, 27(4): 452-461

[56] Király A, Ambrus V, Farkas D, Abdalla N, Dobránszki J. Differential alteration of gene expression by benzyl adenine and meta-topolin in in vitro apple shoots. Plants, 2025, 14(23): 3691

[57] Klimaszewska K, Rutledge RG, Séguin A. Genetic transformation of conifers utilizing somatic embryogenesis. Transgenic plants, 2004Humana Press

[58] Konrádová H, Lipavská H, Albrechtová J, Vreugdenhil D. Sucrose metabolism during somatic and zygotic embryogeneses in Norway spruce: content of soluble saccharides and localisation of key enzyme activities. J Plant Physiol, 2002, 159(4): 387-396

[59] Koo YM, Heo AY, Choi HW. Salicylic acid as a safe plant protector and growth regulator. Plant Pathol J, 2020, 36(1): 1-10

[60] Kopečný D, Končitíková R, Popelka H, Briozzo P, Vigouroux A, Kopečná M, Zalabák D, Šebela M, Skopalová J, Frébort I, Moréra S. Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site. FEBS J, 2016, 283(2): 361-377

[61] Kubeš M, Drážná N, Konrádová H, Lipavská H. Robust carbohydrate dynamics based on sucrose resynthesis in developing Norway spruce somatic embryos at variable sugar supply. In Vitro Cell Dev Biol Plant, 2014, 50(1): 45-57

[62] Kudełko K, Gaj MD. Glutathione (GSH) induces embryogenic response in in vitro cultured explants of Arabidopsis thaliana via auxin-related mechanism. Plant Growth Regul, 2019, 89(1): 25-36

[63] Lelu-Walter MA, 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 Genet Genomes, 2013, 9(4): 883-899

[64] Li CH, Liu BG, Kim TD, Moon HK, Choi YE. Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis. Plant Biotechnol Rep, 2008, 2(4): 259-265

[65] Liao YK, Liao CK, Ho YL. 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, 2008, 93(3): 257-268

[66] Lipavská H, Konrádová H. Somatic embryogenesis in conifers: the role of carbohydrate metabolism. Vitro Cell Dev Biol Plant, 2004, 40(1): 23-30

[67] Lipavská H, Svobodová H, Albrechtová J, Kumstýřová L, Vágner M, Vondráková Z. Carbohydrate status during somatic embryo maturation in Norway spruce. Vitro Cell Dev Biol Plant, 2000, 36(4): 260-267

[68] Liu JL, Qiu GY, Liu C, Li H, Chen XD, Fu QL, Lin YC, Guo B. Salicylic acid, a multifaceted hormone, combats abiotic stresses in plants. Life, 2022, 12(6): 886

[69] Liu C, Fan HR, Zhang JQ, Wu JN, Zhou MB, Cao FL, Tao GY, Zhou XH. Combating browning: mechanisms and management strategies in in vitro culture of economic woody plants. For Res, 2024

[70] Long RW, Adams HD. The osmotic balancing act: when sugars matter for more than metabolism in woody plants. Glob Change Biol, 2023, 29(7): 1684-1687

[71] Lund MN, Ray CA. Control of Maillard reactions in foods: strategies and chemical mechanisms. J Agric Food Chem, 2017, 65(23): 4537-4552

[72] Mamun NHA, Aidun CK, Egertsdotter U. Improved and synchronized maturation of Norway spruce (Picea abies (L.) H.Karst.) somatic embryos in temporary immersion bioreactors. In Vitro Cell Dev Biol Plant, 2018, 54(6): 612-620

[73] Martins JPR, Wawrzyniak MK, Ley-López JM, Mendes MM, Mokhtari AM, Chmielarz P (2025) Regulation of the cytokinin metabolic spectrum and morphogenesis in Quercus robur (L.) in vitro by exogenous cytokinins and activated charcoal. Ind Crops Prod 233: 121362. https://doi.org/10.1016/j.indcrop.2025.121362

[74] Matros A, Peshev D, Peukert M, Mock HP, Van den Ende W. Sugars as hydroxyl radical scavengers: proof-of-concept by studying the fate of sucralose in Arabidopsis. Plant J, 2015, 82(5): 822-839

[75] Miura K, Okamoto H, Okuma E, Shiba H, Kamada H, Hasegawa PM, Murata Y. SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid-induced accumulation of reactive oxygen species in Arabidopsis. Plant J, 2013, 73(1): 91-104

