In vitro Germplasm Conservation of High THC Yielding Elite Clones of Cannabis sativa L. under Slow Growth Conditions

Abstract

Nodal segments of Cannabis sativa L. were excised from in vitro proliferated shoot cultures and encapsulated in high density sodium alginate (5%) hardened by 50 mM CaCl2 [1]. The encapsulated nodal segments were kept at three different storage conditions, refrigerator (5oC), growth chamber (15oC) and tissue culture room (25oC). The cultures were monitored for the re-growth and survival frequency after 8, 16 and 24 weeks under tissue culture (16h photoperiod, 25oC) conditions, on Murashige and Skoog (MS) medium supplemented with TDZ (0.5µM) and plant preservative mixture (PPM, 0.075%, Caisson laboratories, USA). Based on our results, encapsulated nodal segment could be stored at low temperature 15° C up to 24 weeks with a maximum re-growth ability and survival frequency of 83%. The well developed plantlets regenerated from encapsulated nodal segments were hardened off successfully with 90% survival frequency. Furthermore, chemical analysis of cannabinoids, using gas chromatography-flame ionization detection (GC-FID), was done to confirm if qualitative and quantitative differences in the major secondary metabolites exist between the mother plant and in vitro propagated slow growth stored plants. Six major cannabinoids i.e. Δ9-THC, THCV, CBD, CBC, CBG and CBN were identified and compared with the mother plant (MP). Our results clearly show similar cannabinoids profile and insignificant differences in THC contents between the two types of plants.