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Article

Methyl Jasmonate and Ammonium Bicarbonate: Distinct and Synergistic Impacts on Indoor Cannabis Production Dynamics

by
Jose F. Da Cunha Leme Filho
1,2,*,
Spencer Schuchman
2,
Avery Shikanai
3,
Shiksha Sharma
1,
Thais Alberti
1,2,4,
Andre A. Diatta
5,
Alan Walters
2 and
Karla L. Gage
1
1
School of Biological Sciences, Southern Illinois University, 1125 Lincoln Dr, Carbondale, IL 62901, USA
2
School of Forestry and Horticulture, Southern Illinois University, 1125 Lincoln Dr, Carbondale, IL 62901, USA
3
Department of Plant Sciences, North Dakota State University, Fargo, ND 58108, USA
4
Laboratory of Natural and Synthetic Products, Biotechnology Institute, University of Caxias do Sul, 1130, Francisco Getúlio Vargas St., Caxias do Sul 95070-560, Brazil
5
Department of Agronomy, Gaston Berger University, Saint-Louis 234, Senegal
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2025, 16(3), 78; https://doi.org/10.3390/ijpb16030078
Submission received: 14 May 2025 / Revised: 26 June 2025 / Accepted: 1 July 2025 / Published: 8 July 2025
(This article belongs to the Special Issue Challenges in Cannabis sativa: Breeding and Secondary Metabolite Synthesis)
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Abstract

As high-CBD cannabis (Cannabis sativa L.) gains legal and commercial relevance in the United States, studies evaluating how external inputs impact critical traits remain limited. This study investigates the effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and the genetic source (mother plant identity) on the growth and secondary metabolite traits of indoor cannabis. Plants were treated with 1 mM MeJA and/or AB under controlled conditions, and key traits, such as plant height, chlorophyll content, biomass, trichome density, and cannabinoid concentration, were measured. The MeJA treatment led to a significant 32% increase in trichome density. However, it did not significantly alter CBD or THC concentrations. The AB treatment enhanced vegetative growth, increasing chlorophyll content and plant height while reducing CBD concentrations, but the biomass gains could compensate for the lower cannabinoid in the total production. An interaction between MeJA and AB altered the CBD content, suggesting that MeJA may mitigate AB’s negative effect on cannabinoid synthesis. The genetic source significantly influenced most of the measured traits, highlighting the role of the genotype in trait expression and the importance of clonal consistency. These findings highlight the complex dynamics of external inputs and genetic factors in cannabis production, emphasizing the need for further research to optimize cultivation strategies. Future studies should refine input combinations and doses to improve both yield and cannabinoid profiles.

1. Introduction

The 2018 U.S. Farm Bill has allowed licensed institutions and commercial facilities to legally cultivate non-psychoactive Cannabis sativa L. for the first time in decades [1]. This shift enabled cannabis production on a commercial scale. Cannabis sativa L. is a multipurpose crop with applications in nutrition, textiles, construction, and pharmaceuticals. Given its broad utility, there is increasing demand for optimized and validated cultivation practices [2]. While the high-cannabinoid cannabis market is increasingly attractive to growers, the industry lacks scientifically validated production methods. As interest in cultivation practices grows, there is increasing pressure to identify strategies that maximize productivity without sacrificing product quality. Thus, research institutions play a key role in developing agronomic and horticultural strategies for C. sativa cultivation [3,4].
One of the primary goals of C. sativa cultivation is to optimize cannabinoid content, which serves as a central indicator of quality. Cannabis produces over 500 compounds [5], including more than 200 cannabinoids, such as delta-9 tetrahydrocannabinol (THC) and cannabidiol (CBD), which are key plant secondary metabolites [6]. While THC is the most widely known, other cannabinoids such as CBD have attracted attention for their therapeutic potential [7]. Cannabinoids are synthesized and stored in glandular trichomes, which are most abundant on the floral tissues of female plants [8].
Trichomes are extensions of the plant epidermis, which appear across a wide range of terrestrial species [9,10]. They are generally categorized as glandular or non-glandular [11,12]. Glandular trichomes are multicellular and serve as the primary site for the synthesis and storage of secondary metabolites [13,14]. Because cannabinoid synthesis occurs in glandular trichomes, increasing their density has become a target for improving C. sativa productivity and quality [15].
A combination of genetic and environmental factors regulates trichome density. Numerous studies have demonstrated that environmental conditions, such as UV radiation, frost, water-deficit stress, and salinity, can significantly increase trichome density in various plant species [16,17,18,19,20]. Biotic stressors, such as herbivory, have also been shown to stimulate trichome development as part of a defense response [21,22].
Hormonal signaling pathways, particularly those involving phytohormones, such as cytokinin (CK), gibberellin (GA), ethylene, brassinolide (BR), and salicylic acid (SA), influence trichome development [12]. Methyl jasmonate (MeJA), a constituent of the jasmonate hormone family [23], plays a significant role in plant defense and the production of secondary metabolites [24,25]. MeJA has been shown to increase trichome density and stimulate secondary metabolite synthesis in a range of species, including tomato (Solanum lycopersicum L.) and C. sativa [26,27,28,29].
Ammonium bicarbonate (AB), a nitrogen-only fertilizer containing 17% nitrogen (N), has been widely used in China since the mid-20th century [30]. However, AB remains underexplored as a source of nitrogen in cannabis cultivation and is not included in a list of common chemical fertilizers for cannabis [31]. Despite being inexpensive and synthetically produced, AB is not commonly used in U.S. agriculture. Due to its instability and tendency to volatilize, AB’s performance under U.S. conditions requires further evaluation. However, organically derived AB may hold promise as a supplemental nitrogen source for cannabis producers focused on organic inputs. Recently, a few commercial AB products derived from organic sources have been released.
The objective of this study was to assess the effects of exogenous methyl jasmonate (MeJA), ammonium bicarbonate (AB), genotype (mother plants identity), and their interactions on the growth and chemical features of indoor-grown Cannabis sativa L. Specifically, plant height, chlorophyll content (SPAD), biomass, trichome density, and cannabinoid concentrations were measured following treatment with MeJa and/or AB. We hypothesized that the MeJA application would increase trichome density and enhance the accumulation of secondary metabolites, including CBD. We further hypothesized that ammonium bicarbonate, serving as a nitrogen source, would enhance vegetative growth and also support the accumulation of secondary metabolites.

