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

Abstract

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.

Key message

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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Abbreviations

BAP:

6-Benzylaminopurine

BAP9THP:

6-Benzylamino-9-(tetrahydroxypyranyl)purin

DICAMBA:

3,6-Dichloro-o-anisic acid

NAA:

α-Naphthaleneacetic acid

PEO-IAA:

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

KIN:

6-Furfurylaminopurine

m-T:

Meta-topolin

m-TOG:

Meta-topolin O-glucoside

o-T:

Ortho-topolin

p–T:

Para-topolin

CK:

Cytokinin

OG:

Fraction containing O-glucoside

5′MP:

Fraction containing nucleotide

9G:

Fraction containing N9-glucoside

R:

Fraction containing riboside

DHZ:

Dihydrozeatin

iP:

Isopentenyladenine

MSC:

Multiple-shoot culture

AS:

Adenine hemisulphate

tZ:

Trans-zeatin

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Acknowledgements

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). https://doi.org/10.1007/s11240-019-01693-5

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Keywords

  • Hemp
  • Micropropagation
  • Tissue culture
  • Auxin
  • Growth regulators
  • Apical dominance