Temporal cannabinoids profile and biomass yield in cannabigerol dominant industrial hemp under different planting dates in southern Florida

Abstract

Industrial hemp (Cannabis sativa L.) commercialization in Florida is mainly challenged by the lack of suitable essential oil hemp cultivars with Δ-9-tetrahydrocannabinol (THC) concentration below the legal threshold. However, previous studies have only evaluated cannabidiol (CBD)-dominant industrial hemp cultivars but not the cultivars primarily grown for cannabigerol (CBG) production. Hence, field trials were conducted using two CBG dominant cultivars adapted in Kentucky (CBG Gold) and Colorado (Panacea) at three experimental farms located at Okeechobee (Lykes) and Clewiston (Townsite and Ritta) in southern Florida with three planting dates from late April to late June under two different soil types (sandy soil at Lykes and Townsite, and organic soil at Ritta). CBG, CBD, and THC concentrations were analyzed in developing flowers harvested at weekly intervals beginning 2 weeks after flowering until crop maturity. Location and planting date had some effect on cannabinoids profile, but the effect was not consistent in any of the tested cultivars. Both the cultivars contained THC concentration (<0.1%) well below the legal requirement making them suitable for cultivation in Florida. However, both the cultivars adapted in longer photoperiod environments recorded short vegetative growth period in Florida's short-day conditions and consequently produced less floral biomass. Hence, evaluating more cultivars with shorter photoperiod requirements would be helpful in identifying suitable CBG-dominant cultivars for Florida and other locations with similar climatic conditions.

Abbreviations
• CBD
• cannabidiol
• CBDA
• cannabidioalic acid
• CBG
• cannabigerol
• CBGA
• canabigerolic acid
• THC
• Δ-9-tetrahydrocannabinol
• THCA
• tetrahydrocannabinolic acid

INTRODUCTION

Industrial hemp (Cannabis sativa L.) has received considerable attention recently primarily due to important pharmacological applications of some of its cannabinoids. More than 100 cannabinoids have been identified in Cannabis (Nachnani et al., 2021). Among these cannabinoids, Δ-9-tetrahydrocannabinol (THC) is the sole psychoactive cannabinoid, and hence plants with THC concentration above a certain threshold are categorized as marijuana. In the United States, a Cannabis plant with THC concentration below 0.3% is defined as industrial hemp (USDA, 2019). Cannabidiol (CBD) and cannabigerol (CBG) are two widely known nonpsychoactive cannabinoids with therapeutics potential in treating several diseases (Deiana, 2017; Nachnani et al., 2021). CBG was found effective for treating bacterial diseases (Aqawi et al., 2021; Eisohly et al., 2006), neurologic diseases (Nachnani et al., 2021), cancer (Lah et al., 2021), glaucoma (Colasanti, 1990), psoriasis (Wilkinson & Williamson, 2007), and inflammatory bowel disease (Borrelli et al., 2013). Apart from its medicinal uses, CBG is also a precursor molecule for synthesis of other important cannabinoids such as THC and CBD (Deiana, 2017; Nachnani et al., 2021; Pate, 1994).

