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In vitro germplasm conservation of high Δ9-tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions

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

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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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Correspondence to Suman Chandra.

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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

Keywords

  • Cannabinoids
  • GC-FID
  • Germplasm conservation
  • Osmotic agents
  • Synthetic seed