2. Materials and Methods

2.1. Experimental Design

A greenhouse study was conducted during summer 2022 at the Tree Improvement Center in Carbondale, IL, USA (37.70900, 89.26791). The experiment was arranged as a randomized complete block design with 4 replications (n = 4). This study evaluated the effects of MeJA, AB, and mother plant identity (plant 1 and 2) on “Cherry Wine” clones in a greenhouse setting (Table 1). A control group receiving no MeJA or AB was included to establish baseline comparisons. Both mother plants were from the “Cherry Wine” cultivar, a high-CBD type known for its consistent profiles [32], but were obtained from different sources, which may have introduced minor genetic or epigenetic differences. This design enabled an assessment of the influence of the clone source on phenotypic variation. Stock plants were seeded on 6 December 2021 and later used as sources of clonal material. On 26 February 2022, sixteen cuttings were taken from each of the two stock (mother) plants, treated with rooting hormone, and placed in a perlite–vermiculite (1:1) mix [33]. Once rooted, clones were fertilized with 30 mL of a water-soluble, balanced 20-20-20 fertilizer at 400 parts per million (ppm), providing equal concentrations of nitrogen (N), phosphorus (P2O5), and potassium (K2O·). This formulation is widely used to support early-stage vegetative growth in container systems. The plants were then transplanted into 10 cm pots on 12 April 2022. The plants were later transplanted into 18-L containers on 29 April 2022.

2.2. Media and Baseline Fertility

Each container was filled with Pro-Mix BX® (Quebec, QC, Canada) and received Osmocote Plus® (15-9-12) (ICL Group Ltd.—Tel Aviv-Yafo, Israel) at the lowest label-recommended rate (43 g per 18-L pot). A second application was top-dressed on 21 June 2022 to maintain adequate nutrition. The plants were maintained at a 60% field capacity and watered as needed throughout the growing period.

2.3. Treatment Application

The concentrations of methyl jasmonate (1 mM) and ammonium bicarbonate (0.5 g/plant) were selected based on doses previously used in the literature. These concentrations have been reported as effective in eliciting physiological responses and influencing secondary metabolism in various plant species while minimizing phytotoxic effects. They reflect biologically active yet non-phytotoxic ranges for eliciting physiological responses in other species [34]. Our aim was to apply doses that are biologically relevant and within ranges commonly used in similar experimental contexts. The experiment employed a fully crossed design, allowing for the analysis of interaction effects. MeJA was applied three times during flowering using a CO2-pressurized backpack sprayer equipped with a TEEJET AIXR (Glendale Heights, IL, USA) 11002 nozzle at 30 PSI (Table 2). The 1 mM MeJA solution was prepared by stirring for 10 minutes, sonicating for 15 minutes, and then stirring again for an additional 10 minutes. Control plants received a carrier control consisting of water with Tween-20, to account for the surfactant used in the treatment. Ammonium bicarbonate was applied as a soil drench three times per plant during the growing cycle, following the application schedule outlined in Table 2. All applications were timed according to defined phenological stages. Ammonium bicarbonate was applied at the ninth and sixteenth leaf pair stages (BBCH scale) and at the onset of flowering, while methyl jasmonate was applied at the first flowering stage and repeated biweekly for a total of three applications, as detailed in Table 2.

