Planta Med 2010; 76(14): 1629-1633
DOI: 10.1055/s-0030-1249773
Biochemistry, Molecular Biology and Biotechnology
Original Papers
© Georg Thieme Verlag KG Stuttgart · New York

High Frequency Plant Regeneration from Leaf Derived Callus of High Δ 9-Tetrahydrocannabinol Yielding Cannabis sativa L.

Hemant Lata1 , Suman Chandra1 , Ikhlas A. Khan1 , 2 , Mahmoud A. ElSohly1 , 3
  • 1National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS, USA
  • 2Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS, USA
  • 3Department of Pharmaceutics, School of Pharmacy, University of Mississippi, University, MS, USA
Further Information

Publication History

received Nov. 2, 2009 revised January 14, 2010

accepted March 3, 2010

Publication Date:
30 March 2010 (online)


An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC‐FID), high THC yielding elite female clone of a drug-type Cannabis variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy–acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 – MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom.


  • 1 Sirikantaramas S, Taura F, Tanaka Y, Ishikawa Y, Morimoto S, Shoyama Y. Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity is secreted into the storage cavity of the glandular trichomes.  Plant Cell Physiol. 2005;  46 1578-1582
  • 2 Loh W H T, Hartsel S C, Robertson W. Tissue culture of Cannabis sativa L. and in vitro biotransformation of phenolics.  Z Pflanzenphysiol. 1983;  111 395-400
  • 3 Richez-Dumanois C, Braut-Boucher F, Cosson L, Paris M. Multiplication vegetative in vitro du chanvre (Cannabis sativa L.). Application a la conservation des clones selections.  Agronomie. 1986;  6 487-495
  • 4 Mandolino G, Ranalli P. Advances in biotechnological approaches for hemp breeding and industry. Ranalli P Advances in hemp research. New York; Haworth Press 1999: 185-208
  • 5 Slusarkiewicz-Jarzina A, Ponitka A, Kaczmarek Z. Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of Cannabis sativa L.  Acta Biol Craco Series Bot. 2005;  47 145-151
  • 6 Bing X, Ning L, Jinfeng T, Nan G. Rapid tissue culture method of Cannabis sativa for industrial uses. CN Patent 1887043 A 20070103. 2007
  • 7 Lata H, Chandra S, Khan I, ElSohly M A. Thidiazuron induced high frequency direct shoot organogenesis of Cannabis sativa L.  In Vitro Cell Dev Biol Plant. 2009;  45 12-19
  • 8 Lata H, Chandra S, Techen N, Khan I, ElSohly M A. Assessment of genetic stability of micropropagated plants of Cannabis sativa L. by ISSR markers.  Planta Med. 2010;  76 97-100
  • 9 Chandra S, Lata H, Khan I A, Mehmedic Z, ElSohly M A. Assessment of cannabinoids content in micropropagated plants of Cannabis sativa L. and their comparison with conventionally propagated plants and mother plant during developmental stages of growth.  Planta Med. 2009;  DOI: 10.1055/s-0029-1240628
  • 10 Lata H, Chandra S, Khan I, ElSohly M A. Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. – an important medicinal plant.  Physiol Mol Biol Plants. 2009;  15 79-86
  • 11 Chandra S, Lata H, Khan I A, ElSohly M A. Photosynthetic response of Cannabis sativa L. to variations in photosynthetic photon flux densities, temperature and CO2 conditions.  Physiol Mol Biol Plants. 2008;  14 299-306
  • 12 Ross S A, Parker M, Arafat R, Lovett K, ElSohly M A. The analysis of confiscated marijuana samples for different cannabinoids using GC/FID.  Am Lab. 1995;  27 16-17
  • 13 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures.  Physiol Plant. 1962;  15 473-497
  • 14 Huetteman C A, Preece J E. Thidiazuron: a potent cytokinin for woody plant tissue culture.  Plant Cell Tissue Organ Cult. 1993;  33 105-119
  • 15 Al-Juboory K H, Skirvin R M, Williams D J. Callus induction and adventitious shoot regeneration of gardenia (Gardenia jasminoides Ellis) leaf explants.  Sci Hortic. 1998;  72 171-178
  • 16 Lata H, Bedir E, Hosick A, Ganzera M, Khan I A, Moraes R M. In vitro plant regeneration from leaf-derived callus in Black cohosh (Cimicifuga racemosa).  Planta Med. 2002;  68 912-915
  • 17 Thomas T D, Puthur J T. Thidiazuron induced high frequency shoot organogenesis in callus from Kigelia pinnata L.  Bot Bull Acad Sin. 2004;  45 307-313
  • 18 Husain M K, Anis M, Shahzad A. In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using thidiazuron.  In Vitro Cell Dev Biol Plant. 2007;  43 59-64
  • 19 Mok M C, Martin R C, Mok D W S. Cytokinins: biosynthesis, metabolism and perception.  In Vitro Cell Dev Biol Plant. 2000;  36 102-107
  • 20 Khawar K M C, Sancak S U, Zcan S. Effect of thidiazuron on shoot regeneration from different explants of lentil (Lens culinaris Medik.) via organogenesis.  Turk J Bot. 2004;  28 421-426
  • 21 Abrie A L, Staden J V. Micropropagation of the endangered Aloe polyphylla.  Plant Growth Regul. 2001;  33 19-23
  • 22 Fisse J, Braut F, Cosson L, Paris M. Etude in vitro des capacités organogénetiques de tissues de Cannabis sativa L. Effet de differentes substances de croissance.  Planta Med. 1981;  15 217-223
  • 23 Feeney M, Punja Z K. Tissue culture and agrobacterium-mediated transformation of hemp (Cannabis sativa L.).  In Vitro Cell Dev Biol Plant. 2003;  39 578-585

Ph.D. Hemant Lata

National Center for Natural Products Research
Research Institute of Pharmaceutical Sciences
School of Pharmacy
University of Mississippi

University, MS 38677


Phone: + 1 66 29 15 59 28

Fax: + 1 66 29 15 55 87