Abstract
Micropropagation of hemp (Cannabis sativa) is constrained by problems with hyperhydricity and culture decline of microshoots. These problems can be reduced by increasing agar and nutrients in the media during micropropagation stages 1 and 2, respectfully. Performance of microshoots of ‘Abacus’ and ‘Wife’ hemp cultured in Driver and Kuniyuki Walnut medium (DKW) for 15 weeks (6 weeks of stage 1 + 9 weeks of stage 2), with subculturing every 3 weeks during both stages 1 and 2, or in Murashige and Skoog with vitamins medium (MS) for 6 weeks (stage 1) followed by Lubell-Brand Cannabis medium (LBC) for 9 weeks (stage 2), with subculturing every 3 weeks during both stages 1 and 2, was evaluated. In a separate study, microshoot performance of ‘Abacus’ and ‘Wife’ in MS for 3 weeks (stage 1) followed by LBC for 6 weeks (stage 2), with subculturing every 3 weeks, using boxes (Magenta GA-7) with lids featuring a vent with a diameter of 10 mm and a pore size of 0.2 µM or using microboxes (Sac O2 O95/114 + OD95) with lids featuring a filter (Sac O2 #10) were evaluated. Shoot multiplication rate (SMR) and explant height were greater for ‘Abacus’ in LBC than DKW. For ‘Wife’, SMR at 9 weeks was greater in LBC, as LBC provided more nutrients and water than cultures had received in MS initially during stage 1. Culture medium did not influence ex vitro rooting success, which was 75% for ‘Abacus’ and ≥ 90% for ‘Wife’. Microboxes resulted in greater hyperhydricity of shoots and a lower ex vitro rooting percentage than boxes. For cultivars that are highly prone to developing hyperhydricity, like ‘Abacus’, the microboxes were not adequate to control this condition.
Micropropagation is the in vitro multiplication of genetically identical plants (clones) for high-throughput production purposes. Micropropagated plants are uniform and pathogen free, and demonstrate enhanced vigor compared with plants produced from traditional cloning methods (Hartmann et al. 2002). Hemp (Cannabis sativa) growers are interested in micropropagation as an alternative to propagation by stem cuttings from stock mother plants, which has several limitations (Rosslee 2020). Mother plants require a significant amount of grow space and must be replaced every 3 months because they accumulate insects and diseases, and lose vigor because of the serial removal of shoots for cuttings (Lubell-Brand et al. 2021). Micropropagation consists of four primary stages as follows: 1) establishment of sterile shoots in vitro and miniaturization of growth habit, 2) continuous and stable in vitro multiplication of uniform microshoots by subculturing, 3) root formation, and 4) acclimatization of rooted plantlets to greenhouse conditions (Hartmann et al. 2002). The development of a reliable micropropagation protocol for hemp has been problematic as a result of the occurrence of hyperhydricity of shoots during stage 1 and culture decline or the inability of shoot cultures to maintain high-quality growth for an extended period of time during stage 2 (Lubell-Brand et al. 2021; Monthony et al. 2021c).
Published micropropagation studies for hemp, which have evaluated explant source, culture medium formulation, and growth regulators, do not report success at all four micropropagation stages (Monthony et al. 2021c). Monthony et al. (2021c) identified four publications (Lata et al. 2009; Monthony et al. 2021a, Page et al. 2021; Wróbel et al. 2020) that indicate success with stage 2; however, the reported protocols did not translate well to commercial production for the following reasons. Monthony et al. (2021a) and Page et al. (2021) conducted stage 2 for only one subculture, Wróbel et al. (2020) performed hedging, which is not equivalent to stage 2 micropropagation, and the methods of Lata et al. (2009) were determined to be nonreplicable by Monthony et al. (2021b), a finding that we and other propagators have corroborated. Lubell-Brand et al. (2021) demonstrated success at all four stages of micropropagation with a protocol that controls hyperhydricity during stage 1 and includes a novel culture medium [hereafter, Lubell-Brand Cannabis medium (LBC)] for stage 2. Although Lubell-Brand et al. (2021) completed four subculture cycles at 3 weeks per cycle, the Lubell-Brand laboratory has more recently extended stage 2 beyond the 12 weeks originally reported.