2.4. Data Collection

2.4.1. Growth and Chlorophyll Traits

Plant height was measured biweekly beginning 16 May 2022, from the base to the most recently expanded leaf. Chlorophyll content was measured using a SPAD-502 (Konica Minolta—Tokyo, Japan). Readings were taken from the most recently unfolded leaf of the terminal branch. Values in SPAD units provide an indirect measure of chlorophyll content, commonly used to estimate foliar nitrogen status.

2.4.2. Yield Traits

All plants were harvested on 6 October 2022. Fresh weight was recorded using a hanging scale. Plants were then dried to 13% relative humidity, and the total dry weight, “bucked” biomass, and terminal inflorescence weight (top 20 cm) were recorded. Bucked biomass refers to the usable floral and leaf material, separated from stems.

2.4.3. Trichome Density Counting

After drying, the top 20 cm of each inflorescence was sampled. Two samples, one from each of two small subtending leaves from the top 6 cm of the inflorescence, were imaged under a photo dissection microscope. Trichome density was quantified by manually counting glandular trichomes within standardized 1 mm2 fields in ImageJ—1.54p.

2.4.4. Cannabinoid Analysis

Samples were dried, homogenized, and sieved (1.0 mm). A 200 mg sample was extracted in 10 mL of HPLC-grade methanol, sonicated for 30 min, and filtered through a 0.45 μm syringe filter. Extracts were diluted (1:50) and analyzed by HPLC with UV-vis detection at 230 nm using an Agilent 1200 system (Santa Clara, CA, USA) and Poroshell 120 EC-C18 column (Santa Clara, CA, USA). The mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). Elution began at 75% B and reached 95% B at 10 min. The flow rate was 0.5 mL/min, and the injection volume was 10 μL. Analyzed cannabinoids included CBDA, CBD, THCA, and THC—the principal acidic and neutral forms in high-CBD cultivars. The cannabinoid concentration was expressed as a percentage of the dry weight (% dry weight), with the specific cannabinoid compounds identified and quantified using certified cannabinoid standards (Cayman Chemical, 1 mg·mL−1) (Ann Arbor, MI, USA).

2.5. Statistical Analysis

MeJA, AB treatments, and mother plant (clone source) were fully crossed and repeated four times. A hierarchical repeated-measures mixed-effects model was used to evaluate treatment effects, using R version 4.3.2. Fixed effects included MeJA, AB, mother plant, and days since planting; plant nested within block was treated as a random effect. The lme4 and lmerTest packages were used for model fitting [35,36]. Data transformations were applied to stabilize the variance, allowing for the reporting of means on the original scale. Final models were selected by backward elimination or using AIC minimization (MuMIn package). Post hoc comparisons were conducted using emmeans and multcomp with Tukey’s HSD test (α = 0.05).

3. Results

3.1. Plant Height

There were significant interactions between each factor and the number of days since the start of the experiment (Table 3). Plant height was significantly influenced by time, but this effect varied depending on the levels of MeJA, AB, and mother plant (i.e., a three-way interaction). Furthermore, a three-way interaction was observed (AB × time/days since start × MeJA; F1,310.161 = 48.4752, p < 0.001). This means that the effect of AB on plant height varied depending on both the MeJA treatment and the number of days since the experiment began (Figure 1).
There was also a significant interaction between days since the start and the mother plant (F1,310.161 = 69.9679, p < 0.001), indicating that plant height varied with time depending on the mother plant (Figure 2). A significant interaction between the number of days since start and AB was also present (F1,310.161 = 6.7473, p < 0.01), indicating a time-dependent effect of AB on plant height (Table 3). In addition to statistical interactions, time (days since start) had a significant main effect on plant height (F1,310.040 = 2674.7726, p < 0.001). This effect included significant quadratic and cubic effects of days since start (Table 3).

3.2. Soil Plant Analysis Development (SPAD) Chlorophyll Meter

Ammonium bicarbonate had a significant effect on SPAD meter readings (F1,337.10 = 14.4984, p < 0.001), with higher average values observed in the AB-treated individuals. Specifically, AB-treated plants averaged 45.2 SPAD units compared with 43.5 in the non-treated plants. In addition to AB, days since start also significantly affected the SPAD values (F1,337.12 = 966.4076, p < 0.001). The SPAD values decreased progressively throughout the experiment (Figure 3).
There was also a significant effect of the mother plant on the SPAD meter values (F1,337.06 = 9.0811, p < 0.01). Moreover, individuals derived from mother plant “1” had significantly lower SPAD values than those derived from mother plant “2”, averaging 43.7 and 45.0, respectively. MeJA had no effect on SPAD values (F1,337.06 = 0.0619, p = 0.8037316).

