In vivo treatments.

After the cuttings were taken, they were immediately moved from the greenhouse to the laboratory to be treated. A total of 270 cuttings were fully submerged in either colchicine (Sigma-Aldrich, St. Louis, MO, USA) or oryzalin (Surflan AS; United Phosphorus, Trenton, NJ, USA) solutions. The colchicine concentrations were 0% (control), 0.02%, 0.05%, 0.1%, 0.2%. The oryzalin (Surflan) concentrations used were 0% (control), 0.002%, 0.005%, 0.01%, 0.02%. One percent dimethyl sulfoxide (DMSO; Sigma-Aldrich) (v/v) was added to all treatments to improve cellular penetration, and all treatments had their pH adjusted to 5.7. Nine cuttings were placed into each 250-mL beaker filled with 200 mL of respective treatment solution and placed on a rotary shaker at 100 rpm for 3, 6, or 12 h.

After cuttings were removed from their treatments, they were rinsed under running tap water for ∼5 s, and then set into 50-cell trays filled with soilless potting mix (Sunshine-Mix; Sun Gro Horticulture) and perlite (Supreme Perlite Co.; 2:1 by volume) and incorporated with 67.5 g of 18N–2.6P–9.1K controlled-release fertilizer (Harrell’s, Lakeland, FL, USA) per 2 ft³ of soilless potting mix (Sunshine mix) and rooted under humidity domes according to McLeod et al. (2022). The cuttings were rooted indoors under T5 lights (59 µmol·m⁻²·s⁻¹) with 24-h photoperiod. After rooting, the cuttings were transplanted into 2-7/8-in × 5-1/2-in band pots (Anderson Die and Manufacturing Inc., Portland, OR, USA) filled with soilless potting mix (Metro Mix) and perlite (2:1 by volume) and moved into a glasshouse under the conditions described above. Rooted cuttings were grown in the glasshouse for three weeks, until there was sufficient plant material for ploidy analysis. The survival rate of the vegetative cuttings was recorded 3 weeks after the treatments were applied.

In vitro treatments.

Explant cuttings were surface sterilized for 1 min in 70% ethanol, then soaked in a 0.1% plant preservative mixture (PPM; Plant Cell Technology Inc., Washington, DC, USA) for 30 min. Explants were then surface-disinfected in a solution of 2% sodium hypochlorite (Bi-Mart bleach, Eugene, OR, USA; 5% sodium hypochlorite) and 0.1% Tween (VWR Life Science, Radnor, PA, USA; v/v) for 10 m, during which time they were transferred into a laminar flow hood. Following sterilization, explants were rinsed in sterile water at least three times until no suds remained. Sterilized explants were inoculated on 50 mL of solid, sterile tissue culture medium as described by Halstead et al. (2022), using glucose as the carbon source and Driver–Kuniyuki–Walnut (DKW) medium (D2470, PhytoTech Laboratories, Lenexa, KS, USA) as the nutrient source, in 372-mL culture vessels (C2100, PhytoTech Laboratories). The explants were cultured for 5 weeks in the initial medium. Forty new nodal segments containing axillary buds were subcultured from initial plantlets in culture tubes containing the same media as described earlier.

The 50-µM oryzalin treatment was prepared by dissolving oryzalin in 100% ethanol and DMSO (Sigma-Aldrich) for a final concentration of 2.5% ethanol + 1% DMSO (v/v), pH 5.7. After 1 week, treatments were applied to all 40 tubes of one subcultured plantlet per tube representing an experimental unit. Treatments included a control (DKW medium + 2.5% ethanol and 1% DMSO) and the 50-µM oryzalin treatment. The solution was filter sterilized with a 0.45-µM polytetrafluoroethylene syringe filter before being diluted to the final 50-µM oryzalin concentration and applied to the explants, as described in Contreras and Meneghelli (2016). The treatments were applied by pouring 10 mL of solution into each tube, ensuring that all explants were fully submerged (Fig. 3). Treated explants were placed on a rotary shaker for either 12 or 36 h at 100 rpm. After treatment, the liquid solutions were poured out of the culture tubes, all cuttings were rinsed in a 0.1% PPM solution and then transferred to fresh media (as described earlier) in new culture tubes. Plants were grown in 372-mL culture vessels (C2100, PhytoTech Laboratories) in a growth chamber at 25 ± 2 °C with a 16-h photoperiod at ∼21 µmol·m⁻²·s⁻¹. Survival was recorded after 1 week based on tissue culture, with green tissue recorded as a surviving plant and brown tissue recorded as a nonsurvived plant. After 8 weeks, plants with sufficient leaf tissue were evaluated using flow cytometry.

In vivo.

Of 270 cuttings propagated, 85 cuttings (31%) rooted successfully (Table 1). Of the 85 survivors, 22 of the cuttings showed polyploid peaks on their flow cytometry histograms (26% polyploid induction), including 20 2x + 4x cytochimeras (Fig. 1B) and two tetraploids (Fig. 1A).

