BACKGROUND: Hormonal homeostasis plays a critical role in the regulation of microspore embryogenesis (ME). The balance between endogenous phytohormones must be altered to induce microspore reprogramming from the classical pollen-formation pathway to embryogenic development, but too extensive changes may be detrimental. In the present study, the levels of auxins, cytokinins and abscisic acid were monitored in the anthers of two Polish winter wheat F1 lines and the spring cultivar Pavon highly differentiated in terms of ME effectiveness. Analyses were carried out at subsequent steps of the ME induction procedure that combined low temperature, sodium selenate and mannitol tiller pre-treatment. RESULTS: Of all the factors tested, mannitol induced the most profound effect on phytohormones and their homeostasis in wheat anthers. It significantly increased the accumulation of all auxins and decreased the levels of most cytokinins, while the change in ABA content was limited to cv. Pavon. In an attempt to alleviate this hormonal shock, we tested several modifications of the induction medium hormonal composition and found thidiazuron to be the most promising in stimulating the embryogenic development of wheat microspores. CONCLUSIONS: The lack of ABA-driven stress defence responses may be one of the reasons for the low effectiveness of ME induction in winter wheat microspore cultures. Low cytokinin level and a disturbed auxin/cytokinin balance may then be responsible for the morphological abnormalities observed during the next phases of embryogenic microspore development. One possible solution is to modify the hormonal composition of the induction medium with thidiazuron identified as the most promising component.

Department of Chemical Biology Faculty of Science Palacký University Šlechtitelů 27 Olomouc 783 71 Czech Republic

Department of Genetics and Plant Breeding Poznań University of Life Sciences 11 Dojazd St Poznań 60 632 Poland

Institute of Experimental Botany of the Czech Academy of Sciences v v i Šlechtitelů 31 Olomouc 783 71 Czech Republic

Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences Faculty of Science Palacký University Šlechtitelů 27 Olomouc 783 71 Czech Republic

The Franciszek Górski Institute of Plant Physiology Polish Academy of Sciences Niezapominajek 21 Kraków 30 239 Poland

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Knox RB. The pollen grain. In: Johri BM, editor. Embryology of angiosperms. Berlin, Heidelberg: Springer Berlin Heidelberg; 1984. pp. 197–271.

Maraschin SF, de Priester W, Spaink HP, Wang M. Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J Exp Bot. 2005;56(417):1711–26. PubMed

Touraev A, Vicente O, HeberleBors E. Initiation of microspore embryogenesis by stress. Trends Plant Sci. 1997;2(8):297–302.

Zheng MY. Microspore culture in wheat (Triticum aestivum)-doubled haploid production via induced embryogenesis. Plant Cell Tissue Organ Cult. 2003;73(3):213–30.

Soriano M, Li H, Boutilier K. Microspore embryogenesis: establishment of embryo identity and pattern in culture. Plant Reprod. 2013;26:181–96. PubMed PMC

Hand ML, de Vries S, Koltunow AMG. A comparison of in vitro and in vivo asexual embryogenesis. In: Germana M, Lambardi M, editors. Vitro embryogenesis in higher plants. Methods in Molecular Biology. Volume 1359. New York: Humana; 2016. pp. 3–23. PubMed

Kasha KJ, Maluszynski M. Production of doubled haploids in crop plants. An introduction. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I, editors. Doubled haploid production in crop plants: A manual. Dordrecht: Springer Netherlands; 2003. pp. 1–4.

Igrejas G, Branlard G. The importance of wheat. In: Igrejas G, Ikeda TM, Guzmán C, editors. Wheat quality for improving processing and human health. Cham: Springer International Publishing; 2020. pp. 1–7.

Kumlehn J, Lörz H. Monitoring sporophytic development of individual microspores of barley (Hordeum vulgare L). In: Clément C, Pacini E, Audran J-C, editors. Anther and pollen: from biology to biotechnology. Berlin, Heidelberg: Springer Berlin Heidelberg; 1999. pp. 183–90.