3.3. Fresh and Dry Total Biomass

A significant main effect of the mother plant was observed on the total dry biomass of cannabis (F1,23.832 = 11.0534, p < 0.01) (Table 4). Specifically, clones from mother plant “1” produced an average of 0.591 kg of dry biomass, which was significantly greater than the 0.535 kg produced by clones from mother plant “2”. Note that these mean values are not repeated in Table 4, which only includes statistical test results. No significant effects of MeJA or AB were detected on total dry biomass. Likewise, total fresh biomass was not significantly affected by any of the factors evaluated.

3.4. Bucked Biomass

There was a significant three-way interaction between AB, MeJA, and mother plant identity, which influenced the bucked biomass of cannabis (F1,19.997 = 4.4794, p < 0.005). In particular, the effect of AB on the bucked biomass varied according to both MeJA treatment and mother plant (Figure 4). For mother plant “1”, the combination of AB and MeJa led to the highest bucked biomass. In contrast, for mother plant “2”, MeJA alone resulted in the highest bucked biomass, while the addition of AB reduced it.
There was also an interaction between AB and the mother plant identity (F1,19.997 = 7.0540, p < 0.05), where AB influenced the bucked biomass depending on the mother plant (Figure 5). Additionally, there was a significant main effect of the mother plant identity on the bucked biomass of “Cherry Wine” cannabis (F1,19.997 = 8.5363, p < 0.01). Specifically, clones from mother plant “1” produced an average of 132 g, significantly less than the 148 g observed in clones from mother plant “2”.

3.5. Trichome Density

Methyl jasmonate treatment had a significant effect on the trichome density of high-cannabinoid cannabis (F1,27 = 20.1414, p < 0.001) (Table 4). On average, MeJA-treated plants exhibited 22.9 trichomes per mm2 compared with 17.4 trichomes per mm2 in non-treated plants. This represents an average increase of approximately 31.6% in trichome density following MeJA application. Neither AB nor the mother plant had a significant effect on the trichome density (Table 5). No interaction effects were statistically significant and, thus, were excluded from the final model.

3.6. Inflorescence Biomass

A significant interaction influenced inflorescence biomass, where the effect of MeJA treatment on the inflorescence weight depended on the mother plant identity (F1,23.024 = 8.1254, p < 0.01). Post hoc Tukey comparisons revealed that MeJA significantly increased the inflorescence weight in clones from mother plant “1” but did not have a significant effect in clones from mother plant “2” (Figure 6). Additionally, there was a significant main effect of the mother plant on the inflorescence biomass (F1,23.024 = 16.1730, p < 0.003), where clones from mother plant “1” yielded significantly heavier inflorescences compared to those from mother plant “2”, averaging 4.69 g and 3.88 g, respectively.

3.7. Cannabinoid Content (CBD and THC)

There was a significant interaction between AB and MeJA on the total CBD percentage of “Cherry Wine” cannabis (Table 6). The AB treatment increased total CBD content only when MeJA was also applied (Figure 7). Interestingly, the highest CBD levels were observed in the control plants (no AB, no MeJA), suggesting complex interaction effects (Figure 7). In addition, there was also a significant main effect of AB on total CBD percentage (F1,23.188 = 8.4256, p < 0.01). Cannabis treated with AB showed a significantly lower CBD percentage (9.76%) compared with non-treated plants (10.54%).
There was a significant main effect of the mother plant on the total CBD percentage (F1,23.188 = 9.6572, p < 0.01). Clones derived from mother plant “1” had a significantly lower CBD content (9.74%) than those from mother plant “2” (10.57%). There was no significant effect of MeJa on the total CBD percentage of cannabis (F1,23.188 = 0.9229, p = 0.3466). Mother plant had a significant effect on the total THC percentage (F1,23.111 = 44.1664, p < 0.001). THC content was also significantly lower in plants from mother plant “1” (0.442%) compared with mother plant “2” (0.541%). The interaction between AB and MeJA was marginally significant (F1,23.111 = 3.0571, p < 0.1) but did not reach the threshold for statistical significance (p < 0.05). There was also a marginally significant main effect of AB (F1,23.111 = 2.9853, p < 0.1), with the AB-treated plants exhibiting slightly lower THC content. The MeJA treatment had no effect on the total THC (F1,23.111 = 2.3043, p = 0.14258).