Table 1. Response of ‘I3’ hemp stem cuttings after in vivo exposure to colchicine and Surflan for 3, 6, and 12 at various concentrations by submerging the stem cuttings into liquid treatment mixtures. The data are presented as the number of cuttings in each treatment, the percent survival per treatment, the percent of 2x + 4x cytochimeras per number of survivors per treatment, and the percent of tetraploids (4x) per number of survivors per treatment. Eight weeks after treatments, survival and ploidy was evaluated to determine number of cytochimeras and tetraploids.

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Survival ranged from 11% for the 0.1%, 6-h colchicine treatment to 89% for the 0.2%, 3-h colchicine treatments. Schulze and Contreras (2017) experimented with in vivo chromosome doubling on Portuguese Cherrylaurel and observed a range of survival among treatments. Similar to our study, colchicine applied at 0.2% resulted in the highest survival among treatments using seedlings instead of stem cuttings. They also used a different application method in which they treated exposed meristems directly using a semisolid drop vs. our method of submerging the entire stem cutting, which likely had an impact on survival.

Mansouri and Bagheri (2017) performed an in vivo polyploid experiment on cannabis. They used a method like Schulze and Contreras (2017), where they applied drops of colchicine to the apical meristem of growing seedlings. The results showed that 0.2% of colchicine for 24 h was the most efficient for production of polyploid plants. They used seedlings instead of vegetative stem cuttings, and drops were applied to the apical meristem vs. the whole cutting being submerged in the treatment for our experiment. However, these results show that polyploidy was induced in hemp using a 24 h treatment, which was twice as long as my longest treatment duration. This is evidence that we could have had a different result if we treated our cuttings for a longer period, as the Mansouri and Bagheri (2017) experiment treated their plants for 24 and 48 h instead of 3, 6, and 12 h.

The majority (82%) of polyploid cuttings came from colchicine treatments, and the remaining 18% from Surflan treatments. Both tetraploids were the results of colchicine treatments, one coming from a 12-h, 0.2% treatment and the other from a 12-h, 0.05% treatment. Kurtz et al. (2020) also successfully produced tetraploids using a 12-h, 0.05% colchicine treatment in their experiment; this treatment worked on five cultivars. However, they used pregerminated seeds instead of stem cuttings for their experiment, which could influence the survival and number of tetraploid plants. Kurtz et al. (2020) treated pregerminated seeds of five hemp cultivars with 0.05% colchicine for 12 h. This resulted in emergence percentages ranging from 78% to 100% for the five cultivars, as well as confirmed tetraploid plants for each of the cultivars. Their experiment shows evidence that using pregerminated seedlings may be an efficient alternative to vegetative stem cuttings when inducing polyploidy in hemp.

There was not an obvious correlation between colchicine concentrations applied and stem-cutting survival (Fig. 2A) or percent cytochimeras among survivors (Fig. 2B). We expected survival to decline with increasing concentrations and polyploids to increase. This was the case in the Schulze and Contreras (2017) experiment on Prunus laurocerasus and Prunus lusitanica. When colchicine was applied to P. lusitanica at 0.2%, there was a 98.8% survival rate and two cytochimera were recorded. When the treatment was increased to 0.8% colchicine, survival dropped down to 60.7%, but there were four cytochimera. Similar findings have been observed in other crops such as Watsonia lepida (Ascough et al. 2008) and Lychnis senno (Chen et al. 2006) where survival decreases but polyploid induction increases as the mitotic spindle inhibitor concentrations are increased. Between treatment lengths, the 3-h treatment group averaged the highest overall survival. For colchicine concentrations, one of 18 polyploids came from the 0.01% concentration treatment (6%), six of 18 came from the 0.05% concentration treatment (33%), three of 18 came from the 0.1% concentration (17%), and eight of 18 polyploids came from the 0.2% concentration group (44%). One tetraploid resulted from the 12-h, 0.2% concentration treatment, and the other from a 12-h, 0.05% concentration treatment (Table 1). The majority of our polyploids came from 0.2% colchicine treatments, which was our highest concentration. Lelakes (1960) performed a cannabis polyploidy experiment and had the most success inducing polyploids using a 3 h 0.5% colchicine treatment. This treatment concentration was more than double our highest treatment concentration and was still successful. Lelakes’ data show that higher colchicine concentrations can be used to induce polyploidy in hemp. However, like other experiments referenced, Lelakes used a different method of colchicine application. Lelakes immersed seedlings in colchicine at different stages of growth, whereas our experiment submerged vegetative stem cuttings right after they were cut from stock plants. The differences in these two methods could have affected survival and number of tetraploid plants.