[76] Montalbán IA, De Diego N, Moncaleán P. Testing novel cytokinins for improved in vitro adventitious shoots formation and subsequent ex vitro performance in Pinus radiata. Forestry (Lond), 2011, 84(4): 363-373

[77] Montalbán IA, Novák O, Rolčik J, Strnad M, Moncaleán P. Endogenous cytokinin and auxin profiles during in vitro organogenesis from vegetative buds of Pinus radiata adult trees. Physiol Plant, 2013, 148(2): 214-231

[78] Murvanidze N, Doležal K, Plačková L, Werbrouck SPO. Metabolism of fluorinated topolin cytokinins in micropropagated Phalaenopsis amabilis. Horticulturae, 2024, 10(7): 727

[79] Namitha MS, Neema M, Rajesh MK, Fathimath Zaeema MP, Prakash K, Muralikrishna KS, Karun A. Meta-topolin stimulates in vitro plantlet generation from embryonic shoot meristem explants in coconut (Cocos nucifera). Agric Res, 2025

[80] Nawrot-Chorabik K. Plantlet regeneration through somatic embryogenesis in Nordmann’s fir (Abies nordmanniana). J Forestry Res, 2016, 27(6): 1219-1228

[81] Nishizawa A, Yabuta Y, Shigeoka S. Galactinol and raffinose constitute a novel function to protect plants from oxidative damage. Plant Physiol, 2008, 147(3): 1251-1263

[82] Oslovsky VE, Savelieva EM, Drenichev MS, Romanov GA, Mikhailov SN. Distinct peculiarities of in planta synthesis of isoprenoid and aromatic cytokinins. Biomolecules, 2020, 10(1): 86

[83] Pěnčík A, Casanova-Sáez R, Pilařová V, Žukauskaitė A, Pinto R, Micol JL, Ljung K, Novák O. Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis. J Exp Bot, 2018, 69(10): 2569-2579

[84] Pokorná E, Hluska T, Galuszka P, Hallmark HT, Dobrev PI, Záveská Drábková L, Filipi T, Holubová K, Plíhal O, Rashotte AM, Filepová R, Malbeck J, Novák O, Spíchal L, Brzobohatý B, Mazura P, Zahajská L, Motyka V. Cytokinin N-glucosides: occurrence, metabolism and biological activities in plants. Biomolecules, 2021, 11(1): 24

[85] Pullman GS, Buchanan M. Identification and quantitative analysis of stage-specific carbohydrates in loblolly pine (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol, 2008, 28(7): 985-996

[86] Pullman GS, Chase KM, Skryabina A, Bucalo K. Conifer embryogenic tissue initiation: improvements by supplementation of medium with d-xylose and d-chiro-inositol. Tree Physiol, 2009, 29(1): 147-156

[87] Pullman GS, Zeng XY, Copeland-Kamp B, Crockett J, Lucrezi J, May SW, Bucalo K. Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation–reduction agents. Tree Physiol, 2015, 35(2): 209-224

[88] Radchuk R, Neil Emery RJ, Weier D, Vigeolas H, Geigenberger P, Lunn JE, Feil R, Weschke W, Weber H. Sucrose non-fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation. Plant J, 2010, 61(2): 324-338

[89] Rojas-Vargas A, Castander-Olarieta A, do Nascimento AMM, Vélez ML, Pereira C, Martins J, Zuzarte M, Canhoto J, Montalbán IA, Moncaleán P. Testing explant sources, culture media, and light conditions for the improvement of organogenesis in Pinus ponderosa (P. lawson and C. lawson). Plants (Basel), 2023, 12(4): 850

[90] Sakakibara H. Five unaddressed questions about cytokinin biosynthesis. J Exp Bot, 2025, 76(7): 1941-1949

[91] Saleem M, Fariduddin Q, Castroverde CDM. Salicylic acid: a key regulator of redox signalling and plant immunity. Plant Physiol Biochem, 2021, 168: 381-397

[92] Schwarzerová K, Vondráková Z, Fischer L, Boříková P, Bellinvia E, Eliášová K, Havelková L, Fišerová J, Vágner M, Opatrný Z. The role of actin isoforms in somatic embryogenesis in Norway spruce. BMC Plant Biol, 2010, 10(1): 89

[93] Šedivá J, Velebil J, Zahradník D. Micropropagation as a tool for the conservation of autochthonous Sorbus species of Czechia. Plants (Basel), 2023, 12(3): 488

