There is increased interest in the production of hemp (Cannabis sativa) because of its medicinal properties (Small, 2015). For commercial production purposes, hemp is propagated by seed or stem cuttings to take advantage of superior genotypes (Cervantes, 2015). Many indoor hemp production facilities propagate cultivars by taking stem cuttings from stock mother plants, which they must maintain (Bechtel, 2019). Mother plants are large (10-gal container size) and require a significant amount of grow space to provide enough cuttings to meet production quotas. Growers must maintain mother plants in triplicate, with each replicate grown in a separate area of the facility, to reduce the risk of losing valuable cultivars to sudden disease outbreaks. Mother plants lose vigor because of the serial removal of shoots for cuttings, and they must be replaced every 6 months. Additionally, over time, mother plants accumulate insects and diseases, thus limiting their useful life as donors of cuttings. Overall, this propagation process is labor-intensive and inefficient. Hemp growers are interested in micropropagation as an alternative method of generating clones for commercial production (Rosslee, 2020).
Micropropagation provides unique benefits to growers and has several advantages over traditional plant cloning systems. These include the production of a large number of genetically clonal plants, uniform plants with enhanced vigor, disease-free plants, and preservation of maternal germ lines (Hartmann et al., 2002). Micropropagation also requires substantially fewer mother plants to be maintained compared with traditional stem cutting propagation, and in vitro cultures can be stored for longer in a smaller area than mother plants.
There are few published reports of hemp micropropagation. Wang et al. (2009) evaluated the effects of growth regulator additions to Murashige and Skoog (MS) medium on in vitro shoot multiplication and rooting of hemp cultures started from seed. Using nodal stem segments and MS medium, Lata et al. (2009) similarly tested rates of three growth regulators alone and in combination with gibberellic acid (GA3) on shoot multiplication. Lata et al. (2016) published a protocol refinement of their previous work (Lata et al., 2009) and introduced the growth regulator meta-topolin (MT), which was found to be superior to thidiazuron (TDZ) for in vitro shoot multiplication. Unfortunately, these published protocols have not translated well to large-scale micropropagation of clones necessary for commercial production. Noted shortcomings of published micropropagation methods include development of hyperhydricity during establishment of shoots in vitro, lack of consistent shoot elongation in culture, and inability of shoot cultures to maintain quality growth for an extended period of time (Monthony et al., 2021).
Microshoots from in vitro cultures are miniaturized, have altered physiology, and root easily (Hartmann et al., 2002). Nursery producers use a process called retipping to take advantage of and extend the period of time that micropropagated plants retain this miniaturized physiology so that more cuttings can be rooted (Keith and Brand, 1995). Retipping is the repeated harvesting of new shoots from recently micropropagated plants. The retipping process stimulates shoot growth from latent buds originating from the region of the stem that was miniaturized in culture. Retipping is routinely used to substantially increase the yield of propagules for crops such as rhododendron (Rhododendron sp.), mountain laurel (Kalmia latifolia), and lilac (Syringa vulgaris).
The objective of this work was to enhance hemp micropropagation by reducing hyperhydricity, improving in vitro shoot extension and performance through adjustment of the media nutrient content, and developing a method of ex vitro rooting. An additional objective was to evaluate retipping of recently micropropagated plants as a method of obtaining large quantities of clones for commercial-scale hemp production.
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