Murashige and Skoog medium with vitamins [MS (Murashige and Skoog 1962)] has been widely used in studies of hemp micropropagation (Monthony et al. 2021c). Page et al. (2021) found that Driver and Kuniyuki Walnut medium [DKW (Driver and Kuniyuki 1984)] was more suitable for micropropagation of hemp than MS; however, they only evaluated cultures for one subculture period lasting 35 to 43 d, depending on the cultivar. Signs of nutrient deficiency were reported for shoots on DKW, including yellowing along leaf margins and interveinal chlorosis. The first objective of our research was to compare DKW to the media recommended by Lubell-Brand et al. (2021) for stages 1 and 2 using the methods of Lubell-Brand et al. (2021) for micropropagation of hemp. The second objective was to evaluate microboxes (O95/114 + OD95; Sac O2, Deinze, Belgium) with lids featuring a filter (#10, Sac O2) as an alternative vessel for micropropagation of hemp, because the lids featuring a vent with a diameter of 10 mm and a pore size of 0.2 μM (Caisson Laboratories, Smithfield, UT, USA) for boxes (GA-7; Magenta Corp., Lockport, IL, USA) that were used by Lubell-Brand et al. (2021) have been discontinued from manufacture.
Materials and methods
Plant material
‘Abacus’ and ‘Wife’ hemp were used in both the culture medium and micropropagation vessel studies. Plant material was originally obtained from US HempCare Inc., Niantic, CT, USA. ‘Abacus’ was obtained as cuttings from a single female plant, and ‘Wife’ as dioecious seeds. ‘Wife’ seeds were germinated and a female plant was selected. The female ‘Abacus’ and ‘Wife’ plants were maintained as stock in a greenhouse and perpetuated by stem-cutting propagation. Definitive information on the origin of these cultivars is difficult to determine. ‘Abacus’ is likely related to ‘OG Kush’, or plants derived from ‘OG Kush’, and ‘Wife’ appears to be derived from cannabidiol landrace plants (Phylos Bioscience Inc. 2022). ‘Abacus’ is noted for its compact form with dense branching, moderate growth rate, and anthocyanin development on stems and leaves (Kurtz et al. 2020). ‘Wife’ is an upright spreading plant with a fast growth rate and green leaves.
Culture medium study
Stem tips from stock plants of ‘Abacus’ and ‘Wife’ maintained in a greenhouse were disinfected and initiated in vitro according to the protocol of Lubell-Brand et al. (2021). Shoots were cultured in boxes with vented lids containing 45 mL of medium. Shoots were initiated (stage 1) with DKW or MS, plus 3% sucrose, 0.5 mg⋅L–1 metatopolin (MT) and 1% (w/v) agar at pH 5.7. After 3 weeks, sterile shoots were subcultured with the same respective initiation medium. After an additional 3 weeks (or 6 weeks from initiation to aseptic culture), microshoots in DKW were subcultured (stage 2) with DKW supplemented with 3% sucrose, 0.5 mg⋅L–1 MT, 0.1 mg⋅L–1 gibberellic acid (GA), and 0.8% (w/v) agar at pH 5.7. Microshoots in MS were subcultured (stage 2) using LBC (MS + added mesos and vitamins) of Lubell-Brand et al. (2021) plus the same supplements at pH 5.7. Micropropagation stage 1 (initiation) occurred during the first 6 weeks of this study, when the treatment media contained 1% agar. Micropropagation stage 2 (shoot multiplication) occurred from weeks 7 to 15 of this study, when the treatment media agar was reduced to 0.8%. The experimental unit was a vessel containing four microshoots. Boxes were arranged in a randomized complete block design (RCBD) with eight replications during stage 1 and 10 replications during stage 2. Cultures were subcultured every 3 weeks during both stages 1 and 2, and maintained in a growth chamber (Percival, Perry, IA, USA) at 25 °C with an 18-h photoperiod provided by cool-white fluorescent lamps at an intensity of 40 μmol⋅m–2⋅s–1. At each subculture, data were collected on the number of ∼2-cm apical and two-node nonapical microcuttings per experimental unit. The total number of microcuttings per unit was divided by the number of explants per unit, which was four, to determine a shoot multiplication rate (SMR) per unit. In addition, the height, from base to tip, of each microshoot per unit was measured and averaged. After 15 weeks in vitro (stages 1 and 2), microshoots were subcultured with MS with vitamins plus 3% sucrose, 1 mg⋅L–1 indole-3-butyric acid, and 0.8% (w/v) agar at pH 5.7 for prerooting as conducted by Lubell-Brand et al. (2021). The experimental unit was a vessel with eight microcuttings, and units were arranged in an RCBD with four replications. After 2 weeks, microcuttings were transferred ex vitro to rockwool for rooting as described by Lubell-Brand et al. (2021). For ex vitro rooting, we used an RCBD with four replications, each consisting of eight microcuttings. The percent rooting of microcuttings was recorded at 4 weeks after transfer to rockwool. Data were subjected to analysis of variance (PROC GLIMMIX) and mean separation with Tukey’s honestly significant difference test (P ≤ 0.05) using statistical software (SAS version 9.4; SAS Institute Inc., Cary, NC, USA). The interaction of cultivar × medium was significant, and interaction effects by cultivar are shown.