4. Discussion

4.1. Plant Height

Cannabis height was affected by several significant interactions between predictor variables and the number of days since the early stages. A significant three-way interaction among AB, MeJA, and days since the start of the experiment influenced plant height (Figure 1). This interaction suggests that the effects of AB and MeJA on plant growth are interdependent and vary over time, rather than simply additive. Plants treated with AB alone were the tallest overall, regardless of the mother plant’s identity. This result can be reasonably explained by the effect of nitrogen on plant height, which is generally positive unless applied at excessive rates [37]. The second tallest group consisted of plants that received MeJA alone, suggesting that both AB and MeJA individually promote height. This result is particularly interesting as low concentrations of MeJA (0.1 mM) have been shown to yield increases in height, but higher concentrations reduce height [34]. Conversely, when AB and MeJa were applied together, plant height resembled that of the control plants, indicating a possible antagonistic interaction. Thus, while AB and MeJA promote growth independently, their combination may negate each other’s effects (Figure 1). Moreover, the interaction with time suggests that treatment effects evolved over the course of the study period.
A significant interaction between mother plant identity and the number of days since the start of the experiment was also observed (Figure 2). Plant height trajectories differed significantly between the two mother plants over time (Figure 2). Specifically, clones from mother plant “1” were consistently taller than those from mother plant “2”, although this difference varied across time. There was also a similar two-way interaction between AB treatment and days since start (Table 3). The plant’s response to AB also shifted over time. Initially, AB-treated plants were shorter than the controls; however, this trend reversed after day 42, when the AB-treated plants began to grow taller. This pattern may reflect early-stage nitrogen stress followed by a recovery period as nutrients were assimilated.
Lastly, there was a significant effect on the number of days since start on plant height. As expected, plant height increased over time. The high mean square value (65,743) indicates that time was a major driver of the variability in growth. The significance of higher-order polynomial terms further suggests that growth was nonlinear, likely reflecting developmental phases. In summary, plant height was shaped by time in a complex, dynamic manner, influenced by treatment combinations and maternal lineage.

4.2. Soil Plant Analysis Development (SPAD) Meter

A soil plant analysis development meter (SPAD) was utilized in the present study to estimate changes in the leaf chlorophyll content throughout the development of indoor-grown cannabis. SPAD meters assess the relative chlorophyll content or “greenness” by measuring the amount of red and infrared light absorbed by the leaf, which corresponds to the amount of chlorophyll in the leaf [38]. SPAD meter values have been shown to be highly correlated with chlorophyll content in essential-oil-type cannabis [39]. Mother plant identity, MeJA, AB, and the number of days since the start of the experiment presented a variable impact on cannabis chlorophyll content.
Ammonium bicarbonate significantly increased the SPAD meter values compared with the non-treated control. On average, the experimental units treated with AB had approximately 3.9% more estimated chlorophyll content than those that did not receive AB. Although statistically significant, a 3.9% increase in SPAD meter values did not correlate with increased biomass or other key metrics in cannabis, such as yield, trichome density, or cannabinoid content. Further research should investigate whether varying application rates and timings of AB can increase chlorophyll content more substantially, potentially improving yield and other metrics, as SPAD values have been correlated with increased yields in crops such as corn (Zea mays L.) and wheat (Triticum aestivum L.) [40,41].
Ammonium bicarbonate’s effect on the chlorophyll content is likely due to nitrogen’s role in chlorophyll synthesis. Nitrogen deficiency decreases chlorophyll, as shown in rice (Oryza sativa L.) [42]. The mother plant was also found to significantly impact SPAD meter values (Table 3), with individuals from mother plant “2” consistently exhibiting a higher estimated chlorophyll content than those from mother plant “1”. This effect may be due to variations in chlorophyll synthesis and concentrations between genotypes. For instance, variation in SPAD meter values resulting from differences in chlorophyll content across genotypes has been observed in spring wheat [43].
Furthermore, SPAD values significantly declined throughout the experiment (Figure 3). The estimated chlorophyll content declined significantly over time (Figure 3), consistent with natural senescence as plants complete their life cycles [44], which is what was likely observed here. There was no effect of MeJA on the SPAD meter values. The lack of influence of MeJA on SPAD contradicts work conducted on other plant species, where MeJA has been shown to significantly influence chlorophyll content. In canola (Brassica napus L.), for example, MeJa increased chlorophyll under saline [45] and arsenic stress [46], especially when combined with gibberellic acid. These results suggest that the effect of MeJa on chlorophyll content is dependent on environmental conditions, possibly modulated by the presence of other physiologically important plant growth regulators.

4.3. Fresh and Dry Total Biomass

Wasternack & Hause (2013) [47] reported that MeJA triggers defense-related gene expression and can reduce growth and biomass in many species. This inhibition is often attributed to the reallocation of resources from growth to defense. In Arabidopsis thaliana L., exogenous MeJA reduced root and shoot biomass by inhibiting cell expansion and photosynthesis [48].
The total fresh and dry biomass was recorded after harvest. Fresh biomass was not significantly affected by any of the treatments. However, there was a significant main effect of the mother plant identity on the dry biomass (Table 4). This effect was not surprising, considering the strong influence the mother plant exerted on other dependent variables throughout the experiment, and is likely due to genetic variation. These results underscore the importance of genetic consistency and standardized labeling protocols in commercial production, indicating that variation within a cultivar can affect product uniformity.