Environmental factors affecting cannabinoids accumulation, especially CBD and THC, have been documented in several studies. CBD and THC production was generally greater when plants experienced some stresses such as nutrient (Haney & Kutscheid, 1973) and moisture stress (Caplan et al., 2019; Haney & Kutscheid, 1973; Sikora et al., 2011). Temperature had an inconsistent effect on CBD and THC as studies have reported greater CBD and THC production under both lower (Bazzaz et al., 1975; Paris et al., 1975) and higher temperatures (Boucher et al., 1974; Sikora et al., 2011). However, similar information regarding the effect of the environment on CBG accumulation is not available. The CBD and THC accumulation pattern over crop growing season has been documented in both CBD and THC-dominant Cannabis cultivars (Calzolari et al., 2017; De Backer et al., 2012; Latta & Eaton, 1975; Pacifico et al., 2008; Sandhu et al., 2022; Chiluwal et al., 2023). However, CBG accumulation pattern in CBG-dominant cultivars is still unknown. The CBG accumulation pattern in fiber, CBD, and THC-dominant cultivars differed between the studies. Yang et al. (2020) using CBD-dominant hemp cultivars reported CBG content increased gradually and reached its peak at seventh week after anthesis and then started decreasing. Calzolari et al. (2017) reported inconsistent CBG accumulation curve in the three hemp cultivars in their study. De Backer et al. (2012) using THC-dominant marijuana cultivars found CBG concentration gradually increased with plant ages until maturity. Pacifico et al. (2008) using fiber, CBD, and dual-purpose cultivars reported CBG concentration increased gradually and then started declining at maturity. However, as none of the cultivars in their study was CBG dominant, the highest CBG concentration recorded in those studies was only 1.07%. Hence, the CBG accumulation pattern across crop growing season in the hemp cultivars primarily grown for CBG production is still unknown.

Although industrial hemp has received a lot of attention in many subtropical and tropical areas such as Florida. Due to its photoperiod sensitivity coupled with comparatively shorter day length in Florida, cultivars from the northern latitude begin the reproductive phase very early in the season without accumulating much vegetative biomass in Florida's environment (Zhang et al., 2021). Similarly, most of the industrial hemp cultivars adapted in other agroclimatic conditions often exceed THC concentration above the limit (>0.3%) when grown in Florida (Chiluwal et al., 2023; Sandhu et al., 2022; Yang et al., 2020). Hence, lack of suitable cultivars with acceptable legal THC concentration and less biomass yield remains the major challenge for its commercialization in Florida and similar other climatic conditions. However, only CBD-dominant hemp cultivars have been studied and none of the previous studies have attempted to evaluate the potential of growing CBG-dominant hemp cultivars in Florida and similar other environments. Hence, this study was conducted to assess the temporal cannabinoid accumulation pattern and biomass yield of two CBG-dominant hemp cultivars adapted in different geographical regions under different planting dates, locations, and soil types in southern Florida.

Core Ideas

• Hemp commercialization in Florida is mainly challenged by lack of cultivars with Δ-9-tetrahydrocannabinol level below the legal limit.
• THC concentration in cannabigerol-dominant industrial hemp cultivars was well below the legal limit.
• Cultivars adapted in longer photoperiod environments produced less biomass in Florida's short-day conditions.
• Evaluating more cultivars with shorter photoperiod requirement would help identify suitable hemp cultivars.

MATERIALS AND METHODS Experimental sites

Field experiments were conducted at three experimental farms in southern Florida: Lykes Bros. farm in Glades County near Okeechobee located at 27°12′44.6″ N and 81°05′08.9″ W (hereafter called Lykes), and two United States Sugar Corporation (USSC) farms, one in Hendry County near Clewiston located at 26°44′25.3″ N and 80°58′56.3″ W (hereafter called Townsite) and the other located at 26°43′0.6672″ N and 80°50′58.2468″ W (hereafter called Ritta). The soil in the experimental fields at Lykes and Townsite was classified as Immokalee sand, and at Ritta as Terra Ceio series, Histosol (Euic, hyperthermic Typic Haplosaprists) (USDA-NRCS, 2021).

Experimental design and crop management

The experiment was laid out in split–split plot randomized complete block design with three replications. Planting date was considered as main plot, fumigation as subplot, and cultivars as sub-sub plot. There were three planting dates: first, last week of April or second week of May; second, last week of May; and third, last week of June, two fumigation treatments (fumigated or control), and two cultivars (CBG Gold from Kentucky and Panacea from Colorado) in the study (Table 1).