Kasha KJ, Simion E, Miner M, Letarte J, Hu TC. Haploid wheat isolated microspore culture protocol. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I, editors. Doubled haploid production in crop plants: A manual. Dordrecht: Springer Netherlands; 2003. pp. 77–81.

Pauk J, Mihály R, Puolimatka M. Protocol for wheat (Triticum aestivum L.) anther culture. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I, editors. Doubled haploid production in crop plants: A manual. Dordrecht: Springer Netherlands; 2003. pp. 59–64.

Touraev A, Indrianto A, Wratschko I, Vicente O, HeberleBors E. Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature. Sex Plant Reprod. 1996;9(4):209–15.

Tuvesson S, von Post R, Ljungberg A. Wheat anther culture. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I, editors. Doubled haploid production in crop plants: A manual. Dordrecht: Springer Netherlands; 2003. pp. 71–6.

Dubas E, Krzewska M, Surówka E, Kopeć P, Springer A, Janowiak F, et al. New prospects for improving microspore embryogenesis induction in highly recalcitrant winter wheat lines. Plants-Basel. 2024;13(3):363. PubMed PMC

Bajguz A, Piotrowska A. Conjugates of auxin and cytokinin. Phytochemistry. 2009;70(8):957–69. PubMed

Gomes GLB, Scortecci KC. Auxin and its role in plant development: structure, signalling, regulation and response mechanisms. Plant Biol. 2021;23(6):894–904. PubMed

Dubas E, Janowiak F, Krzewska M, Hura T, Żur I. Endogenous ABA concentration and cytoplasmic membrane fluidity in microspores of oilseed rape (Brassica Napus L.) genotypes differing in responsiveness to androgenesis induction. Plant Cell Rep. 2013;32(9):1465–75. PubMed PMC

Żur I, Dubas E, Krzewska M, Janowiak F. Current insights into hormonal regulation of microspore embryogenesis. Front Plant Sci. 2015;6:424. PubMed PMC

Bari R, Jones J. Role of plant hormones in plant defence responses. Plant Mol Biol. 2009;69(4):473–88. PubMed

Nordström A, Tarkowski P, Tarkowska D, Norbaek R, Åstot C, Dolezal K, et al. Auxin regulation of cytokinin biosynthesis in Arabidopsis thaliana:: A factor of potential importance for auxin-cytokinin-regulated development. Proc Natl Acad Sci USA. 2004;101(21):8039–44. PubMed PMC

Wani SH, Kumar V, Shriram V, Sah SK. Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. Crop J. 2016;4(3):162–76.

Dubas E, Castillo AM, Żur I, Krzewska M, Vallés MP. Microtubule organization changes severely after mannitol and n-butanol treatments inducing microspore embryogenesis in bread wheat. BMC Plant Biol. 2021;21(1):586. PubMed PMC

Moubayidin L, Di Mambro R, Sabatini S. Cytokinin-auxin crosstalk. Trends Plant Sci. 2009;14(10):557–62. PubMed

Romanov GA, Schmülling T. On the biological activity of cytokinin free bases and their ribosides. Planta. 2022;255(1). PubMed PMC

Yamamuro C, Zhu JK, Yang ZB. Epigenetic modifications and plant hormone action. Mol Plant. 2016;9(1):57–70. PubMed PMC

Zalabák D, Pospísilová H, Smehilová M, Mrízová K, Frébort I, Galuszka P. Genetic engineering of cytokinin metabolism: prospective way to improve agricultural traits of crop plants. Biotechnol Adv. 2013;31(1):97–117. PubMed

Kohli A, Sreenivasulu N, Lakshmanan P, Kumar PP. The phytohormone crosstalk paradigm takes center stage in Understanding how plants respond to abiotic stresses. Plant Cell Rep. 2013;32(7):945–57. PubMed

Juzoń-Sikora K, Nowicka A, Plačková L, Doležal K, Żur I. Hormonal homeostasis associated with effective induction of triticale microspore embryogenesis. Plant Cell Tissue Organ Cult. 2023;152(3):583–604.

