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.
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.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Fraction containing O-glucoside
Fraction containing nucleotide
Fraction containing N9-glucoside
Fraction containing riboside
Andre ChM, Hausman J-F, Guerriero G (2016) Cannabis sativa: the plant of the thousand and one molecules. Front Plant Sci 19:1–17
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
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
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
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
Braemer R, Paris M (1987) Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L. Plant Cell Rep 6:150–152
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)
Chandra S, Lata H, El Sohly MA (2017) Cannabis sativa L.—botany and biotechnology. Springer, Basel
Davies PJ (2004) Plant hormones: biosynthesis, signal transduction, action. Springer, Dordrecht. ISBN 1-4020-2684-6
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
Farag S (2014) Cannabinoids production in Cannabis sativa L.: An in vitro approach. Dissertation, Technical University of Dortmund, Germany. https://doi.org/10.17877/DE290R-16424
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
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
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)
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497
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
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
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
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
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
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
Ranalli P, Venturi G (2004) Hemp as a raw material for industrial applications. Euphytica 140:1–6
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)
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
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
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
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
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
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
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
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).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Communicated by Ranjith Pathirana.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
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). https://doi.org/10.1007/s11240-019-01693-5
- Tissue culture
- Growth regulators
- Apical dominance