TABLE 1 Tested cultivars and their source, planting and flowering dates in the study

		Planting date			Date of 50% floweringa
Variety	Source	Planting I	Planting II	Planting III	Planting I	Planting II	Planting III
CBG Gold	Kentucky	05/12/2020	05/29/2020	06/25/2020	5/24/2020	6/10/2020	7/6/2020
Panacea	Colorado	04/29/2020	05/29/2020	06/25/2020	5/13/2020	6/15/2020	7/10/2020

Abbreviation: CBG, cannabigerol.

a

Flowering dates was averaged across all three locations as there was no significant differences in flowering times among the locations.

Raised beds around 20 cm in height were prepared in the field. Drip tape was placed on the beds for irrigation and then covered with white plastic for weed control. Beds were 76 cm wide and 1.8 m (6 ft) apart. Beds assigned for fumigation treatment were fumigated with Tripic 100 at 134 kg per treated ha. Seedlings were transplanted on the beds at 1.2 m (4 ft) plant spacing. Each plot had 20 plants. N and K fertilizers were applied at weekly interval via drip tape at 0.84 kg/ha per day during the early crop growth stage. The fertilization dose was increased to 3.36 kg/ha per day 1 month after transplanting.

Data collection and analysis

The date of flowering was recorded when more than 50% plants in the plot begin flowering. Two weeks after flowering, top 25 cm plant parts were harvested at weekly interval until crop maturity. The samples were oven dried and analyzed for canabigerolic acid (CBGA), tetrahydrocannabinolic acid (THCA), cannabidioalic acid (CBDA), CBG, THC, and CBD using ultra-high-performance liquid chromatography. Total CBG, total THC, and total CBD were calculated as the sum of CBG plus 0.877 × CBGA, sum of THC plus 0.877 × THCA, and sum of CBD plus 0.877 × CBDA, respectively. Three plants were harvested from each replication at maturity to estimate the final floral biomass yield.

Data were analyzed with ANOVA using Proc Glimmix in SAS version 9.4 (SAS Institute). The effect of fumigation was nonsignificant in both locations, hence data across both fumigation treatments were combined, analyzed, and presented in the results. Location, planting date, cultivar, and their interactions were considered fixed factors. Block or replication and its interaction with other fixed factors were considered random factors. Means were separated by Tukey's honestly significant difference, when treatments and interactions were significant at p ≤ 0.05.

RESULTS Cannabinoids accumulation pattern

CBG and THC concentrations in both cultivars under all planting conditions gradually increased and reached their peak and then decreased at the end of the growing season (Figure 1). However, there was no consistent CBD accumulation pattern in both cultivars (Figure S1). The stage when CBD concentration was maximum varied between the planting dates and locations in both cultivars (Figure S1). Total CBD concentration was very low (>0.2%) in both CBG-dominant hemp cultivars (Figure S1). Total THC concentration never exceeded the legal US threshold (0.3%) for the tested cultivars and was always below 0.1% in both cultivars in all planting conditions (Figure 1).

FIGURE 1. Cannabigerol (CBG) and Δ-9-tetrahydrocannabinol (THC) concentration curves within each planting date and location in CBG Gold (A) and Panacea (B).

There was a significant positive association between total CBG and total THC in both cultivars (Figure 2A,B). However, there was no significant association between total CBD and total CBG in both cultivars (Figure 2C,D).

FIGURE 2. Relationship between cannabigerol (CBG) and Δ-9-tetrahydrocannabinol (THC) in CBG Gold (A) and Panacea (B), and between CBG and cannabidiol (CBD) in CBG Gold (C) and Panacea (D) across all three locations.

Final CBG concentration

CBG concentration at harvest was significantly affected by location and cultivar, two-way interaction between location and planting date, and planting date and cultivar, and three-way interactions between location, planting date, and cultivar (Table 2).