Żur I, Dubas E, Krzewska M, Waligórski P, Dziurka M, Janowiak F. Hormonal requirements for effective induction of microspore embryogenesis in triticale (x Triticosecale Wittm.) anther cultures. Plant Cell Rep. 2015;34(1):47–62. PubMed PMC

Antolić S, Kveder M, Klaic B, Magnus V, Kojic-Prodic B. Recognition of the folded conformation of plant hormone (auxin, IAA) conjugates with glutamic and aspartic acids and their amides. J Mol Struct. 2001;560(1–3):223–37.

Zhao YD. Essential Roles of Local Auxin Biosynthesis in Plant Development and in Adaptation to Environmental Changes. In: Merchant SS, editor. Annual Review of Plant Biology. Annual Review of Plant Biology. 692018. pp. 417– 35. PubMed

Bao DF, Chang SQ, Li XD, Qi YH. Advances in the study of auxin early response genes: Aux/IAA, GH3, and SAUR. Crop J. 2024;12(4):964– 78.

Hayashi K, Arai K, Aoi Y, Tanaka Y, Hira H, Guo RP et al. The main oxidative inactivation pathway of the plant hormone auxin. Nat Commun. 2021;12(1). PubMed PMC

Rosquete MR, Barbez E, Kleine-Vehn J. Cellular auxin homeostasis: gatekeeping is housekeeping. Mol Plant. 2012;5(4):772–86. PubMed

Slater SMH, Yuan HY, Lulsdorf MM, Vandenberg A, Zaharia LI, Han XM, et al. Comprehensive hormone profiling of the developing seeds of four grain legumes. Plant Cell Rep. 2013;32(12):1939–52. PubMed

Raspor M, Motyka V, Ninkovic S, Dobrev PI, Malbeck J, Cosic T et al. Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics. Sci Rep. 2020;10(1). PubMed PMC

Schäfer M, Brütting C, Meza-Canales ID, Grosskinsky DK, Vankova R, Baldwin IT, et al. The role of cis-zeatin-type cytokinins in plant growth regulation and mediating responses to environmental interactions. J Exp Bot. 2015;66(16):4873–84. PubMed PMC

Gajdosová S, Spíchal L, Kamínek M, Hoyerová K, Novák O, Dobrev PI, et al. Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J Exp Bot. 2011;62(8):2827–40. PubMed

Perez-Pinar T, Hartmann A, Bössow S, Gnad H, Mock HP. Metabolic changes during wheat microspore embryogenesis induction using the highly responsive cultivar Svilena. J Plant Physiol. 2024;294. PubMed

Seifert F, Bössow S, Kumlehn J, Gnad H, Scholten S. Analysis of wheat microspore embryogenesis induction by transcriptome and small RNA sequencing using the highly responsive cultivar Svilena. BMC Plant Biol. 2016;16. PubMed PMC

Nishiyama R, Watanabe Y, Fujita Y, Le DT, Kojima M, Werner T, et al. Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis. Plant Cell. 2011;23(6):2169–83. PubMed 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):343–51.

Guzmán M, Arias FJZ. Increasing anther culture efficiency in rice (Oryza sativa L.) using anthers from ratooned plants. Plant Sci. 2000;151(2):107–14. PubMed

Imamura J, Harada H. Effects of abscisic acid and water stress on the embryo and plantlet formation in anther culture of Nicotiana tabacum Cv. Samsun Z Für Pflanzenphysiologie. 1980;100(4):285–9.

Reynolds TL, Crawford RL. Changes in abundance of an abscisic acid-responsive, early cysteine-labeled Metallothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum). Plant Mol Biol. 1996;32(5):823–9. PubMed

van Bergen S, Kottenhagen MJ, van der Meulen RM, Wang M. The role of abscisic acid in induction of androgenesis:: A comparative study between Hordeum vulgare L-cvs. Igri and digger. J Plant Growth Regul. 1999;18(3):135–43. PubMed

Wang M, van Bergen S, van Duijn B. Insights into a key developmental switch and its importance for efficient plant breeding. Plant Physiol. 2000;124(2):523–30. PubMed PMC

Żur I, Krzewska M, Dubas E, Gołębiowska-Pikania G, Janowiak F, Stojalowski S. Molecular mapping of loci associated with abscisic acid accumulation in triticale (xTriticosecale Wittm.) anthers in response to low temperature stress inducing androgenic development. Plant Growth Regul. 2012;68(3):483–92.

