The effect of different concentrations of TDZ and BA on in vitro regeneration of Iranian cannabis (Cannabis sativa) using cotyledon and epicotyl explants

Document Type : Original research paper


1 Plant Breeding, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Genetics and Agricaltural Biotechnology Institute of Tabarestan, Sari Agricaltural Sciences and Natural Resources University, Sari, Iran

3 Department of Plant Breeding and Biotechnology, Science and Research, Islamic Azad University, Tehran, Iran


The present study was carried out to investigate micropropagation possibility and determine the optimal medium composition and plant growth regulators (PGRs) combinations under in vitro conditions. The cotyledon and epicotyl explants obtained from 1 month old in vitro grown seedlings were used in MS medium containing BA (0.1, 0.2, 0.5, 1, 2 and 3 mg-1) and TDZ (0.1, 0.2, 0.5, 1, 2 and 3 mg-1) either alone or in combination with 0.5 mg-1 IBA. The response of cannabis explants to PGRs treatments was much different from those observed in most of plant species. That is, callus formation had priority over direct regeneration in most of the PGRs treatments. Comparing the two explants, cotyledon had higher callus formation frequency and the largest callus volume was obtained for this explant in MS medium supplemented with 3 mg-1 TDZ + 0.5 mg-1 IBA. The highest callus fresh weight (3.15 gr) was obtained for cotyledon explant treated with 2 mg-1 TDZ+ 0.5 mg-1 IBA. In shoot formation step, the highest rate of shoot regeneration was achieved in the calli produced from epicotyl explant treated with 2 mg-1 BA + 0.5 mg-1 IBA; and the highest length of regenerated shoots (1.23 cm) was observed in 2 mg-1 BA + 0.5 mg-1 IBA treatment. In general, cotyledon was the best explant and TDZ in combination with IBA was the best treatment for callus formation. Epicotyl explant also showed better regeneration compared to cotyledon.


[1]    Alhadi, M. 2011. Micropropagation of Stevia Rebaudiana Bertoni a new sweetening crop in Egypt. Glob. J Biotechnol Biochem. 6(4):178-182.
[2]    Appendino, G., Chianese, G. and Taglialatela-Scafati,; O. 2011. Cannabinoids: occurrence and medicinal chemistry. Curr Med Chem. 18: 1085-1099.
[3]    Duncan, D. B. (1955). "Multiple range and multiple F tests". Biometrics 11: 1–42.
[4]    Flores-Sanchez, I. J., Verpoorte, R. 2008. PKS activities and biosynthesis of cannabinoids and flavonoids in Cannabis sativa L. plants. Plant Cell Physiol. 49, 1767–1782.
[5]    Feeney, M., Punja, Z. K. 2003. Tissue culture and Agrobacterium-mediated transformation of hemp (Cannabis sativa L.).  In Vitro Cell Dev Biol Plant. 39(6):578-585.
[6]    Gomez-Leyva, J.F., Martinez-Acosta, L.A., Lopez- Muraira, I.G., Silos-Espino, H., Ramirez-Cervantes, F. and Andrade-Gonzalez, I., 2008. Multiple shoot regeneration of roselle (Hibiscus sabdariffa L.) from a shoot apex culture system. Int J Botany, 4: 326–330.
[7]         Hendricks H., T.M. Malingre, S. Batterman and R. Bos. 1975. Mono- and sesquiterpene hydrocarbons of the essential oil of Cannabis sativa. Phytochemistry. 14: 814-15.
[8]         Hazekamp A, Grotenhermen F. 2010. Review on Clinical Studies with Cannabis and Cannabinoids 2005-2009. Cannabinoids. 5:1-21.
[9]         Huetteman A., and Preece J. 1993. Thidiazuron a potent cytokinin for woody plant tissue culture. Plant cell, tissue and organ culture 37:105-119.
[10]    Lata H, Chandra S, Khan Ia, Elsohly Ma. 2009. Propagation Through Alginate Encapsulation of Axillary Buds of Cannabis Sativa L. An Important Medicinal Plant. Physiol Mol Biol Plants. 15(1):79-86.
[11]    Lata, H., Chandra, S., Khan, I. A. and Elsohly, M. A. 2010. High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.. Planta Med. 76(14): 1629-1633.
[12]    Kostic M, Pejic B, Skundric P. 2008. Quality of chemically modified hemp fibres. Bioresource Tech. 99: 94-99.
[13]    Mechoulam R. 2005. Plant cannabinoids: a neglected pharmacological treasure trove. Br J Pharmacol; 146: 913-915.
[14]    Metheson, S. L., Nowak, J., and Maclean, N. 1990. Selection of regenerative genotypes from highly productive cultivars of alfalfa. Euphytica, 45(2):105-112.
[15]    Mujib, A., and Samaj, J. 2005. Somatic Embryogenesis. Springer-Verlag Berlin Heidelberg.
[16]    Pate, D.W.1994. Chemical ecology of Cannabis. J Int Hemp Assoc. 2: 32-37.
[17]    Raharjo, T. J., Eucharia, O., Chang, W.T., Verpoorte, R. (2006) Callus induction and  phytochemical characterization of Cannabis Sativa cell suspension culture. Indonesian Journal of Chemistry. 6 (1), 70-74.
[18]    Sharma, S., Kumar, N. and Reddy, M.P. 2011. Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration. Industrial Crops Product, 34: 943-951.
[19]    Singh, P. and Dwivedi, P. 2014. Two-stage culture procedure using thidiazuron for efficient micropropagation of Stevia rebaudiana, an anti-diabetic medicinal herb. Biotech. 4:431–437. 
[20]    Slusarkiewicz- Jarzina, A., Ponitka, A. and Kaczmarek, Z. 2005. Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of Cannabis Sativa. Acta Biologica Cracoviensia Series Botanica,15147(2): 145-151.
[21]    Wang X, Tang C, Yang X and Gao W. 2008. Characterization, amino acid composition and vitro digestibility of hemp (Cannabis Sativa L) proteins. Food Chemistry. 107, 11-18.
[22]    Ware MA, Tawfik VL. 2005. Safety issues concerning the medical use of cannabis and cannabinoids. Pain Research Management 10, Supplement A, 31–37.
Volume 3, Issue 2 - Serial Number 2
December 2015
Pages 20-27
  • Receive Date: 17 May 2015
  • Revise Date: 15 November 2015
  • Accept Date: 21 November 2015
  • First Publish Date: 01 December 2015