An In Vitro–Ex Vitro Micropropagation System for Hemp

Abstract

Hyperhydricity of shoots initiated in vitro, poor shoot extension, inability of shoot cultures to maintain good growth over an extended time, and unsuccessful ex vitro rooting have limited the development of a commercial scale micropropagation system for hemp (Cannabis sativa). We present a culture initiation method that prevents shoot hyperhydricity using vented-lid vessels with 0.2-µm pores and medium containing agar at 1% (w/v). To optimize shoot multiplication in vitro, a control medium (medium A) and four treatment media (medium B, C, D, and E), with varying inorganic nutrients and vitamins were tested. Control medium A consisted of 1× Murashige and Skoog (MS) with vitamins plus 3% (w/v) sucrose, 0.5 mg·L−1 metatopolin, 0.1 mg·L−1 gibberellic acid, and 0.8% agar (w/v) at pH 5.7. The four treatment media differed from the control medium as follows: medium B, 2.5× MS with vitamins; medium C, 1× MS with vitamins plus added mesos [calcium chloride (anhydrous), magnesium sulfate (anhydrous), and potassium phosphate (monobasic) nutrients]; medium D, 1× MS with vitamins plus added vitamins; and medium E, 1× MS with vitamins plus added mesos and vitamins. Medium C and medium E produced more microcuttings than the control at 6 weeks after the initial subculture with shoot multiplication media and all other treatments at 9 and 12 weeks. Shoots grown on these two media displayed optimal extension and leaf lamina development; however, they exhibited slight chlorosis by 12 weeks after subculture with shoot multiplication media. In a separate experiment, medium E was supplemented with ammonium nitrate at 0, 500, 1000, or 1500 mg·L−1, and cultures grown with 500 mg·L−1 produced the most microcuttings and exhibited the best combination of shoot extension and leaf lamina development. We provide a method of prerooting microshoots in vitro that has resulted in 75% to 100% rooting ex vitro in rockwool. Using 10 recently micropropagated plants, ≈300 retip cuttings (cuttings taken from new shoots from recently micropropagated plants) were harvested over 10 weeks. The average weekly rooting was more than 90%. Retipping can produce nine-times as many plants in a similar amount of floor space as stem cuttings derived from traditional stock mother plants. The micropropagation/retipping method proposed can be a more efficient way to generate clonal liner plants for commercial-scale production.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

“…Meristems have not been previously tested as an explant source in either hemp or cannabis, although shoot tips were used for direct regeneration of hemp (Wang et al, 2009 ), and stem tips were used for micropropagation and retipping studies on hemp (Lubell-Brand et al, 2021 ). Meristems are important starting material, as they contain a lower frequency of internally-borne microbes and viruses (Wang and Hu, 1980 ).…”

“…Wang et al ( 2009 ) used an MS basal salts medium with 30 g/L sucrose, 6.8 g/L phytagel, and 1 μM of TDZ to produce axillary buds from shoot tips of hemp during micropropagation. Lubell-Brand et al ( 2021 ) used an MS salts medium described by Lata et al ( 2016 ) in which TDZ was replaced with the growth regulator meta-topolin (mT). They reported that hyperhydricity was reduced by modifying the agar content of the medium, coupled with improved vessel ventilation and enhanced nitrogen levels.…”

“…Therefore, both DKW and MS salts media can support short-term growth in tissue culture media during the initiation of cultures. However, continuous subcultures and multiplication on a DKW-based medium appear to yield healthier and more vigorous plants (Page et al, 2021 ) or on an MS medium supplemented with enhanced levels of nitrogen (Lubell-Brand et al, 2021 ).…”

“…When compared with conventionally propagated tea plants using tissue culture, the ex vitro rooted micro shoots produced superior plants. Similarly, an in vitro – ex vitro micropropagation system was recently described for hemp (Lubell-Brand et al, 2021 ).…”

“…The interest in micropropagation through tissue culture is to produce genetically identical, pathogen-free plants year-round in a limited amount of space (Yancheva and Kondakova, 2018 ; Lubell-Brand et al, 2021 ; Monthony et al, 2021 ). The results of this study, and those of previous investigators (Lata et al, 2009 ; Wang et al, 2009 ; Page et al, 2021 ) show that it is possible to obtain plantlets from tissue cultures of drug-type C. sativa , but the process requires a detailed assessment of the genotypic response, evaluation of the effect of basal salts medium, establishment of conditions promoting shoot growth and rooting frequency, and achievement of success in acclimatization.…”