TABLE 2 Analysis of variance for CBG, THC, CBG/THC, and total CBD content at harvest

Source	Total CBG	Total THC	CBG/THC	Total CBD	Yield
Location	*	NS	NS	NS	***
Planting date	NS	*	NS	NS	*
Cultivar	*	*	NS	NS	***
Location × planting date	*	*	*	NS	NS
Location × cultivar	NS	*	*	*	***
Planting date × cultivar	*	**	NS	NS	NS
Location × planting date × cultivar	**	**	*	NS	NS

Abbreviations: CBG, cannabigerol; NS, not significant; THC, Δ-9-tetrahydrocannabinol.

*, **, and *** significant at the 0.05, 0.01 and 0.001 probability level, respectively.

No significant differences in CBG concentration among the locations were observed in the first planting. In the second planting, CBG concentration in both cultivars was higher at Ritta than at Lykes (Figure 3A,B). In the third planting, CBG concentration was higher in Lykes than Townsite in CBG Gold (Figure 3A) while higher at Townsite than Lykes in Panacea (Figure 3B). Across both cultivars in all planting dates, CBG concentration was lower at Lykes (4.4%) compared to Townsite (6.0%) and Ritta (6.7%).

FIGURE 3. Effect of location on total cannabigerol (CBG) (A,B), total Δ-9-tetrahydrocannabinol (THC) (C,D), total CBG/total THC (E,F) and total cannabidiol (CBD) (G,H) concentration at harvest in each cultivar within a planting date. NS, not significant, and bars with different letters within a group indicate significant difference at 0.05 probability level. Error bars represent standard error of the mean.

CBG concentration in CBG Gold was higher under the third planting than other plantings at Lykes, while higher under the second planting than other plantings at Townsite and Ritta (Figure 4A). Similarly, CBG concentration of Panacea was higher under the second than third planting at Lykes, lower under the second planting than other plantings at Townsite, and lower under the third than other plantings at Ritta (Figure 4B).

FIGURE 4. Effect of planting dates on total cannabigerol (CBG) (A,B), total Δ-9-tetrahydrocannabinol (THC) (C,D), total CBG/total THC (E,F) and total cannabidiol (CBD) (G,H) concentration at harvest in each cultivar within a location. NS, not significant, and bars with different letters within a group indicate significant difference at 0.05 probability level. Error bars represent standard error of the mean.

CBG concentration was higher in Panacea than CBG Gold in the first planting at Townsite and Ritta, the second planting at Lykes, and the third planting at Townsite (Figure 5A–C). In contrast, CBG Gold had higher CBG concentration than Panacea in the second planting at Townsite and third planting at Lykes (Figure 5A–C). Across all planting dates and locations, Panacea (6.5%) recorded higher CBG concentration than CBG Gold (4.9%).

FIGURE 5. Varietal differences in total cannabigerol (CBG) (A,B,C), total Δ-9-tetrahydrocannabinol (THC) (D,E,F), total CBG/total THC (G,H,I) and total cannabidiol (CBD) (J,K,L) concentrations at harvest in each planting date within a location. *Cultivar with significantly higher value than other at 0.05 probability level. Error bars represent standard error of the mean.

Final THC concentration

Except the main effect of location, THC concentration at harvest was significantly affected by all three dependent variables (location, planting date, and cultivar) and their two- and three-way interactions (Table 2). There was no significant differences in THC concentration among the locations in the first and third planting in both cultivars (Figure 3C,D). However, in the second planting, THC concentration was higher at Townsite than other locations in CBG Gold (Figure 3C), and higher at Ritta than other locations in Panacea (Figure 3D). There was no significant effect of planting date in both cultivars at Lykes (Figure 4C,D). However, THC concentration was higher in the first and second planting in Panacea (Figure 4D) and CBG Gold (Figure 4C), respectively, than in other plantings at Townsite. In Ritta, planting date had no significant effect on CBG Gold (Figure 4C), while the second planting resulted in higher THC concentration than other planting in Panacea (Figure 4D). THC concentration was higher in Panacea than CBG Gold in the second planting at Lykes (Figure 5D), the first planting at Townsite (Figure 5E), and the first and second planting in Ritta (Figure 5F). However, CBG Gold had higher THC concentration than Panacea in the second planting at Townsite (Figure 5E).