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From the Surflan treatments we recovered two mixoploids from the 0.002%, 12-h treatment group; one mixoploid from the 0.002%, 6-h treatment group; and one from the 0%, 12-h control group. In Fig. 2C, there is a negative correlation between the Surflan concentration and the survival of ‘I3’ stem cuttings, the survival of stem cuttings decreases as the Surflan concentration increases. The same can be observed in relation to the cytochimera per number of survivors (Fig. 2d), where cytochimeras decrease as the concentration increases.

Using 12-h colchicine treatments at 0.05% and 0.2% concentrations, we were able to successfully double the diploid ‘I3’ hemp genome using in vivo techniques. None of the Surflan treatments were successful in yielding homogeneous tetraploids, but we did recover four mixoploids that have been shown to be useful in breeding if the LII histogenic layer is tetraploid (Olsen et al. 2006); however, we did not identify the ploidy levels of constituent histogenic layers. The 3- and 6-h treatment lengths were successful in producing mixoploids, but no tetraploids were produced from these treatments.

In vitro.

Thirty-five percent of the explants displayed green tissue 1 week after treatment: 7.5% of survivors came from the 12-h control group; 12.5% came from the 36-h control group; 12.5% came from the 12-h, 50-µM oryzalin group; and 2.5% came from the 36-h, 50-µM oryzalin treatment. The explants treated for the in vitro part of this experiment are shown in Fig. 3. Four of the 14 surviving explants developed enough leaf tissue for flow cytometric analysis after 8 weeks (29%). These included two 12-h control treatments, one 50-µM oryzalin 12-h treatment, and one 50-µM oryzalin 36-h treatment. Of these four, the 12-h, 50-µM oryzalin treatment yielded a tetraploid plant, and the 36-h, 50-µM oryzalin treatment yielded a cytochimera (mixoploid) plant (Table 2). The other two evaluated plants from the control group were diploids.

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Table 2. In vitro treatments and quantity of ploidy altered ‘I3’ hemp explants.

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The data show low survival numbers in the in vitro experiment. This could be due to the subculturing method used in combination with the genotype. Subcultured cuttings were not vigorous and had frail, thin stems. Furthermore, plantlets in the initial culture were not vigorous, producing few leaves and nodes. The low vigor of the subculture cuttings is likely the cause of the poor survival outcomes observed. Subcultures were employed in this case to ensure plant sterility so that posttreatment survival could be evaluated with other factors impacting survival counts as controlled as possible. However, this approach had the opposite of the intended effect, and it is difficult to evaluate whether plant survival was due to a treatment effect, both because of the small sample size and the varying but generally poor vigor of the subcultured cuttings. Further experiments to evaluate the effects of these treatments on subcultured cuttings would do well to use a hemp genotype known to perform vigorously in a tissue culture setting. Otherwise, based on the results of this trial, subculturing may not be a reliable method to produce vigorous cuttings. Although subculturing can provide some control of external factors, it is not necessary for the evaluation of ploidy manipulation treatments in hemp tissue culture, as demonstrated by Parsons et al. (2019), who successfully treated axillary bud explants with oryzalin and eventually grew the cuttings into full plants transitioned back into the greenhouse. In hindsight, we may have been able to achieve the desired vigorous growth in our explants if we used the “hedging” technique described by Murphy and Adelberg (2021) in their in vitro cannabis experiment instead of subculturing.

Parsons et al. (2019) performed one of their in vitro polyploid experiment trials using 20-, 40-, and 60-µM as the oryzalin treatment concentrations. They had the highest number of tetraploids result from the 20- and 40-µM treatments. In our in vitro experiment, there was only one treatment concentration (50 µM) besides the control. Their experiment was successful in producing multiple tetraploids at both 20- and 40-µM oryzalin treatment concentrations, so it is possible that we could have had better results if we used a lower oryzalin concentration on our explants.

Most explants did not have enough plant material to sample for flow cytometry; this could be due to the allotted growth period. Parsons et al. (2019) stated that in their experiment, sterile shoots emerged after 1 to 5 months. We only allowed the explants 8 weeks to grow before starting flow cytometry. If the explants were allowed a longer growth period, there may have been more leaf tissue produced that would have allowed us to test more plant.

In conclusion, we were able to double ‘I3’ hemp chromosomes successfully in both in vivo and in vitro environments by using different mitotic spindle inhibitors at various concentrations and treatment durations. We were limited by time constraints when attempting to evaluate the survival of the in vitro explants but were still able to induce polyploidy within this group. Further optimization is possible to create a more efficient protocol for inducing polyploids, such as evaluating how well our methods work when dealing with other cultivars, as well as evaluating whether using the “hedging” technique would have resulted in more vigorous growth in the explants which could have raised our survival rate. However, for generating polyploids of individual genotypes to be used in interploidy crossing to generate triploids, our methods may prove useful to other breeders.