Micropropagation of Hemp (Cannabis sativa L.)

Abstract

Hemp (Cannabis sativa L.) is commonly grown for the medicinal secondary metabolites produced by pistillate inflorescences. Micropropagation is a valuable method of propagating hemp plants because of the aseptic process and the production of true-to-type propagules. The hemp cultivar TJ’s CBD was used for a series of experiments to compare media inputs and practices for the clonal micropropagation of hemp. For stage I, shoot tips harvested from stock plants that were grown in a growth chamber produced less endogenous contamination in newly established cultures than shoot tips harvested from the greenhouse. In addition, stage I disinfection treatments with 20%, 40%, and 60% bleach (7.5% sodium hypochlorite) for 10 minutes had no differences in surface contamination rates. All concentrations were able to clean explants equally, and no damage to the explants was observed. For stage II, there were no differences in growth and multiplication rate between shoot tip or nodal explants. In addition, no differences were observed between the gelling agent’s agar, agargellan, and gellan gum at standard rates. When basal nutrient formulations were compared at standard rates and with their respective vitamins, Murashige and Skoog, Linsmaier & Skoog, and Driver & Kuniyuki Walnut media were found to be superior to Lloyd & McCown Woody Plant Medium. Media pH levels of 4.0, 5.0, 5.8, 6.0, and 7.0 were compared, and no differences were observed in final fresh weights, shoot lengths, or quality ratings. The pH levels of 5.8, 6.0, and 7.0 generated a greater number of lateral nodes. Sucrose levels of 0%, 1.5%, 3.0%, 4.5%, and 6.0% (wt/vol) were also compared, with the 1.5% and 3.0% rates showing greater fresh weights, shoot lengths, and quality ratings. Growth room temperatures of 22, 24, 26, and 28 °C were compared, with temperatures of 28 and 26 °C generating greater fresh weights, shoot lengths, numbers of nodes, and quality ratings compared with cooler temperatures. The cytokinins 6-enzylaminopurine (BA), 6-(γ,γ-dimethylallylamino) purine (2iP), and thidiazuron (TDZ) were compared at 1.0, 5.0, and 10.0 μM concentrations. The 5.0-μM TDZ treatment generated greater fresh weights and numbers of lateral nodes; however, it also produced the shortest shoot lengths and lowest quality ratings. The 2iP treatments at 1.0 and 5.0 μM, and the BA treatment at 1.0 μM produced the greatest quality ratings. The 5.0-μM 2iP level was considered the best treatment for stage II multiplication based on high ratings, in addition to the greater final fresh weights, shoot lengths, and numbers of nodes that were produced. For stage III experiments, the auxins indole 3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) were compared at concentrations of 0.25, 0.5, and 2.5 μM. Auxin treatments of 0.25 μM NAA, 0.5 μM NAA, and 2.5 μM IBA generated the greatest final shoot fresh weights, root fresh weights, and numbers of nodes. However, the 2.5-μM IBA treatment resulted in a higher overall rating. For stage IV, ex vitro rooting and acclimation trials compared a dome and an intermittent mist system, as well as treated the unrooted cuttings with an externally applied auxin. Acclimating with a dome produced greater shoot heights, fresh shoot weights, and overall ratings compared with the mist system. The auxin treatment mildly increased fresh root weight, but was not as important to acclimation success as the domed environment. It has been concluded that a micropropagation system that uses lower rates of sucrose, higher growing temperatures, and lower rates of the cytokinins BA and 2iP are optimal for the micropropagation of hemp. In addition, when acclimating hemp plants from tissue culture, an in vitro stage III can be bypassed and plants can be rooted ex vitro during stage IV acclimation with a dome with or without additional auxin treatments.