Examining the stages of micropropagation for hemp (Cannabis sativa)

The five stages of in vitro micropropagation were investigated for Cannabis sativa 'TJs CBD' (hemp). This valuable plant species is used for food, fiber and its medicinal properties produced on pistillate inflorescences. Hemp is a dioecious plant, with the stamens and pistils developing on separate plants. Because of the dioecious nature of Cannabis and the importance of female plants, it is important to propagate female clones. The aseptic nature of plant micropropagation and the ability to clone large numbers of plants in vitro makes it a valuable process for Cannabis sativa. For phase 1 of micropropagation, bleach concentration were tested for their effect on disinfection and establishment success. For Stage 2, numerous trials were conducted to determine optimal inputs. Sucrose levels of 0, 1.5, 3.0, 4.5 and 6.0% were compared. The gelling agents agar (7 g L-1), agargellan (4 g L-1), and gellan gum (2 g L-1) were evaluated. Four basal media were assessed: Murashige and Skoog Medium (MS), Linsmaier & Skoog (LS), Driver and Kuniyaki Walnut medium (DKW), and Woody Plant Medium ( WPM). Media pH levels of 4, 5, 5.8, 6 and 7 were evaluated. Growth room temperatures of 22, 24, 26, and 28 degrees C were compared. The cytokinins 6-benzylaminopurine (BA), 6-(gamma, gamma-dimethylallylamino) purine (2iP), and thidiazuron (TDZ) were compared at 1.0, 5.0 and 10.0 uM concentrations. For Stage 3 micropropagation, the auxins indole3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) were tested at concentrations of 0, 0.25, 0.5 and 2.5 uM. Stage 4 acclimation was conducted by removing shoot tips from cultured plants and rooting them ex vitro in a greenhouse. A dome and mist system were compared, as well as auxin and no auxin treatments. The results from these experiments will be explained along with a recommended protocol for the micropropagation of Cannabis sativa 'TJs CBD'.