4.4. Bucked Biomass

A significant three-way interaction influenced the bucked biomass among MeJA, AB, and mother plant identity (Figure 5). The effect of MeJA on bucked biomass depended on the AB treatment, and the interaction varied by genotype. In the case of mother plant “1”, MeJA alone yielded slight increases in the bucked biomass compared with the control. However, when the AB treatment was also present, there was a substantial increase in the bucked biomass compared with the control. Conversely, in mother plant “2”, MeJA alone resulted in the highest biomass, while the combination with AB produced the lowest. These opposing trends suggest that the interactive effects of AB and MeJA are modulated by genotype.
In addition to the three-way interaction, a significant two-way interaction was also observed between AB and mother plant identity (Figure 5), where the effect of AB depended on the genotype. Furthermore, the results show that for mother plant “1”, AB had a substantial positive effect on the bucked biomass. In contrast, plants of mother plant “2” that received AB produced less bucked biomass, although this effect was less remarkable. This differential response is likely due to genotypic variation in nitrogen uptake and utilization. Additionally, considering the total cannabinoid production, the biomass gains promoted by the AB application may compensate for the lower CBD concentration, which is likely due to the biomass dilution (Figure 5 and Figure 7). Ammonium bicarbonate can affect plant biomass through its role as a nitrogen (N) source; however, its impact depends on the dosage, crop type, and environmental conditions. According to Liu et al. (2013) [49], AB applied at low to moderate concentrations can enhance biomass by supplying readily available ammonium nitrogen, which supports vegetative growth and improves rice shoot and root biomass under controlled conditions. The application of AB as a nitrogen source increased the biomass and yield in corn and wheat when used properly with organic amendments [49].
There was also a significant main effect of the mother plant identity on the bucked biomass. Clones from mother plant “2” produced significantly more bucked biomass than those from mother plant “1”. Although clones are genetically identical, variation in biomass may arise from physiological factors, rooting success, or epigenetic differences. Druege et al. (2016) [50] stated that apical cuttings tend to root faster and produce more vigorous biomass than basal or middle cuttings due to higher auxin content and better hormonal balance. Even genetically identical clones may exhibit epigenetic changes due to stress or microenvironmental influences during propagation, resulting in variability in biomass, as reported in [51]. Also, differences in rooting speed and initial nutrient uptake can influence later biomass accumulation [52]. Therefore, cannabis clones with poor rooting could lag in shoot growth and yield. Neither MeJA nor AB showed significant main effects on bucked biomass, suggesting that under the conditions of this study, these treatments are not primary drivers of biomass allocation in indoor-grown cannabis.

4.5. Trichome Density

Consistent with our hypothesis, the exogenous application of MeJA significantly increased the trichome density of high-CBD cannabis. On average, plants treated with 1 mM MeJA exhibited ~32% more trichomes per mm2 than untreated controls.
Similar studies have also investigated the effect of MeJA on the trichome density of cannabis; however, none have observed a significant stimulatory effect on trichome density [34,53]. Although this contradicts previous findings in cannabis, MeJA has consistently enhanced trichome density in other species [27,28,29,54,55].
Genetic differences may explain the discrepancies between our results and previous cannabis studies. Transcription factors (TFs) are specialized proteins that regulate gene expression by binding to specific DNA sequences. TFs can be signaled hormonally to initiate the expression of genes related to secondary metabolites [56]. Methyl jasmonate is a phytohormone that has been shown to regulate trichome formation and the expression of biosynthesis genes via the modulation of TFs [26]. This hormonal signaling likely underlies MeJA’s influence on glandular trichome development and cannabinoid biosynthesis in Cannabis sativa, although the specific TFs involved remain poorly characterized [26]. The transcriptional factor HDG5 is suggested as a potential starting point for this investigation and may be one of the first TFs endogenous to C. sativa that directly influences trichome formation and development [26].

4.6. Inflorescence Weight

There was a significant interactive effect on inflorescence weight between MeJA and mother plant (Figure 6). The response to MeJA depended on the genotype. In the case of mother plant “1”, the application of MeJA resulted in a notable 21.23% increase in inflorescence weight compared with the non-treated counterpart, which is statistically significant. However, an opposite effect was observed in clones from mother plant “2”, where the application of MeJA resulted in a 6.25% decrease in the inflorescence weight compared with the non-treated analog. Notably, the difference in inflorescence weight among the MeJA-treated genotypes reached 37%.
Despite these opposing trends, MeJA did not show a significant main effect, reinforcing that its influence is genotype-dependent and likely mediated by genetic mechanisms. There was, however, a significant main effect of the mother plant on the inflorescence weight, where clones of mother plant “1” had notably higher inflorescence weights than mother plant “2”. MeJA could reduce the overall inflorescence size due to its trade-off between growth and defense, but it often enhances quality traits, for example, MeJA treatments reduced the petunia (Petunia Juss) flower size and biomass by suppressing cell elongation [57]. Also, in tomato (Solanum lycopersicum L.), MeJA foliar application reduced flower and fruit size, especially at higher concentrations [58]. These findings suggest that MeJA may modulate floral biomass through conserved growth-regulating pathways, though the direction and magnitude of the response vary by species and genotype.

