In vitro germplasm conservation of high Delta(9)-tetrahydrocannabinol yielding elite clones of Cannabis sativa L. under slow growth conditions

作者：Lata Hemant; Chandra Suman^*; Mehmedic Zlatko; Khan Ikhlas A; ElSohly Mahmoud A

来源：Acta Physiologiae Plantarum, 2012, 34(2): 743-750.

DOI：10.1007/s11738-011-0874-x

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

Germplasm conservation of a high Delta(9)-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 %26apos;encapsulated%26apos; (synthetic seeds) and %26apos;non-encapsulated%26apos; nodal segments were stored at 5, 15 and 25A degrees C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25A degrees C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 mu M). %26apos;Encapsulated%26apos; nodal segments could be stored at low temperature 15A degrees C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to %26apos;encapsulated%26apos; cultures, the highest re-growth in %26apos;non-encapsulated%26apos; cultures was observed in the explants kept at 15A degrees 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. 25A degrees 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 %26apos;encapsulated%26apos; 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, Delta(9)-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from %26apos;encapsulated%26apos; 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.

• 出版日期2012-3