Micropropagation vessel study
‘Abacus’ and ‘Wife’ were initiated (stage 1) in vitro as described for the culture medium study. The initiation medium was MS plus 3% sucrose, 0.5 mg⋅L–1 MT, and 1% (w/v) agar at pH 5.7. Two different micropropagation vessels were evaluated: 1) boxes with lids featuring a vent and 2) microboxes with lids featuring a filter. The experimental unit was a micropropagation vessel with 45 mL of medium and four microshoots. Units were arranged in an RCBD with 10 replications. After 3 weeks, stage 2 was begun by subculturing the shoots to the same type of vessel with LBC plus 3% sucrose, 0.5 mg⋅L–1 MT, 0.1 mg⋅L–1 GA, and 0.8% (w/v) agar at pH 5.7. Cultures were subcultured every 3 weeks, during both stages 1 and 2, and maintained in a growth chamber as described previously. Data collection occurred at 6 and 9 weeks in vitro, just before subculturing. Therefore, at the first data collection time, cultures had already spent 6 weeks in vitro total (3 weeks in stage 1 followed by subculturing to stage 2, where they grew for another 3 weeks). Data were collected on the number of microcuttings per unit, and SMR was calculated as described for the culture medium study. At 9 weeks in vitro, percent hyperhydricity of microshoots per unit was also recorded. A microshoot was considered hyperhydric when the leaves appeared thick, translucent, and brittle. After 9 weeks in vitro, shoots were subcultured to the same respective vessel for prerooting with medium as described for the culture medium study. The experimental unit remained a vessel with four microcuttings. After 2 weeks, microcuttings were transferred ex vitro to rockwool for rooting as described previously, and the experimental unit was four microcuttings. The percent rooting of microcuttings was recorded at 4 weeks after transfer to rockwool. Data were subjected to analysis of variance (PROC GLIMMIX) and mean separation with Tukey’s honestly significant difference test (P ≤ 0.05) using statistical software (SAS version 9.4). The interaction of cultivar × micropropagation vessel was not significant; therefore, main treatment effects are shown.
Results
Culture medium study
SMR and explant height for ‘Abacus’ were statistically similar in MS and DKW at 3 and 6 weeks in vitro (Table 1). ‘Abacus’ had a greater SMR at 9, 12, and 15 weeks, and a greater explant height at 12 and 15 weeks in LBC. ‘Wife’ in DKW and MS/LBC had a similar SMR and explant height throughout the study, except at 9 weeks, when SMR was greater on LBC than DKW. Ex vitro rooting was similar for microshoots cultured in LBC and DKW for both cultivars.
Shoot multiplication rate (SMR) and microshoot height at 3, 6, 9, 12, and 15 weeks in vitro, and percent rooting ex vitro of ‘Abacus’ and ‘Wife’ hemp cultured on Driver and Kuniyuki Walnut medium (DKW) for all 15 weeks in vitro culture, or Murashige and Skoog with vitamins medium (MS) for weeks 0 to 6 in culture followed by Lubell-Brand Cannabis medium (LBC) for weeks 7 to 15 in culture.