Hoekstra S, van Bergen S, van Brouwershaven IR, Schilperoort RA, Wang M. Androgenesis in hordeum vulgare L: effects of mannitol, calcium and abscisic acid on anther pretreatment. Plant Sci. 1997;126(2):211–8.

Wang M, Hoekstra S, van Bergen S, Lamers GE, Oppedijk BJ, van der Heijden MW, et al. Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Mol Biol. 1999;39(3):489–501. PubMed

Żur I, Dubas E, Golemiec E, Szechyńska-Hebda M, Janowiak F, Wędzony M. Stress-induced changes important for effective androgenic induction in isolated microspore culture of triticale (x Triticosecale Wittm). Plant Cell Tissue Organ Cult. 2008;94(3):319–28.

Liu H, Xing MY, Yang WB, Mu XQ, Wang X, Lu F et al. Genome-wide identification of and functional insights into the late embryogenesis abundant (LEA) gene family in bread wheat (Triticum aestivum). Sci Rep. 2019;9. PubMed PMC

Rai MK, Shekhawat NS, Harish, Gupta AK, Phulwaria M, Ram K, et al. The role of abscisic acid in plant tissue culture: a review of recent progress. Plant Cell Tissue Organ Cult. 2011;106(2):179–90.

Nowicka A, Kovacik M, Maksylewicz A, Kopećć P, Dubas E, Krzewska M, et al. The transcriptional landscape of the developmental switch from regular pollen maturation towards microspore-derived plant regeneration in barley. Crop J. 2024;12(4):1064–80.

Gupta M, Gupta S. An overview of selenium uptake, metabolism, and toxicity in plants. Front Plant Sci. 2017;7. PubMed PMC

Lanza M, dos Reis AR. Roles of selenium in mineral plant nutrition: ROS scavenging responses against abiotic stresses. Plant Physiol Biochem. 2021;164:27–43. PubMed

Stoop JMH, Williamson JD, Mason Pharr D. Mannitol metabolism in plants: a method for coping with stress. Trends Plant Sci. 1996;1(5):139–44.

Shen B, Jensen RG, Bohnert HJ. Increased resistance to oxidative stress in Transgenic plants by targeting mannitol biosynthesis to chloroplasts. Plant Physiol. 1997;113(4):1177–83. PubMed PMC

Shen B, Jensen RG, Bohnert HJ. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiol. 1997;115(2):527–32. PubMed PMC

Patel TK, Williamson JD. Mannitol in plants, fungi, and Plant-Fungal interactions. Trends Plant Sci. 2016;21(6):486–97. PubMed

Attree SM, Fowke LC. Embryogeny of Gymnosperms - Advances in synthetic seed technology of conifers. Plant Cell Tissue Organ Cult. 1993;35(1):1–35.

Diamond JM, Wright EM. Biological Membranes - Physical basis of ion and nonelectrolyte selectivity. Annu Rev Physiol. 1969;31:581–646. PubMed

Okonkwo SNC. Studies on Striga senegalensis Iv. In vitro culture of seedlings. Effect of various sugars and glutamine. Am J Bot. 1966;52(7):687–93.

Roberts-Oehlschlager SL, Dunwell JM. Barley anther Culture - Pretreatment on mannitol stimulates production of Microspore-Derived embryos. Plant Cell Tissue Organ Cult. 1990;20(3):235–40.

Cistué L, Ramos A, Castillo AM. Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars. Plant Cell Tissue Organ Cult. 1998;55(3):159–66.

