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The effects of novel synthetic cytokinin derivatives and endogenous cytokinins on the in vitro growth responses of hemp (Cannabis sativa L.) explants

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The biotechnological utilization (genetic transformation, gene editing) of industrial hemp (Cannabis sativa L.) has been hampered by a lack of robust regeneration/in vitro multiplication protocols. In order to break this barrier we propose an approach combining standard application of exogenous growth regulators (auxins, cytokinins) with the knowledge of (1) endogenous cytokinin concentrations in primary explants based on latest analytical techniques and (2) the exogenous application of novel synthetic cytokinins or the regulators of their activity/function. We have tested eight explant types isolated from aseptically germinated seedlings in order to induce shoot regeneration/multiple shoot formation. The main problems appeared were callus formation and strong apical dominance. The latter phenomenon was suppressed using the auxin antagonist α-(2-oxo-2-phenylethyl)-1H-indole-3-acetic acid (PEO-IAA), and multiple shoot cultures were established from isolated apical meristems using media containing the novel synthetic cytokinin derivative 6-benzylamino-9-(tetrahydroxypyranyl)purin (BAP9THP). Endogenous cytokinin concentrations were measured in primary explants and explants cultured under diverse conditions to clarify the interactions between endogenous and exogenously applied cytokinins and their synthetic derivatives. These measurements were subsequently used to optimize the applied concentrations and timing of application of specific cytokinin derivatives to achieve desirable in vitro responses and regeneration including inhibition of callus formation, single shoot formation, and induction of multiple shoots. The well-developed shoots were rooted on media containing auxin (α-naphthaleneacetic acid). The protocol was developed using the variety USO-31 and has been successfully applied to four other industrial hemp varieties: Tygra, Monoica, Bialobrzeskie, Fibrol.

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Biotechnological production of cannabis (Cannabis sativa L.) is currently of great interest. Analysis of endogenous cytokinins in different types of explants of germinated hemp seedlings and application of new cytokinin derivatives and other growth regulators helped to create a functional protocol for hemp multiplication in vitro on a selected hemp genotype. The protocol was developed as a basic biotechnological method for other procedures, such as transformation techniques.

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3,6-Dichloro-o-anisic acid


α-Naphthaleneacetic acid


α-(2-Oxo-2-phenylethyl)-1H-indole-3-acetic acid






Meta-topolin O-glucoside








Fraction containing O-glucoside


Fraction containing nucleotide


Fraction containing N9-glucoside


Fraction containing riboside






Multiple-shoot culture


Adenine hemisulphate




  • Andre ChM, Hausman J-F, Guerriero G (2016) Cannabis sativa: the plant of the thousand and one molecules. Front Plant Sci 19:1–17

    Google Scholar 

  • Aremu AO, Bairu MW, Doležal K, Finnie JF, Van Staden J (2012a) Topolins: a panacea to plant tissue culture challenges? Plant Cell Tissue Org Cult 108:1–16

    Article  CAS  Google Scholar 

  • Aremu AO, Bairu MW, Novák O, Plačková L, Zatloukal M, Doležal K et al (2012b) Physiological responses and endogenous cytokinin profiles of tissue-cultured ‘Williams’ bananas in relation to Roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments. Planta 236:1775–1790

    Article  CAS  PubMed  Google Scholar 

  • Barahmand F, Beizaee N, Nayyeri MD, Sharafi A, Manjili HK, Danafar H, Sohi HH (2016) Cannabis sativa L. genetically transformed root based culture via Agrobacterium rhizogenes. Pharm Biomed Res 2:13–18

    Article  Google Scholar 

  • Blagoeva E, Dobrev PI, Malbeck J, Motyka V, Strnad M, Hanuš J, Vaňková R (2004) Cytokinin N-glucosylation inhibitors suppress deactivation of exogenous cytokinins in radish, but their effect on active endogenous cytokinins is counteracted by other regulátory mechanisms. Physiol Plant 121:215–222

    Article  CAS  PubMed  Google Scholar 

  • Braemer R, Paris M (1987) Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L. Plant Cell Rep 6:150–152

    CAS  PubMed  Google Scholar 

  • Braut-Boucher F, Petiard V (1981) Sur la mise en culture in vitro de tissus de différents types chimiques du Cannabis sativa L. C R Acad Sci Paris sér III 292:833–838 (in Frecnh)

    CAS  Google Scholar 

  • Chandra S, Lata H, El Sohly MA (2017) Cannabis sativa L.—botany and biotechnology. Springer, Basel

    Book  Google Scholar 

  • Davies PJ (2004) Plant hormones: biosynthesis, signal transduction, action. Springer, Dordrecht. ISBN 1-4020-2684-6

    Google Scholar 

  • Faiss M, Zalubilová J, Strnad M, Schmülling T (1997) Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. Plant J 12:401–415

    Article  CAS  PubMed  Google Scholar 

  • Farag S (2014) Cannabinoids production in Cannabis sativa L.: An in vitro approach. Dissertation, Technical University of Dortmund, Germany.

