Adventitious shoot regeneration in woody species is regulated by interactions between plant growth regulators, endogenous hormone metabolism, and environmental cues such as light quality. Here, we investigated the effects of thidiazuron (TDZ) and the cytokinin oxidase/dehydrogenase (CKX) inhibitors INCYDE and phenyladenine (PA), in combination with different light spectra, on morphogenesis in Melia volkensii leaf explants. TDZ induced the highest frequencies of callus formation and adventitious shoot regeneration, particularly under white light. INCYDE promoted localized regeneration responses, including activation of dormant meristematic regions in secondary leaf axils, whereas PA showed limited regeneration efficiency. Light quality significantly influenced morphogenesis, with white and blue light favoring organized shoot development, while red and far-red light suppressed shoot regeneration and promoted callus formation. Cytokinin profiling revealed treatment-dependent shifts in endogenous cytokinin composition, most notably in isopentenyladenine (iP)-type cytokinins, which is consistent with altered cytokinin degradation dynamics. Cis-zeatin-type cytokinins were abundant across treatments, likely reflecting regulation associated with in vitro culture conditions. These findings indicate that cytokinin metabolism and light quality jointly influence organogenic competence in Melia volkensii Gürke, providing a physiological basis for optimizing regeneration strategies in woody plants. This study provides the first integrated analysis of cytokinin-modulating compounds and light spectra on adventitious shoot regeneration in Melia volkensii. The findings establish a physiological basis for improving regeneration protocols in recalcitrant woody species and support future biotechnological applications, including genetic improvement and advanced propagation strategies.

Department of Plants and Crops Faculty of Bioscience Engineering Ghent University 9000 Ghent Belgium

Laboratory of Growth Regulators Palackỳ University and Institute of Experimental Botany AS CR Šlechtitelů 11 783 71 Olomouc Czech Republic

Zobrazit více v PubMed

Sugiyama M. Organogenesis in vitro. Curr. Opin. Plant Biol. 1999;2:61–64. doi: 10.1016/S1369-5266(99)80012-0. PubMed DOI

George E.F., Hall M.A., De Klerk G.J. Plant Propagation by Tissue Culture. 3rd ed. Springer; Dordrecht, The Netherlands: 2008.

Plíhalová L., Vylíčilová H., Doležal K., Strnad M. Synthesis of aromatic cytokinins for plant biotechnology. New Biotechnol. 2016;33:614–624. doi: 10.1016/j.nbt.2015.11.009. PubMed DOI

Schmülling T., Werner T., Riefler M., Krupková E., Bartrina y Manns I. Structure and function of cytokinin oxidase/dehydrogenase genes. J. Plant Growth Regul. 2003;21:131–140. doi: 10.1007/s10265-003-0096-4. DOI

Werner T., Schmülling T. Cytokinin action in plant development. Curr. Opin. Plant Biol. 2009;12:527–538. doi: 10.1016/j.pbi.2009.07.002. PubMed DOI

Thomas J.C., Katterman F.R. Thidiazuron-induced cytokinin-like responses in plant tissues. Plant Physiol. 1986;81:681–683. doi: 10.1104/pp.81.2.681. PubMed DOI PMC

Nisler J., Zatloukal M., Popa I., Doležal K., Strnad M., Spíchal L. Novel thidiazuron-derived inhibitors of cytokinin oxidase/dehydrogenase. Plant Mol. Biol. 2016;92:235–248. doi: 10.1007/s11103-016-0509-0. PubMed DOI

Motte H., Galuszka P., Spíchal L., Tarkowski P., Plíhal O., Šmehilová M., Jaworek P., Vereecke D., Werbrouck S., Geelen D. Phenyl-adenine is a potent promoter of shoot regeneration through CKX inhibition. Plant Physiol. 2013;161:1229–1241. doi: 10.1104/pp.112.210716. PubMed DOI PMC