[94] Singleton VL, Rossi JAJr. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic, 1965, 16(3): 144-158

[95] Sompornpailin K, Khunchuay C. Synergistic effects of BAP and kinetin media additives on regeneration of vetiver grass (Vetiveria zizanioides L. Nash). Aust J Crop Sci, 2016, 10(5): 726-731

[96] Spíchal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, Strnad M, Schmülling T. Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol, 2004, 45(9): 1299-1305

[97] Spitzer M, Wildenhain J, Rappsilber J, Tyers M. BoxPlotR: a web tool for generation of box plots. Nat Methods, 2014, 11(2): 121-122

[98] Stasolla C, Yeung EC. Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult, 2003, 74(1): 15-35

[99] Stirk WA, Novák O, Žižková E, Motyka V, Strnad M, van Staden J. Comparison of endogenous cytokinins and cytokinin oxidase/dehydrogenase activity in germinating and thermoinhibited Tagetes minuta achenes. J Plant Physiol, 2012, 169(7): 696-703

[100] Strnad M. The aromatic cytokinins. Physiol Plant, 1997, 101(4): 674-688

[101] Svačinová J, Novák O, Plačková L, Lenobel R, Holík J, Strnad M, Doležal K. A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods, 2012, 8(1): 17

[102] Svobodová H, Albrechtová J, Kumstýřová L, Lipavská H, Vágner M, Vondráková Z. Somatic embryogenesis in Norway spruce: anatomical study of embryo development and influence of polyethylene glycol on maturation process. Plant Physiol Biochem, 1999, 37(3): 209-221

[103] Teixeira da Silva JA, Gulyás A, Magyar-Tábori K, Wang MR, Wang QC, Dobránszki J. In vitro tissue culture of apple and other Malus species: recent advances and applications. Planta, 2019, 249(4): 975-1006

[104] Tikkinen M, Varis S, Aronen T. Development of somatic embryo maturation and growing techniques of Norway spruce emblings towards large-scale field testing. Forests, 2018, 9(6): 325

[105] Välimäki S, Paavilainen L, Tikkinen M, Salonen F, Varis S, Aronen T. Production of Norway spruce embryos in a temporary immersion system (TIS). In Vitro Cell Dev Biol Plant, 2020, 56(4): 430-439

[106] Viktor Nørgaard J, Krogstrup P. Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk. Plant Cell Rep, 1991, 9(9): 509-513

[107] von Aderkas P, Lelu MA, Label P. Plant growth regulator levels during maturation of larch somatic embryos. Plant Physiol Biochem, 2001, 39(6): 495-502

[108] Vondrakova Z, Dobrev PI, Pesek B, Fischerova L, Vagner M, Motyka V. Profiles of endogenous phytohormones over the course of Norway spruce somatic embryogenesis. Front Plant Sci, 2018, 9: 1283

[109] Wang M, Le Gourrierec J, Jiao FC, Demotes-Mainard S, Perez-Garcia MD, Ogé L, Hamama L, Crespel L, Bertheloot J, Chen JT, Grappin P, Sakr S. Convergence and divergence of sugar and cytokinin signaling in plant development. Int J Mol Sci, 2021, 22(3): 1282

[110] Weibel ER. Practical methods for biological morphometry, 1979, London, Academic Press

[111] Werbrouck SP, van der Jeugt B, Dewitte W, Prinsen E, Van Onckelen HA, Debergh PC. The metabolism of benzyladenine in Spathiphyllum floribundum ‘Schott Petite’ in relation to acclimatisation problems. Plant Cell Rep, 1995, 14(10): 662-665

[112] Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC. Meta-topolin, an alternative to benzyladenine in tissue culture?. Physiol Plant, 1996, 98(2): 291-297

[113] Zheleznichenko T, Karakulov A, Asbaganov S, Kochetov A. Effect of thidiazuron and meta-topolin on in vitro shoot multiplication and development of the Populus davidiana × P. bolleana hybrid. Plant Cell Tissue Organ Cult, 2025, 163(1): 35

[114] Zhong QL, Hu HL, Fan BF, Zhu C, Chen ZX. Biosynthesis and roles of salicylic acid in balancing stress response and growth in plants. Int J Mol Sci, 2021, 22(21): 11672

Funding

Czech Ministry of Education, Youth and Sports(LUC23150)

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