4.7. Cannabinoid Content (CBD and THC)

The “dilution effect” in cannabis cultivation refers to the phenomenon where increasing plant biomass leads to a decrease in the concentration of cannabinoids like cannabidiol (CBD) per unit of dry weight. Dilena et al. (2025) [59] demonstrated that nitrogen application increased from 3% to 5% of dry weight, the total biomass of cannabis plants approximately doubled, but CBD and THC concentrations decreased by ~25%. This indicates that while the plants grew larger, the concentration of cannabinoids in their tissues became more diluted. Other studies have found that increasing fertilizer concentrations leads to higher accumulation of plant dry matter and inflorescence dry matter. However, CBD concentration tends to plateau or decline after a certain fertilization threshold is exceeded [60,61]. Other works found nitrogen application influences cannabinoid concentration in a quadratic fashion [62], indicating that excessive nitrogen application (N > 50 ppm) may limit CBD concentrations in cannabis [39].
Although both mother plants were of the same accession (cultivar “Cherry Wine”), the apparent differences in total THC and CBD between them align with previous findings that genetics play a primary role in cannabinoid content [63], and genetic variation both between and within cultivars is not uncommon in cannabis [64], as the breeding industry is still developing consistent genetic lines. In addition, factors such as cutting position, epigenetic variation, rooting success, and mother plant stress or health may have contributed to these differences, even within a single accession.
There was also an interactive effect between AB and MeJA treatments that influenced the CBD content, as well as a marginal interaction for THC (Table 6). Experimental units that received no AB or MeJA produced the highest CBD concentrations (Figure 7), while plants treated with AB alone had the lowest CBD values. This supports the idea that AB alone may suppress CBD synthesis, consistent with its negative main effect with more plant biomass and diluted cannabinoid concentration.
Interestingly, MeJA appeared to mitigate the inhibitory effect of AB. Excessive nitrogen is known to stimulate vegetative over reproductive growth [65], which may explain AB’s negative impact on CBD. MeJA, a phytohormone that activates defense and secondary metabolite pathways [25], may have counteracted this effect when applied in combination with AB. The combined treatment may have shifted metabolic allocation back toward secondary metabolism, including the production of cannabinoids.
The absence of a direct effect of MeJA on CBD content, despite promoting a higher trichome density and its role in defense signaling, underscores the complex regulation of cannabinoid biosynthesis [66]. Further research is needed to elucidate the complex mechanisms that influence cannabinoid content in response to external stimuli such as MeJA and AB.

5. Conclusions

This study demonstrated that cannabis growth and development are highly responsive to complex interactions among genotype, treatments with MeJA, AB, and time (days since start). While both AB and MeJA independently promoted plant height, their combination nullified these positive effects, indicating a non-additive, antagonistic interaction. Temporal patterns also revealed that the effects of AB evolved over time, initially stunting growth but later enhancing height—likely due to nitrogen dynamics across developmental stages. Mother plant identity consistently determined both plant height and chlorophyll content, reinforcing the importance of genetic background in cultivation outcomes. Biomass analyses further highlighted genotype-specific responses to treatment. While AB and MeJA influenced bucked and inflorescence biomass, their effects varied depending on the mother plant, and their combination often led to inconsistent or even opposing results.
Ultimately, this research underscores the importance of considering genotype–treatment interactions when evaluating cultivation inputs for cannabis. While AB shows promise as a nitrogen source—particularly in organic systems—and MeJA may enhance certain growth parameters, their combined application requires careful optimization. These findings offer important preliminary insights for optimizing input strategies in cannabis cultivation. This initial evidence lays the groundwork for future studies aimed at optimizing the use of AB and MeJA in diverse cultivation settings. Further studies examining application timing, dosage, and environmental conditions are warranted to better harness these compounds for optimized cannabis production.

Author Contributions

Conceptualization, J.F.D.C.L.F.; data curation and writing—original draft preparation, S.S. (Spencer Schuchman); formal statistical analysis and validation, A.S.; revision and data curation, S.S. (Shiksha Sharma), T.A., and A.A.D.; supervision and methodology, J.F.D.C.L.F., A.W., and K.L.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The authors are willing to provide any type of raw data to support their findings.