Hoekstra S, van Zijderveld MH, Louwerse JD, Heidekamp F, van der Mark F. Anther and microspore culture of Hordeum-vulgare L Cv Igri. Plant Sci. 1992;86(1):89–96. PubMed

Ziauddin A, Marsolais A, Simion E, Kasha KJ. Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA). Plant Cell Rep. 1992;11(10):489–98. PubMed

Cistué L, Soriano M, Castillo AM, Vallés MP, Sanz JM, Echávarri B. Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture. Plant Cell Rep. 2006;25(4):257–64. PubMed

Labbani Z, de Buyser J, Picard E. Effect of mannitol pretreatment to improve green plant regeneration on isolated microspore culture in triticum turgidum ssp durum Cv. ‘Jennah Khetifa’. Plant Breeding. 2007;126(6):565–8.

Asif M, Eudes F, Randhawa H, Amundsen E, Spaner D. Induction medium osmolality improves microspore embryogenesis in wheat and triticale. Vitro Cell Dev Biology - Plant. 2014;50(1):121–6. PubMed

Hu TC, Ziauddin A, Simion E, Kasha KJ. Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment, isolation methods, and hormones. In Vitro Cellular & Developmental Biology - Plant. 1995;31(2):79–83.

Indrianto A, Heberle-Bors E, Touraev A. Assessment of various stresses and carbohydrates for their effect on the induction of embryogenesis in isolated wheat microspores. Plant Sci. 1999;143(1):71–9.

Hoekstra S, van Zijderveld MH, Heidekamp F, van der Mark F. Microspore culture of Hordeum vulgare L.: the influence of density and osmolality. Plant Cell Rep. 1993;12(12):661–5. PubMed

Li H, Devaux P. Enhancement of microspore culture efficiency of recalcitrant barley genotypes. Plant Cell Rep. 2001;20(6):475–81.

Wojnarowiez G, Caredda S, Devaux P, Sangwan R, Clément C. Barley anther culture:: assessment of carbohydrate effects on embryo yield, green plant production and differential plastid development in relation with albinism. J Plant Physiol. 2004;161(6):747–55. PubMed

Yu B, Chao DY, Zhao Y. How plants sense and respond to osmotic stress. J Integr Plant Biol. 2024;66(3):394–423. PubMed

Yu SM. Cellular and genetic responses of plants to sugar starvation. Plant Physiol. 1999;121(3):687–93. PubMed PMC

Hu TC, Kasha KJ. A cytological study of pretreatments used to improve isolated microspore cultures of wheat (Triticum aestivum L.) Cv. Chris Genome. 1999;42(3):432–41.

Zoriniants S, Tashpulatov AS, Heberle-Bors E, Touraev A. The role of stress in the induction of haploid microspore embryogenesis. In: Don Palmer CE, Keller WA, Kasha KJ, editors. Haploids in crop improvement II. Berlin, Heidelberg: Springer Berlin Heidelberg; 2005. pp. 35–52.

Corral-Martínez P, Parra-Vega V, Seguí-Simarro JM. Novel features of Brassica Napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning. J Exp Bot. 2013;64(10):3061–75. PubMed

Bárány I, Berenguer E, Solís MT, Pérez-Pérez Y, Santamaría ME, Crespo JL, et al. Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley. J Exp Bot. 2018;69(6):1387–402. PubMed PMC

Berenguer E, Minina EA, Carneros E, Bárány I, Bozhkov PV, Testillano PS. Suppression of Metacaspase- and Autophagy-Dependent cell death improves Stress-Induced microspore embryogenesis in Brassica Napus. Plant Cell Physiol. 2020;61(12):2097–110. PubMed PMC

Pérez-Pérez Y, Barany I, Berenguer E, Carneros E, Risueño MC, Testillano PS. Modulation of autophagy and protease activities by small bioactive compounds to reduce cell death and improve stress-induced microspore embryogenesis initiation in rapeseed and barley. Plant Signal Behav. 2018;14(2). PubMed PMC