  • Feeney M, Punja ZK (2003) Tissue culture and Agrobacterium–mediated transformation of hemp (Cannabis sativa L.). In Vitro Cell Dev Biol—Plant 39:578–585

    Article  CAS  Google Scholar 

  • Feeney M, Punja ZK (2006) Hemp (Cannabis sativa L.). In: Wang K (ed) Methods in molecular biology, vol 2. Series 344: Agrobacterium protocols. Humana Press Inc, Totowa, pp 373–382

    Google Scholar 

  • Fissi J, Boucher F, Cosson L, Paris M (1981) Etude in vitro des capacités organogénétiques des tissus du Cannabis sativa L. Effet des différentes substances de croissance. Pl Médet Phyto 15:217–223 (in French)

    Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    Article  CAS  PubMed  Google Scholar 

  • Griga M, Bjelková M (2013) Flax (Linum usitatissimum L.) and hemp (Cannabis sativa L.) as fibre crops for phytoextraction of heavy metals: Biological, agro-technological and economical point of view. In: Gupta DK (ed) Plant-based remediation processes, soil biology, vol 35. Springer-Verlag, Berlin, pp 199–237

    Chapter  Google Scholar 

  • Grulichova M, Mendel P, Lalge AB, Slamova N, Trojan V, Vyhnanek T, Winkler J, Vaverková MD, Adamcova D, Đorđevic B (2017) Effect of different phytohormones on growth and development of micropropagated Cannabis sativa L. Mendelnet November 8–9, Brno, Czech Republic, pp 618–623

  • Hartsel SC, Loh WH-T, Robertson LW (1983) Biotransformation of cannabidiol to cannabielsoin by suspension cultures of Cannabis sativa and Saccharum officinarum. Planta Med 48:17–19

    Article  CAS  PubMed  Google Scholar 

  • Hayashi K, Neve J, Hirose M, Kuboki A, Shimada Y, Kepinski S, Nozaki H (2012) Rational design of an auxin antagonist of the SCFTIR1 auxin receptor complex. ACS Chem Biol 7:590–598

    Article  CAS  PubMed  Google Scholar 

  • Hemphill JK, Turner JC, Mahlberg PG (1978) Studies on growth and cannabinoid composition of callus derived from different strains of Cannabis sativa. Lloydia 41:453–462

    CAS  Google Scholar 

  • Jekkel Z, Heszky LE, Ali AH (1989) Effect of different cryoprotectants and transfer temperatures on the survival rate of hemp (Cannabis sativa L.) cell suspension in deep freezing. Acta Biol Hungarica 40:127–136

    CAS  Google Scholar 

  • Lata H, Chandra S, Khan IA, El Sohly MA (2009a) Thidiazuron induced high frequency direct shoot organogenesis of Cannabis sativa L. In Vitro Cell Dev Biol- Plant 45:12–19

    Article  CAS  Google Scholar 

  • Lata H, Chandra S, Khan IA, El Sohly MA (2009b) Propagation through alginate encapsulation of axillary buds of Cannabis sativa L.—an important medicinal plant. Physiol Mol Biol Plants 15:79–86

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lata H, Chandra S, Khan IA, El Sohly MA (2010a) High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L. Planta Med 76:1629–1633

    Article  CAS  PubMed  Google Scholar 

  • Lata H, Chandra S, Techen N, Khan IA, El Sohly MA (2010b) Assessment of the genetic stability of micropropagated plants of Cannabis sativa by ISSR markers. Planta Med 76:97–100

    Article  CAS  PubMed  Google Scholar 

  • Lata H, Chandra S, Techen N, Khan IA, El Sohly MA (2011) Molecular analysis of genetic fidelity in Cannabis sativa L. Plants grown from synthetic (encapsulated) seeds following in vitro storage. Biotechnol Lett 33:2503–2508

    Article  CAS  PubMed  Google Scholar 

  • Lata H, Chandra S, Techen N, Khan IA, El Sohly MA (2016) In vitro mass propagation of Cannabis sativa L.: a protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants. J Appl Res Med Aromat Plants 3:18–26

    Google Scholar 

  • Loh WH-T, Hartsel SC, Robertson LW (1983) Tissue culture of Cannabis sativa L. and in vitro biotransformation of phenolics. Z Pflanzenphysiol 111:395–400

    Article  CAS  Google Scholar 

  • MacKinnon L, McDougall G, Aziz N, Millam S (2001) Progress towards transformation of fibre hemp. In: Macfarlane Smith MH, Heilbronn TD (eds) Annual report of the Scottish Crop Research Institute. SCRI Invergowrie, Dundee, pp 84–86

    Google Scholar 

  • Mandolino G, Ranalli P (1999) Advances in biotechnological approaches for hemp breeding and industry. In: Ranalli P (ed) Advances in hemp research. Haworth Press, New York, pp 185–208