Total CBG/total THC ratio

CBG/THC ratio at harvest was affected by interaction between location and planting date, and location and cultivar, and also by the three-way interaction between location, planting date, and cultivar (Table 2). The effect of location on the ratio was observed only in CBG Gold in the third planting where Lykes recorded higher ratio than other locations (Figure 3E). Planting date also had no significant effect on the ratio in most cases except in CBG Gold at Lykes (Figure 4E) and Panacea at Townsite (Figure 4F) where the third planting had higher ratio than earlier plantings. Similarly, significant differences in ratio between the cultivars were observed only in the third planting at Lykes (Figure 5G) and Townsite (Figure 5H) where CBG Gold had higher ratio at Lykes while Panacea had higher ratio at Townsite.

Final CBD concentration

Total CBD concentration at harvest was affected only by location and cultivar interaction (Table 2). Panacea recorded higher CBD concentration than CBG Gold at Lykes (Figure 5J), while CBG Gold recorded higher CBD concentration than Panacea at Townsite (Figure 5K).

Final floral biomass yield

Final floral biomass yield was affected by the location, planting date, cultivar, and location by cultivar interaction (Table 2). Across all planting dates, Panacea recorded higher yield compared to CBG Gold in each location (Figure 6). On average, biomass yield was higher at Lykes (556 kg ha⁻¹), followed by Ritta (400 kg ha⁻¹) and Townsite (238 kg ha⁻¹). Averaged across both cultivars, biomass yield was higher in the second planting (460 kg ha⁻¹) than the first planting (352 kg ha⁻¹), which was higher than in the third planting (264 kg ha⁻¹).

FIGURE 6. Differences in biomass yield between the cultivar in each location across all planting date. *Cultivar with significantly higher biomass yield than other at 0.05 probability level. Error bars represent standard error of the mean.

DISCUSSION

To our knowledge, there are no previous studies that evaluated cannabinoid accumulation pattern in CBG-dominant industrial hemp cultivars. However, CBG and THC accumulation pattern observed in this study was consistent with a recent study with CBD-dominant cultivars at Quincy, FL (Yang et al., 2020), where they also observed their concentration decreased after reaching its peak. In contrast to their study where they found a similar accumulation pattern for CBD, we did not observe any consistent CBD accumulation pattern in this study. Pacifico et al. (2008) using different (fiber, drug type, and hybrid) industrial hemp cultivars also reported CBD, CBG, and THC increased gradually and then started declining after reaching the peak. Latta and Eaton (1975) in their study with marijuana also found both CBD and THC concentrations decreased at the end of crop growth after reaching their peak. De Backer et al. (2012) also found THC concentrations in marijuana followed a similar pattern in some cultivars but also observed its concentration remained plateaued and did not drop in other cultivars. Similarly, Calzolari et al. (2017) reported CBG, CBD, and THC concentration patterns were not consistent between the environments and cultivars in their study as their concentration either gradually increased until maturity or decreased after reaching the peak.

We found some effect of location and planting date on cannabinoids profile and floral biomass yield in both the cultivars. However, the effect of the location was inconsistent among the planting dates (Figure 3). Similarly, the effect of planting date was also not consistent among the locations (Figure 4). In other studies with CBD-dominant industrial hemp cultivars, we also found that the effect of location and planting date was not consistent across the cultivars (Chiluwal et al., 2023; Sandhu et al., 2022). We did not see significant location and planting date effect in most of the tested cultivars possibly due to similar environmental conditions during cannabinoid accumulation across all planting dates and locations in the experimental year.