Luo P, Zhao ZF, Yang F, Zhang L, Li SY, Qiao Y, et al. Stress-Induced autophagy is essential for microspore cell fate transition to the initial cell of androgenesis. Plant Cell Environ. 2025;48(1):421–34. PubMed

Brunoni F, Pencík A, Zukauskaite A, Ament A, Kopecná M, Collani S, et al. Amino acid conjugation of OxIAA is a secondary metabolic regulation involved in auxin homeostasis. New Phytol. 2023;238(6):2264–70. PubMed

Canonge J, Roby C, Hamon C, Potin P, Pfannschmidt T, Philippot M. Occurrence of albinism during wheat androgenesis is correlated with repression of the key genes required for proper Chloroplast biogenesis. Planta. 2021;254(6). PubMed

Zheng MY, Konzak CF. Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L). Plant Cell Rep. 1999;19(1):69–73. PubMed

Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmülling T. Cytokinin-deficient Transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell. 2003;15(11):2532–50. PubMed PMC

Su YH, Liu YB, Bai B, Zhang XS. Establishment of embryonic shoot–root axis is involved in auxin and cytokinin response during Arabidopsis somatic embryogenesis. Front Plant Sci. 2015;5. PubMed PMC

Sakakibara H. Five unaddressed questions about cytokinin biosynthesis. J Exp Bot. 2024. PubMed

Dinani ET, Shukla MR, Turi CE, Sullivan JA, Saxena PK. Thidiazuron: modulator of morphogenesis in vitro. In: Ahmad N, Faisal M, editors. Thidiazuron: from Urea derivative to plant growth regulator. Singapore: Springer; 2018. pp. 1–36.

Lu CY. The use of Thidiazuron in Tissue-Culture. Vitro Cell Dev Biology-Plant. 1993;29P(2):92–6.

Erland LAE, Giebelhaus RT, Victor JMR, Murch SJ, Saxena PK. The morphoregulatory role of Thidiazuron: Metabolomics-Guided hypothesis generation for mechanisms of activity. Biomolecules. 2020;10(9). PubMed PMC

Esteves P, Clermont I, Marchand S, Belzile F. Improving the efficiency of isolated microspore culture in six-row spring barley: II-exploring novel growth regulators to maximize embryogenesis and reduce albinism. Plant Cell Rep. 2014;33(6):871–9. PubMed

Jia JX, Zhang Y, Cui LW, Feng H. Effect of thidiazuron on microspore embryogenesis and plantlet regeneration in Chinese flowering cabbage (Brassica rapa. var. parachinenis). Plant Breeding. 2019;138(6):916– 24.

Zou JQ, Zou X, Gong ZC, Song GX, Ren J, Feng H. Thidiazuron Promoted Microspore Embryogenesis and Plant Regeneration in Curly Kale (Brassica oleracea L. convar. acephala var. sabellica). Horticulturae. 2023;9(3).

Wędzony M. Protocol for anther culture in hexaploid triticale (x triticosecale Wittm). In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I, editors. Doubled haploid production in crop plants: A manual. Dordrecht: Springer Netherlands; 2003. pp. 123–8.

Wang P, Chen Y. Preliminary study on prediction of height of pollen H2 generation in winter wheat grown in the field. Acta Agron Sinica. 1983;9(4):283–4.

Kumlehn J, Serazetdinova L, Hensel G, Becker D, Loerz H. Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnol J. 2006;4(2):251–61. PubMed

Svacinová J, Novák O, Placková L, Lenobel R, Holík J, Strnad M, et al. A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods. 2012;8:1–14. PubMed PMC

Novák O, Hauserová E, Amakorová P, Dolezal K, Strnad M. Cytokinin profiling in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry. 2008;69(11):2214–24. PubMed

Pencík A, Casanova-Sáez R, Pilarová V, Zukauskaite A, Pinto R, Micol JL, et al. Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis. J Exp Bot. 2018;69(10):2569–79. PubMed PMC

Pencík A, Rolcík J, Novák O, Magnus V, Barták P, Buchtík R, et al. Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction. Talanta. 2009;80(2):651–5. PubMed