    Google Scholar 

  • Movahedi M, Ghasemiomran V, Torabi S (2016) Effect of explants type and plant growth regulators in vitro callus induction and shoot regeneration of Cannabis sativa L. Iran J Med Aromatic Plants 32:758–768

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Mve MDS, Mergeai G, Druart P, Baudoin JP, Toussaint A (2013) In vitro micropropagation of Jatropha curcas L. from bud aggregates. J Technol Innov Renew Energy 2:145–154

    Google Scholar 

  • Novák O, Hauserová E, Amakorová P, Doležal K, Strnad M (2008) Cytokinin profilig in plant tissues using ultra-performance liquid chromatography-electrospray tandem mass spectrometry. Phytochemistry 69:2214–2224

    Article  CAS  PubMed  Google Scholar 

  • Plawuszewski M, Lassocinski W, Wielgus K (2006) Regeneration of Polish cultivars of monoecious hemp (Cannabis sativa L.) grown in in vitro. In: Kozlowski R, Gennady E, Pudel F (eds) Renewable resources and plant biotechnology. Nova Science Publishers, New York, pp 42–131

    Google Scholar 

  • Plíhal O, Szučová L, Galuszka P (2013) N9-substituted aromatic cytokinins with negligible side effects on root development are an emerging tool for in vitro culturing. Plant Signal Behav 8:24392

    Article  CAS  Google Scholar 

  • Plíhalová L, Vylíčilová H, Doležal K, Zahajská L, Zatloukal M, Strnad M (2016) Synthesis of aromatic cytokinins for plant biotechnology. N Biotechnol 33:614–624

    Article  CAS  PubMed  Google Scholar 

  • Preneux C, Braut-Boucher F, Van Tran Thahn K (1985) Expression in vitro de la variabilité au niveau de la différentiation morphogénétique et des capacités biosynthétiques d´explants de Cannabis sativa L. placés dans différentes conditions d´environment. Bull Soc Bot Fr, Actualités botaniques 132:3

    Google Scholar 

  • Ranalli P, Venturi G (2004) Hemp as a raw material for industrial applications. Euphytica 140:1–6

    Article  Google Scholar 

  • Richez-Dumanois Ch, Braut-Boucher F, Cosson L, Paris M (1986) In vitro propagation of hemp: application to selected clones of Cannabis sativa L. for preservation of plants. Agronomie 6:487–495 (in French)

    Article  Google Scholar 

  • Salentijn EMJ, Zhang Q, Amaducci S, Yang M, Trindade LM (2015) New developments in fiber hemp (Cannabis sativa L.) breeding. Industr Crops Prod 68:32–41

    Article  Google Scholar 

  • Ślusarkiewicz-Jarzina A, Ponitka A, Kaczmarek Z (2005) Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of Cannabis sativa L. Acta Biol Crac Ser Bot 47:145–151

    Google Scholar 

  • Szučová L, Spíchal L, Doležal K, Zatloukal M, Greplová J, Galuszka P, Kryštof V, Voller J, Popa I, Massino FJ, Jørgensen J-E, Strnad M (2009) Synthesis, characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives. Bioorg Med Chem 17:1938–1947

    Article  CAS  PubMed  Google Scholar 

  • Verzár-Petri G, Ladocsy T, Oroszlán P (1982) Differentiation and production of cannabinoids in tissue cultures of Cannabis sativa. Acta Bot Acad Sci Hung 28:279–290

    Google Scholar 

  • Wahby I, Caba JM, Ligero F (2013) Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures. J Plant Interact 8:312–320

    Article  CAS  Google Scholar 

  • Wang R, He LS, Xia B, Tong JF, Li N, Peng F (2009) A micropropagation system for cloning of hemp (Cannabis sativa L.) by shoot tip culture. Pak J Bot 41:603–608

    CAS  Google Scholar 

  • Zatloukal M, Gemrotová M, Doležal K, Havlíček L, Spíchal L, Strnad M (2008) Novel potent inhibitors of A. thaliana cytokinin oxidase/dehydrogenase. Bioorg Med Chem 16:9268–9275

    Article  CAS  PubMed  Google Scholar 

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This study was supported by a Grant (TA04010331) from the Technology Agency of the Czech Republic, institutional support from the Ministry of Agriculture of the Czech Republic, a Grant (51834/2017-MZE-17253/6.2.8) from the National Programme on Conservation and Utilization of Plant Genetic Resources and Agro-biodiversity Institutional support of Ministry of Agriculture of Czech Republic, and by the Ministry of Education, Youth and Sports of the Czech Republic an ERDF project entitled “Development of Pre- Applied Research in Nanotechnology and Biotechnology” (No. CZ.02.1.01/0.0/0.0/17_048/0007323).

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Correspondence to Iva Smýkalová.

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Communicated by Ranjith Pathirana.

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Smýkalová, I., Vrbová, M., Cvečková, M. et al. The effects of novel synthetic cytokinin derivatives and endogenous cytokinins on the in vitro growth responses of hemp (Cannabis sativa L.) explants. Plant Cell Tiss Organ Cult 139, 381–394 (2019).

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