THC concentration exceeding a legal threshold in CBD-dominant hemp cultivars has become a bottleneck for hemp cultivation for essential oil production in Florida (Chiluwal et al., 2023; Sandhu et al., 2022; Yang et al., 2020). However, previous studies have only evaluated CBD-dominant hemp cultivars and there is no information regarding the potentiality of growing CBG-dominant hemp cultivars in Florida and similar other locations. In contrast to most of the CBD-dominant hemp cultivars from previous studies, both CBG-dominant cultivars in this study had low THC concentration (<0.1%), well below the legal limit (0.3%). Canabigerolic acid (CBGA) is the precursor molecule for CBG synthesis and also for tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), which in turn produces THC and CBD, respectively (Nachnani et al., 2021; Sirikantaramas et al., 2004). Furthermore, CBG can also be converted into several metabolites including THC and CBD (Deiana, 2017; Nachnani et al., 2021; Pate, 1994). Hence, the transformation of CBGA and CBG could possibly largely determine the final CBD and THC concentration in industrial hemp. Both THC (<0.1%) and CBD (<0.2%) concentration was very low in both cultivars in this study suggesting there was no or little transformation from CBGA and CBG into THC and CBD in all planting conditions and locations. Hence, the results of this study suggest that CBG-dominant industrial hemp cultivars could be better suited in Florida's environment as most of the CBD-dominant cultivars often exceed acceptable THC concentration (Chiluwal et al., 2023; Sandhu et al., 2022; Yang et al., 2020;). However, floral biomass yield was low in both cultivars. Both the cultivars in this study were photoperiod sensitive, hence reproductive phase begins when the day length or photoperiod decreases to a certain threshold. Since these cultivars are from the northern latitude (Colorado and Kentucky) where summer day length is comparatively longer than in Florida, they are adapted to longer photoperiod requirement. As a result, they started flowering very early in their growth stage (11–17 days after transplanting) (Table 1) in Florida's short-day environment, which resulted in less biomass accumulation. Identifying CBG-dominant hemp cultivars with shorter photoperiod requirement would provide an extended vegetative growth period and consequently higher biomass yield. Hence, screening more cultivars adapted in similar photoperiod as Florida would pave the way toward essential oil hemp commercialization in Florida.

CONCLUSIONS

The results from this study showed that location and planting date had some effect on total CBG and total THC concentration. However, their effect was not consistent within each and between two genotypes for any definitive conclusion. THC concentration never exceeded 0.1% in both the cultivars under all planting conditions, which was well below the 0.3% federal threshold for industrial hemp cultivation. The results showed both CBG dominant cultivars met the legal THC requirement making them suitable for cultivation in Florida. However, both the tested cultivars which are adapted to longer photoperiod in northern latitude had comparatively shorter vegetative growth period in Florida's short-day environments, which resulted in less biomass accumulation. Hence, evaluating more cultivars with shorter photoperiod requirement would be helpful in identifying suitable cultivars in Florida's environments and consequently industrial hemp commercialization in Florida and similar other locations.

AUTHOR CONTRIBUTIONS

Anuj Chiluwal: Data curation; Formal analysis; writingOriginalDraft; writingReviewEditing. Saroop S. Sandhu: Investigation; Project administration; writingReviewEditing. Mike Irey: Conceptualization; Funding acquisition; Project administration; Supervision; Validation; writingReviewEditing. Flint Johns: Investigation; Project administration; writingReviewEditing. Richard Sanchez: Investigation; Project administration; writingReviewEditing. Hardev Sandhu: Conceptualization; Funding acquisition; Project administration; Supervision; Validation; writingReviewEditing.

ACKNOWLEDGMENTS

The authors are thankful to the farm crew at United States Sugar Corporation and Lykes Bros. farm for their help in crop establishment, management, and related farm operations. This research was funded by United States Sugar Corporation and Highlands Hemp Farms, LLC, an affiliate of Lykes Bros. farm. This project was also supported by USDA-NIFA Hatch project (FLA-ERC-006028).

CONFLICTS OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest.