2,4-D is a broadly used auxin herbicide. The presence of the 2,4-D synthetic auxin in the medium is imperative for long-term BY-2 tobacco suspension viability. The precise mechanism of this symbiosis of the suspension and the synthetic auxin remains unclear. Our goal was to study the hormonal regulation of the growth of the cell suspension; and to describe the experiments clarifying the interaction between the chosen growth regulators and phytohormones on the cellular level, specifically between the 2,4-D synthetic auxin and the native stress phytohormone - ethylene. This study examined the influence of low 2,4-D concentrations stimulating cell growth in vitro as well as the influence of high herbicide concentrations on the model tobacco BY-2 suspension. The culture took 6 days. Different parameters were evaluated, including the influence of different 2,4-D concentrations on the production of the phytohormone ethylene and its precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) in the tobacco cells. The content of 2,4-D in the cells and the medium was established. The observations of the morphological changes showed that a heavy impregnation of the cell walls taking place depending on the concentration of 2,4-D. A dramatic increase in protective polysaccharides and a remodulation of the cell walls by the formation of a pectin shield in artificial conditions were expected and observed. At the same time, massive production of the stress phytohormone ethylene took place, and, because of that, plant mutagenicity, anomalous tumour-type proliferation growth, and the production of supercells were observed. The hypothesis of the protective shield is discussed.

Brno University of Technology Faculty of Mechanical Engineering Brno Czech Republic

Global Change Research Institute Academy of Sciences of the Czech Republic Brno Czech Republic

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Nagata T, Sakamoto K, Shimizu T (2004) Tobacco BY-2 cells: the present and beyond. Vitro Cell Dev Biology-Plant 40(2):163–166

Hanamata S, Kurusu T, Kuchitsu K (2020) Cell cycle-dependence of autophagic activity and inhibition of autophagosome formation at M phase in Tobacco BY-2 cells. Int J Mol Sci 21(23):9166 PubMed PMC

Buntru M, Vogel S, Spiegel H, Schillberg S (2014) Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnol 14(1):1–11

Brookwell A, Oza JP, Caschera F (2021) Biotechnology applications of cell-free expression systems. Life 11(12):1367 PubMed PMC

Armero Gimenez J, Wilbers R, Schots A, Williams C, Finnern R Rapid screening and scaled manufacture of immunogenic virus-like particles in a tobacco BY-2 cell free protein synthesis system. Front Immunol, 14, 296

Grossmann K (2010) Auxin herbicides: current status of mechanism and mode of action. Pest Manage Science: Former Pesticide Sci 66(2):113–120

Garcia C, Furtado de Almeida AA, Costa M, Britto D, Valle R, Royaert S, Marelli JP (2019) Abnormalities in somatic embryogenesis caused by 2, 4-D: an overview. Plant Cell Tissue Organ Cult (PCTOC) 137(2):193–212

Iakimova ET, Yordanova ZP, Cristescu SM, Harren FJ, Woltering EJ (2019) Cell deathsignaling and morphology in chemical-treatedtobacco BY-2 suspensionculturedcells. Environ Exp Bot 164:157–169

Liang X, Wang H, Hu Y, Mao L, Sun L, Dong T, Bi Y (2015) Silicon does not mitigate cell death in culturedtobacco BY-2 cellssubjected to salinity without ethylene emission. Plant Cell Rep 34(2):331–343 PubMed

Tichati L, Trea F, Ouali K (2020) Potential role of selenium against hepatotoxicity induced by 2, 4-dichlorophenoxyacetic acid in albino Wistar rats. Biol Trace Elem Res 194(1):228–236 PubMed

Bernat P, Nykiel-Szymańska J, Gajewska E, Różalska S, Stolarek P, Dackowa J, Słaba M (2018) Trichoderma Harzianum diminished oxidative stress caused by 2, 4-dichlorophenoxyacetic acid (2, 4-D) in wheat, with insights from lipidomics. J Plant Physiol 229:158–163 PubMed

Colzi I, Doumett S, DelBubba M, Fornaini J, Arnetoli M, Gabbrielli R, Gonnelli C (2011) On the role ofthe cell wall in thephenomenonofcopper tolerance in Sileneparadoxa L. Environ Exp Bot 72(1):77–83

Wang R, Liang R, Dai T, Chen J, Shuai X, Liu C (2019) Pectin-based adsorbents for heavy metal ions: a review. Trends Food Sci Technol 91:319–329

Mehes-Smith M, Nkongolo K, Cholewa E (2013) Copingmechanismsofplants to metal contaminatedsoil. Environmentalchange Sustain 54:53–90

Torres M, Jiquel A, Jeanne E, Naquin D, Dessaux Y, Faure D (2022) Agrobacterium tumefaciens fitness genes involved in the colonization of plant tumors and roots. New Phytol 233(2):905–918 PubMed

Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the HeLa cell in the cell biology of higher plants. International review of cytology, vol 132. Academic, pp 1–30

Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18(1):100–127

Franek M, Kolar V, Granatova M, Nevorankova Z (1994) Monoclonal ELISA for 2, 4-dichlorophenoxyacetic acid: characterization of antibodies and assay optimization. J Agric Food Chem 42(6):1369–1374

Fišerová H, Mikušová Z, Klemš M (2008) Estimation of ethylene production and 1-aminocyclopropane-1-carboxylic acid content in plants by means of gas chromatography. Plant Soil Environ 54(2):55–60

Masuda H, Ozeki Y, Amino SI, Komamine A (1984) Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture. Physiol Plant 62(1):65–72

Masuda H, Ozeki Y, Amino SI, Komamine A (1985) Changes in the activities of various glycosidases during carrot cell elongation in a 2, 4-D-free medium. Plant Cell Physiol 26(6):995–1001

McCann MC, Shi J, Roberts K, Carpita NC (1994) Changes in pectin structure and localization during the growth of unadapted and NaCl-adapted tobacco cells. Plant J 5(6):773–785

Miyazawa Y, Nakajima N, Abe T, Sakai A, Fujioka S, Kawano S, Yoshida S (2003) Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents. J Exp Bot 54(393):2669–2678 PubMed

Toyooka K, Sato M, Kutsuna N, Higaki T, Sawaki F, Wakazaki M, Matsuoka K (2014) Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells. Plant Cell Physiol 55(9):1544–1555 PubMed

Orchard CB, Siciliano I, Sorrell DA, Marchbank A, Rogers HJ, Francis D, Onckelen HV (2005) Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle. Plant J 44(2):290–299 PubMed

Suchomelová-Mašková P, Novák O, Lipavská H (2008) Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application. Plant Physiol Biochem 46(7):673–684 PubMed

Campanoni P, Nick P (2005) Auxin-dependent cell division and cell elongation. 1-Naphthaleneacetic acid and 2, 4-dichlorophenoxyacetic acid activate different pathways. Plant Physiol 137(3):939–948 PubMed PMC

Kariya K, Demiral T, Sasaki T, Tsuchiya Y, Turkan I, Sano T, Yamamoto Y (2013) A novel mechanism of aluminium-induced cell death involving vacuolar processing enzyme and vacuolar collapse in tobacco cell line BY-2. J Inorg Biochem 128:196–201 PubMed

Iakimova ET, Yordanova ZP, Cristescu SM, Harren FF, Woltering EJ (2020) Cell death associated release of volatile organic sulphur compounds with antioxidant properties in chemical-challenged tobacco BY-2 suspension cultured cells. J Plant Physiol 251:153223 PubMed

Kobylińska A (2017) Exogenous quercetin as a proliferation stimulator in tobacco BY-2 cells. J Elementology, 22(1)

Liang X, Wang H, Hu Y, Mao L, Sun L, Dong T, Bi Y (2015) Silicon does not mitigate cell death in cultured tobacco BY-2 cells subjected to salinity without ethylene emission. Plant Cell Rep 34:331–343 PubMed

Klíma P, Laňková M, Vandenbussche F, Van Der Straeten D, Petrášek J (2018) Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells. Plant Cell Rep 37(5):809–818 PubMed

Kieffer F, Lherminier J, Simon-Plas F, Nicole M, Paynot M, Elmayan T, Blein JP (2000) The fungal elicitor cryptogein induces cell wall modifications on tobacco cell suspension. J Exp Bot 51(352):1799–1811 PubMed

Issawi M, Muhieddine M, Girard C, Sol V, Riou C (2017) Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition. Glycoconj J 34(5):585–590 PubMed

Hensel G, Kunze G, Kunze I (2002) The influence of 2, 4-dichlorophenoxyacetic acid on localisation of the PR-proteins CBP20 and class I chitinase in tobacco suspension cell cultures. Plant Sci 163(6):1099–1106

Lherminier J, Benhamou N, Larrue J, Milat ML, Boudon-Padieu E, Nicole M, Blein JP (2003) Cytological characterization of elicitin-induced protection in tobacco plants infected by Phytophthora Parasitica or Phytoplasma. Phytopathology 93(10):1308–1319 PubMed

Krzyzanowska J, Czubacka A, Pecio L, Przybys M, Doroszewska T, Stochmal A, Oleszek W (2012) The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in Mentha× piperita cell suspensioncultures. Plant cell Tissue Organ Cult (Pctoc) 108(1):73–81

Yang JG, Uchiyama T (2000) Hydroxycinnamicacids and theirdimersinvolved in thecessation of cell elongation in Menthasuspensionculture. Biosci Biotechnol Biochem 64(8):1572–1579 PubMed

Yang JG, Miyao S, Uchiyama T (1999) Responses of Mentha suspension-cultured cells to 2, 4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls. Biosci Biotechnol Biochem 63(9):1522–1527 PubMed

Gómez B, Gullon B, Remoroza C, Schols HA, Parajo JC, Alonso JL (2014) Purification, characterization, and prebiotic properties of pecticoligosaccharides from orangepeel wastes. J Agric Food Chem 62(40):9769–9782 PubMed

Zhexenbay N, Akhmetsadykova S, Nabiyeva Z, Kizatova M, Iskakova G (2020) Usingpectin as heavy metals detoxification agent to reduce environmental contamination and health risks. Procedia Environ Sci Eng Manage 7(4):551–562

Ogura H (1990) Chromosome variation in plant tissue culture. Somaclonal variation in crop improvement I. Springer, Berlin, Heidelberg, pp 49–84

Doležel J, Novák FJ (1985) Karyological and cytophotometric study of callus induction in Allium sativum L. J Plant Physiol 118(5):421–429 PubMed

Dolezel J, Novak FJ (1984) Effect of plant tissue culture media on the frequency of somatic mutations in Tradescantia Stamen hairs. Z für Pflanzenphysiologie 114(1):51–58