Micropropagation vessel study
‘Wife’ had a significantly greater SMR than ‘Abacus’ at 6 weeks in vitro, but at 9 weeks in vitro SMR did not differ statistically for the cultivars (Table 2). ‘Abacus’ microshoots had significantly greater hyperhydricity and significantly less ex vitro rooting compared with ‘Wife’. SMR at 6 weeks did not differ statistically for micropropagation vessel, but at 9 weeks cultures in microboxes had a lower SMR than boxes. Cultures in microboxes had greater hyperhydricity and less ex vitro rooting.
Shoot multiplication rate (SMR) at 6 and 9 weeks in vitro, percent hyperhydricity of microshoots at 9 weeks in vitro, and percent rooting ex vitro for ‘Abacus’ and ‘Wife’ hemp cultured in boxes (GA-7; Magenta Corp., Lockport, IL, USA) with lids featuring a vent with a diameter of 10 mm and a pore size of 0.2 μM (Caisson Laboratories, Smithfield, UT, USA) or microboxes (O95/114 + OD95; Sac O2, Deinze, Belgium) with lids featuring a filter (#10, Sac O2), containing Lubell-Brand Cannabis medium.
Discussion
The goal for optimizing hemp micropropagation is to obtain the greatest continuous SMR while producing quality microshoots that will root and acclimatize successfully to ex vitro conditions. ‘Abacus’ and ‘Wife’ responded differently to the culture media tested, which was not surprising, because variability in cultivar performance has been identified in other tissue culture studies of hemp that evaluated media and growth regulators (Monthony et al. 2021c). ‘Abacus’ grew more vigorously in LBC, which provided more nitrogen (N), phosphorous (P), and magnesium (Mg) compared with DKW (Table 3, Fig. 1). Using LBC, a greater SMR was achieved for ‘Abacus’ while maintaining adequate rooting ability (75%) of microshoots (Table 1). In addition, microshoots of ‘Abacus’ in LBC were 32% and 50% taller at 12 and 15 weeks, respectively, than microshoots in DKW (Fig. 1). Larger microshoots can contribute to an increased SMR because they may be cut into two or more stem pieces (apical and two-node microcuttings) during subculture (stage 2). Single-node stem pieces are not recommended for subculturing hemp because they do not grow well (Page et al. 2021). Larger microshoots, in addition, are easier to handle as microcuttings for prerooting and ex vitro rooting in rockwool. ‘Wife’ performed well in both LBC and DKW, and had a high ex vitro rooting percentage (≥90%) (Table 1, Fig. 2).
In vitro cultures of ‘Abacus’ hemp in Driver and Kuniyuki Walnut medium [DKW (Driver and Kuniyuki 1984)] for all 15 weeks in vitro culture, or Murashige and Skoog with vitamins medium (Murashige and Skoog 1962) for weeks 0 to 6 in culture followed by Lubell-Brand Cannabis medium [LBC (Lubell-Brand et al. 2021)] for weeks 7 to 15 in culture, after (A) 3 weeks, (B) 6 weeks, (C) 9 weeks, (D) 12 weeks, and (E) 15 weeks.
Citation: HortTechnology 33, 2; 10.21273/HORTTECH05179-22
In vitro cultures of ‘Wife’ hemp in Driver and Kuniyuki Walnut medium [DKW (Driver and Kuniyuki 1984)] for all 15 weeks in vitro culture, or Murashige and Skoog with vitamins medium (Murashige and Skoog 1962) for weeks 0 to 6 in culture followed by Lubell-Brand Cannabis medium [LBC (Lubell-Brand et al. 2021)] for weeks 7 to 15 in culture, after (A) 3 weeks, (B) 6 weeks, (C) 9 weeks, (D) 12 weeks, and (E) 15 weeks.
Citation: HortTechnology 33, 2; 10.21273/HORTTECH05179-22
Percentage change in nutrient content (milligrams per liter) of medium from Murashige and Skoog with vitamins medium (MS) to Driver and Kuniyuki Walnut medium (DKW), MS to Lubell-Brand Cannabis medium (LBC), and DKW to LBC.
