Abstract
Germplasm conservation of a high Δ9-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230 mM) hardened by 50 mM CaCl2. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 μM). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2% sorbitol and 2% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, Δ9-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.
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Abbreviations
- Δ9-THC:
-
Δ9-Tetrahydrocannabinol
- CBD:
-
Cannabidiol
- CBC:
-
Cannabichromene
- CBG:
-
Cannabigerol
- CBN:
-
Cannabinol
- DP:
-
Donor plant
- GC-FID:
-
Gas chromatography-flame ionization detection
- IBA:
-
Indole-3-butyric acid
- ½ MS:
-
Half strength Murashige and Skoog medium
- MS:
-
Murashige and Skoog medium
- PPFD:
-
Photon flux density
- PPM:
-
Plant preservative mixture
- RH:
-
Relative humidity
- TDZ:
-
Thidiazuron
- THCV:
-
Tetrahydrocannabivarin
References
Anand Y, Bansal YK (2002) Synthetic seeds: a novel approach of in vitro plantlet formation in vasaka (Adhatoda vasica Nees). Plant Biotech 19:159–162
Banerjee N, De Langhe E (1985) A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (Banana and plantain). Plant Cell Rep 4:351–354
Bhat SR, Chandel KPS (1993) In vitro conservation of Musa germplasm: effects of mannitol and temperature on growth and storage. J Hort Sci 68:841–846
Brenneisen R, Egli A, ElSohly MA, Henn V, Spiess Y (1996) The effect of orally and rectally administered Δ9-tetrahydrocannabinol on spasticity: a pilot study with patients. Int J Clin Pharmacol Ther 34(10):446–452
Chandra S, Lata H, Khan IA, ElSohly MA (2010a) Propagation of elite Cannabis sativa for the production of Δ9-tetrahydrocannabinol (THC) using biotechnological tools. In: Rajesh Arora (ed) Medicinal Plant Biotechnology. CAB International Press, UK, pp 98–114
Chandra S, Lata H, Mehmedic Z, Khan IA, ElSohly MA (2010b) 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 76:743–750
Dodds JH, Roberts LW (1995) Experiments in plant tissue culture. Cambridge University Press, New York
Fadel D, Kintzios S, Economou AS, Moschopoulou G, Constantinidou HIA (2010) Effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (Mentha spicata). The Open Hort J 3:31–35
Faisal M, Anis M (2007) Regeneration of plants from alginate encapsulated shoots of Tylophora indica (Burm.f.) Merrill, an endangered medicinal plant. J Hort Sci Biotechnol 82:351–354
Formukong EA, Evans AT, Evans F (1989) The medicinal uses of Cannabis and its constitutents. Phytother Res 3:219–231
Grinspoon L, Bakalar JB (1997) Marihuana the forbidden medicine. Yale University Press, New Haven and Yale
Lamrioui AM, Louerguioui A, Abousalim A (2009) Effect of the medium culture on the micro cutting of material resulting from adult cuttings of wild cherry trees (Prunus Avium L.) and of in vitro germination. European J Sci Res 25 (2):345–352
Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214
Lata H, Chandra S, Khan I, ElSohly 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 I, ElSohly MA (2009b) Propagation through alginate encapsulation of axillary buds of Cannabis sativa L.–an important medicinal plant. Physiol Mol Biol Plants 15(1):79–86
Lata H, Moraes RM, Bertoni B, Pereira AMS (2009c) In vitro germplasm conservation of Podophyllum peltatum L. under slow growth conditions. In Vitro Cell Dev Biol Plant 46:22–27
Mandal J, Patnaik S, Chand PK (2000) Alginate encapsulation of axillary buds of Ocimum americanum L. hoary basil), O. basilicum L. (sweet basil), O. gratissimum L. (shrubby basil), and O. sanctum L. (sacred basil). In Vitro Cell Dev Biol Plant 36:287–292
Mattes RD, Shaw LM, Eding-Owens J, Egelman K, ElSohly MA (1993) By passing the first pass effect for therapeutic use of cannabinoids. Pharmacol Biochem Behav 44(3):745–747
Mattes RD, Egelman K, Shaw LM, ElSohly MA (1994) Cannabinoids appetite stimulation. Pharmacol Biochem Behav 49(1):187–195
Mechoulam R (1986) The pharmacohistory of Cannabis sativa. In: Mechoulam R (ed) Cannabinoids as therapeutic agents. CRC Press, Florida, pp 1–19
Mechoulam R, Ben-Shabat A (1999) From gan-zi-gun-nu to anandamide and 2-arachidonoylglycerol: the ongoing story of Cannabis. Nat Prod Rep 16:131–143
Murashige T, Skoog FA (1962) Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Naik SK, Chand PK (2006) Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange. Sci Hortic 108:247–252
Narula A, Kumar S, Srivastava PS (2007) Genetic fidelity of in vitro regenerants encapsulation of shoot tips and high diosgenin content in Dioscorea bulbifera L., a potential alternative source of diosgenin. Biotechnol Lett 29:623–629
Negash A, Krens F, Schaart J, Visse B (2001) In vitro conservation of enset under slow-growth conditions. Plant Cell Tiss Organ Cult 66:107–111
Ray A, Bhattacharyaa S (2008) Storage and plant regeneration from encapsulated shoot tips of Rauvolfia serpentine-an effective way of conservation and mass propagation. S Afr J Bot 74:776–779
Redenbaugh K, Slade D, Viss P, Fujii JA (1987) Encapsulation of somatic embryos in synthetic seed coats. Horti Sci 22:803–809
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 16:16–17
Schumann E, Peil A, Weber WE (1999) Preliminary results of a German trial with different hemp (Cannabis sativa L.) accessions. Genet Resour Crop Evol 46:399–407
Singh AK, Sharma M, Varshney R, Agarwal SS, Bansal KC (2006a) Plant regeneration from alginate to encapsulated shoot tips of Phyllanthus amarus Schum and Thonn, a medicinally important plant species. In Vitro Cell Dev Biol Plant 42:109–113
Singh AK, Varshney R, Sharma M, Agarwal SS, Bansal KC (2006b) Regeneration of plants from alginate to encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species. J. Plant Physiol 163:220–223
Sirikantaramas S, Taura F, Morimoto Y, Shoyama Y (2007) Recent advances in Cannabis sativa research: biosynthetic studies and its potential in biotechnology. Curr Pharm Biotechnol 8:237–243
Van den Houwe I, Smet de K, Tezenas du MH, Swennen R (1995) Variability in storage potential of banana shoot cultures under medium term storage conditions. Plant Cell Tissue Organ Cult 42:269–274
West TP, Ravindra MB, Preece JE (2006) Encapsulation, cold storage, and growth of Hibiscus moscheutos nodal segments. Plant Cell Tissue Organ Cult 87:223–231
Westcott RJ (1981) Tissue culture storage of potato germplasm: use of growth retardants. Potato Res 24:343–352
Withers LA (1986) In vitro approaches to the conservation of plant genetic resources. In: Withers LA, Alderson PG (eds) Plant tissue culture and its agriculture applications. Butterworths, London, pp 261–276
Acknowledgments
This work was supported in part by 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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Communicated by B. Borkowska.
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Lata, H., Chandra, S., Mehmedic, Z. et al. In vitro germplasm conservation of high Δ9-tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions. Acta Physiol Plant 34, 743–750 (2012). https://doi.org/10.1007/s11738-011-0874-x
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DOI: https://doi.org/10.1007/s11738-011-0874-x