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In Vitro Propagation of Cannabis sativa L. and Evaluation of Regenerated Plants for Genetic Fidelity and Cannabinoids Content for Quality Assurance

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Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, Second Edition

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1391))

Abstract

Cannabis sativa L. (Marijuana; Cannabaceae), one of the oldest medicinal plants in the world, has been used throughout history for fiber, food, as well as for its psychoactive properties. The dioecious and allogamous nature of C. sativa is the major constraint to maintain the consistency in chemical profile and overall efficacy if grown from seed. Therefore, the present optimized in vitro propagation protocol of the selected elite germplasm via direct organogenesis and quality assurance protocols using genetic and chemical profiling provide an ideal pathway for ensuring the efficacy of micropropagated Cannabis sativa germplasm. A high frequency shoot organogenesis of C. sativa was obtained from nodal segments in 0.5 μM thidiazuron medium and 95 % in vitro rhizogenesis is obtained on half-strength MS medium supplemented with 500 mg/L activated charcoal and 2.5 μM indole-3-butyric acid. Inter Simple Sequence Repeats (ISSR) and Gas Chromatography-Flame Ionization Detection (GC-FID) are successfully used to monitor the genetic stability in micropropagated plants up to 30 passages in culture and hardened in soil for 8 months.

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References

  1. Hammond CT, Mahlberg PG (1977) Morphogenesis of capitate glandular hairs of Cannabis sativa (Cannabaceae). Am J Bot 64:1023–1031

    Article  Google Scholar 

  2. ElSohly MA, Gul W (2014) Constituents of Cannabis sativa. In: Pertwee RG (ed) Handbook of Cannabis. Oxford University Press, Oxford, pp 3–22

    Chapter  Google Scholar 

  3. Sirikantaramas S, Taura F, Morimoto S, Shoyama Y (2007) Recent advances in Cannabis sativa research: biosynthetic studies and its potential in biotechnology. Curr Pharm Biotechnol 8:237–243

    Article  CAS  PubMed  Google Scholar 

  4. Faisal M, Anis M (2002) Rapid in vitro propagation of Rauvolfia tetraphylla L.—an endangered medicinal plant. Physiol Mol Biol Plants 8(2):295–299

    Google Scholar 

  5. Patial V, Devi K, Sharma M, Bhattacharya A, Ahuja PS (2012) Propagation of Picrorhiza kurroa Royle ex Benth: an important medicinal plant of Western Himalaya. J Med Plant Res 6(34):4848–4860

    Google Scholar 

  6. Sivanesan I (2007) Direct regeneration from apical bud explants of Withania somnifera Dunal. Indian J Biotechnol 6:125–127

    CAS  Google Scholar 

  7. Husain MK, Anis M, Shahzad A (2007) In vitro propagation of Indian kino (Pterocarpus marsupium Roxb.) using Thidiazuron. In Vitro Cell Dev Biol Plant 43:59–64

    Article  CAS  Google Scholar 

  8. Shahzad A, Ahmad N, Anis M (2006) An improved method of organogenesis from cotyledonary callus of Acacia sinuate (Lour.) Merr. using Thidiazuron. J Plant Biotechnol 8:15–19

    Google Scholar 

  9. Lata H, Bedir E, Hosick A, Ganzera M, Khan IA, Moraes RM (2002) In vitro plant regeneration from leaf-derived callus in Black cohosh (Cimicifuga racemosa). Planta Med 68:912–915

    Article  CAS  PubMed  Google Scholar 

  10. Shahzad A, Parveen S, Fatema M (2010) Development of a regeneration system via nodal segment culture in Veronica anagallis-aquatica L.—an amphibious medicinal plant. J Plant Interact 5:115–121

    Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Loh WHT, Hartsel SC, Robertson W (1983) Tissue culture of Cannabis sativa L. and in vitro biotransformation of phenolics. Z Pflanzenphsiol: 395–400

    Google Scholar 

  13. Richez-Dumanois C, Braut-Boucher F, Cosson L, Paris M (1986) Multiplication vegetative in vitro du chanvre (Cannabis sativa L.). Application a la conservation des clones selectionnes. Agronomie 6:487–495

    Google Scholar 

  14. 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 

  15. Slusarkiewicz-Jarzina A, Ponitka A, Kaczmarek Z (2005) Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of C annabis sativa L. Acta Biol Cracov Bot 47(2):145–151

    Google Scholar 

  16. Bing X, Ning L, Jinfeng T, Nan G (2007) Rapid tissue culture method of Cannabis sativa for industrial uses. CN 1887043 A 20070103 Patent. pp 9

    Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Ross SA, Parker M, Arafat R, Lovett K, ElSohly MA (1996) The analysis of confiscated marijuana samples for different cannabinoids using GC/FID. Am Lab:16–17

    Google Scholar 

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Acknowledgements

This work was supported in part through the National Institute on Drug Abuse (NIDA), National Institute of Health (NIH), Department of Health and Human Services, USA, contract no. N01DA-10-7773.

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Correspondence to Hemant Lata .

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Lata, H., Chandra, S., Khan, I.A., ElSohly, M.A. (2016). In Vitro Propagation of Cannabis sativa L. and Evaluation of Regenerated Plants for Genetic Fidelity and Cannabinoids Content for Quality Assurance. In: Jain, S. (eds) Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, Second Edition. Methods in Molecular Biology, vol 1391. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3332-7_19

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  • DOI: https://doi.org/10.1007/978-1-4939-3332-7_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3330-3

  • Online ISBN: 978-1-4939-3332-7

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