SMR for ‘Wife’ decreased progressively throughout the course of the 15-week culture medium study to about half the initial rate, except for at week 9, when SMR peaked at 2.3 in LBC (Table 1). ‘Wife’ microshoots in LBC at 9 weeks were observed visually to have better shoot extension and leaf coloration than microshoots in DKW (Fig. 2). Performance of ‘Wife’ in LBC at 9 weeks was superior because LBC provides ∼150% more calcium, Mg, P, and sulfur (S), and 21% more N than MS, which was used in the initiation stage (Table 3). The move from initiation medium to LBC increased production of microshoots by 31% for ‘Abacus’ and 35% for ‘Wife’ (Table 1). In general, the visual quality of ‘Wife’ cultures in both media declined throughout the course of the study (Fig. 2). This observation is consistent with previous work showing ‘Wife’ declines more rapidly in culture than other cultivars, including Abacus (Kurtz et al. 2022). At 12 and 15 weeks in vitro, leaf color was observed to be a darker green for microshoots in DKW compared with those in LBC (Fig. 1). This may be because DKW contains significantly more S (+68%; Table 3) than LBC, and this nutrient is important for chlorophyll formation (Bidlack and Jansky 2021).
SMR at 6 weeks in vitro was 2.4 for ‘Abacus’ and 3.7 for ‘Wife’ in the micropropagation vessel study, whereas in the culture medium study, it was only 1.3 for ‘Abacus’ and 1.7 for ‘Wife’ in LBC (Tables 1 and 2). This is likely because explants were subcultured to LBC at only 3 weeks postinitiation for the vessel study instead of the recommended 6 weeks postinitiation as was conducted for the culture study (Lubell-Brand et al. 2021). LBC not only provided cultures with more nutrients, but also it likely provided more available water, because LBC has less agar than initiation medium. Although a greater SMR may be achieved with a shorter duration initiation stage, this approach will result in significant hyperhydricity for cultivars such as Abacus, which have a strong propensity for developing hyperhydricity. The high incidence of hyperhydricity for Abacus, in the micropropagation vessel study (Fig. 3) likely contributed to the low ex vitro rooting success for this cultivar (38%), because hyperhydric microshoots do not root as well (Nairn et al. 1995). In the culture medium study, 75% rooting was achieved for ‘Abacus’ because hyperhydricity was controlled, likely by the extended period in initiation medium with greater agar content.
In vitro cultures of ‘Abacus’ and ‘Wife’ hemp in boxes (GA-7; Magenta Corp., Lockport, IL, USA) with lids featuring a vent with a diameter of 10 mm (0.394 inch) and a pore size of 0.2 μM (Caisson Laboratories, Smithfield, UT, USA) or microboxes (O95/114 + OD95; Sac O2, Deinze, Belgium) with lids featuring a filter (#10, Sac O2) after (A) 3 weeks and (B) 6 weeks.
Citation: HortTechnology 33, 2; 10.21273/HORTTECH05179-22
The use of vented lids to increase air exchange and decrease the relative humidity inside the micropropagation vessel has been shown to reduce the occurrence of hyperhydricity for hemp (Lubell-Brand et al. 2021). This is especially important for cultivars such as Abacus that are likely to develop hyperhydricity during initiation to in vitro culture. We found that microboxes with lids featuring a filter did not control hyperhydricity as well as boxes with lids featuring a vent (Fig. 3). Microboxes with lids featuring filters with greater air exchange capacity than the ones used in this study are available, and these may offer more control of hyperhydricity.
We found that shoots of ‘Abacus’ were more likely to develop hyperhydricity and take longer to acclimatize to in vitro conditions than ‘Wife’. However, once miniaturized, ‘Abacus’ is easier to maintain by subculturing in stage 2 for a longer period of time than ‘Wife’. SMRs reported here for ‘Abacus’ and ‘Wife’ are similar to those reported by Wróbel et al. (2020) and Page et al. (2021) for stage 2 micropropagation of hemp. However, a direct comparison with these studies is not possible because the cultures they evaluated were started from seed and were of unknown age in vitro, respectively. For cultivars such as Abacus that are likely to develop hyperhydricity during initiation, we recommend a longer initiation stage and micropropagation vessels that provide adequate air exchange. For cultivars such as Wife that are less susceptible to hyperhydricity, propagators may consider using a shorter initiation stage and moving cultures to stage 2 medium sooner to maximize SMRs before cultures decline.
Units
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