Kami C., Lorrain S., Hornitschek P., Fankhauser C. Light-regulated plant growth and development. Curr. Opin. Plant Biol. 2010;13:476–485. doi: 10.1016/j.pbi.2010.05.004. PubMed DOI

Paik I., Huq E. Plant photoreceptors: Multi-functional sensory proteins and their signaling networks. Semin. Cell Dev. Biol. 2019;92:114–121. doi: 10.1016/j.semcdb.2019.03.007. PubMed DOI PMC

Folta K.M., Maruhnich S.A. Green light: A signal to slow down or stop. J. Exp. Bot. 2007;58:3099–3111. doi: 10.1093/jxb/erm130. PubMed DOI

Briggs W.R., Huala E. Blue-light photoreceptors in higher plants. Annu. Rev. Cell Dev. Biol. 1999;15:33–62. doi: 10.1146/annurev.cellbio.15.1.33. PubMed DOI

Murthy B.N.S., Murch S.J., Saxena P.K. Thidiazuron: A potent regulator of in vitro plant morphogenesis. In Vitro Cell. Dev. Biol.—Plant. 1998;34:267–275. doi: 10.1007/BF02822732. DOI

Dewir Y.H., Naidoo Y., da Silva J.A.T. Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep. 2018;37:1451–1470. doi: 10.1007/s00299-018-2326-1. PubMed DOI

Zatloukal M., Gemrotová M., Doležal K., Havlíček L., Spíchal L., Strnad M. Novel potent inhibitors of cytokinin oxidase/dehydrogenase with strong cytokinin activity in plants. Bioorg. Med. Chem. 2008;16:9268–9275. doi: 10.1016/j.bmc.2008.09.008. PubMed DOI

Sakakibara H. Cytokinins: Activity, biosynthesis, and translocation. Annu. Rev. Plant Biol. 2006;57:431–449. doi: 10.1146/annurev.arplant.57.032905.105231. PubMed DOI

Gajdošová S., Spíchal L., Kamínek M., Hoyerová K., Novák O., Doležal K., Galuszka P., Klíma P., Strnad M., Motyka V. Distribution, biological activities, metabolism, and the conceivable function of cis-zeatin-type cytokinins in plants. J. Exp. Bot. 2011;62:2827–2840. doi: 10.1093/jxb/erq457. PubMed DOI

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. 2001;13:2539–2550. doi: 10.1105/tpc.014928. PubMed DOI PMC

Franklin K.A., Quail P.H. Phytochrome functions in Arabidopsis development. J. Exp. Bot. 2010;61:11–24. doi: 10.1093/jxb/erp304. PubMed DOI PMC

Batista D.S., Dias L.L.C., Rêgo M.M., Silva P.O., Otoni W.C. Light quality in plant tissue culture: Does it matter? In Vitro Cell. Dev. Biol.—Plant. 2018;54:195–215. doi: 10.1007/s11627-018-9902-5. DOI

Guan Y., Li S.G., Fan X.F., Su Z.H. Applications of somatic embryogenesis and organogenesis in woody plants. Plant Cell Front. Plant Sci. 2016;7:938. doi: 10.3389/fpls.2016.00938. PubMed DOI PMC

Li H., Xu Z., Tang C. Effect of LEDs on growth and morphogenesis of cotton plantlets in vitro. Plant Cell Tissue Organ Cult. 2010;103:155–163. doi: 10.1007/s11240-010-9763-z. DOI

Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. DOI

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;8:17. doi: 10.1186/1746-4811-8-17. PubMed DOI PMC

Novák O., Hauserová E., Amakorová P., Doležal K., Strnad M. Cytokinin profiling in plant tissues using ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Phytochemistry. 2008;69:2214–2224. doi: 10.1016/j.phytochem.2008.04.022. PubMed DOI

Tallarida R.J., Murray R.B. Manual of Pharmacologic Calculations with Computer Programs. 2nd ed. Springer; New York, NY, USA: 1987.