Acknowledgments

This research was made possible by the contributions of Southern Illinois University—Carbondale.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Response of plant height over time to the interaction of methyl jasmonate treatment, ammonium bicarbonate, and days since the start of the experiment. Blue lines represent MeJA-treated plants (Y), and red lines represent untreated plants (N). Curved lines represent cubic polynomial regression models bounded by standard errors. A significant three-way interaction was observed (p < 0.001). Polynomial regression was used to model the progression of plant height over time.
Figure 1. Response of plant height over time to the interaction of methyl jasmonate treatment, ammonium bicarbonate, and days since the start of the experiment. Blue lines represent MeJA-treated plants (Y), and red lines represent untreated plants (N). Curved lines represent cubic polynomial regression models bounded by standard errors. A significant three-way interaction was observed (p < 0.001). Polynomial regression was used to model the progression of plant height over time.
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Figure 2. Plant height over time as influenced by the clone source (mother plant “1” or “2”). Curved lines represent cubic polynomial regression models bounded by standard errors. A significant interaction was observed between days since start and mother plants (p < 0.001). Polynomial regression was used to capture growth dynamics over time.
Figure 2. Plant height over time as influenced by the clone source (mother plant “1” or “2”). Curved lines represent cubic polynomial regression models bounded by standard errors. A significant interaction was observed between days since start and mother plants (p < 0.001). Polynomial regression was used to capture growth dynamics over time.
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Figure 3. Soil plant analysis development (SPAD) meter readings (chlorophyll content) over time. The solid line represents the model prediction (SPAD = 53.11 − 0.15 × days since start), and individual points represent SPAD readings from experimental units. A significant negative effect of time on the SPAD values was observed (p < 0.001). Data were modeled using linear regression to capture this temporal trend.
Figure 3. Soil plant analysis development (SPAD) meter readings (chlorophyll content) over time. The solid line represents the model prediction (SPAD = 53.11 − 0.15 × days since start), and individual points represent SPAD readings from experimental units. A significant negative effect of time on the SPAD values was observed (p < 0.001). Data were modeled using linear regression to capture this temporal trend.
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Figure 4. Interaction effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on the bucked biomass (g). The red and blue lines represent mother plant “1” and mother plant “2”, respectively. The treatment combinations were as follows: NN = no MeJA, no AB; YN = yes MeJA, no AB; NY = no MeJA, yes AB; and YY = yes MeJA, yes AB. Letters indicate significant differences (p ≤ 0.05) based on Tukey-adjusted pairwise comparisons within each genotype.
Figure 4. Interaction effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on the bucked biomass (g). The red and blue lines represent mother plant “1” and mother plant “2”, respectively. The treatment combinations were as follows: NN = no MeJA, no AB; YN = yes MeJA, no AB; NY = no MeJA, yes AB; and YY = yes MeJA, yes AB. Letters indicate significant differences (p ≤ 0.05) based on Tukey-adjusted pairwise comparisons within each genotype.
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Figure 5. Interaction effects of ammonium bicarbonate (N = no application and Y = application) and mother plant accessions/identity on the bucked biomass (g). Letters indicate significant differences at p ≤ 0.05 based on Tukey-adjusted pairwise comparisons.
Figure 5. Interaction effects of ammonium bicarbonate (N = no application and Y = application) and mother plant accessions/identity on the bucked biomass (g). Letters indicate significant differences at p ≤ 0.05 based on Tukey-adjusted pairwise comparisons.
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Figure 6. Interaction effects of methyl jasmonate (MeJA) (N = no application and Y = application) and mother plant identity on the inflorescence weight (g). Letters indicate significant differences at p ≤ 0.05 based on Tukey-adjusted pairwise comparisons.
Figure 6. Interaction effects of methyl jasmonate (MeJA) (N = no application and Y = application) and mother plant identity on the inflorescence weight (g). Letters indicate significant differences at p ≤ 0.05 based on Tukey-adjusted pairwise comparisons.
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Figure 7. Interactive effect of methyl jasmonate (MeJA) and ammonium bicarbonate (AB) (N = no application and Y = application) on total CBD concentration in cannabis. The means are shown as either crosses or circles, and the 95% confidence limits are indicated by error bars. Treatments not sharing the same letter are significantly different at p ≤ 0.05 based on interaction effects.
Figure 7. Interactive effect of methyl jasmonate (MeJA) and ammonium bicarbonate (AB) (N = no application and Y = application) on total CBD concentration in cannabis. The means are shown as either crosses or circles, and the 95% confidence limits are indicated by error bars. Treatments not sharing the same letter are significantly different at p ≤ 0.05 based on interaction effects.
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Table 1. Overview of treatments for the greenhouse-grown “Cherry Wine” accession.
Table 1. Overview of treatments for the greenhouse-grown “Cherry Wine” accession.
FactorTreatments
Methyl jasmonate (MeJA)
-
1 mM MeJA + 0.1% Tween-20
-
Water only
Ammonium bicarbonate (AB)
-
0.5 g Ammonium bicarbonate
-
None
“Cherry Wine” mother plant
(clones from two distinct “Cherry Wine” stock plants)
-
Mother plant “1”
-
Mother plant “2”
Table 2. Treatment applications of methyl jasmonate and ammonium bicarbonate.
Table 2. Treatment applications of methyl jasmonate and ammonium bicarbonate.
TreatmentTimingRate
Methyl jasmonate (MeJA)
-
First flowering
-
Biweekly (total of 3 applications)
1 mM MeJA + 0.1% Tween-20
Ammonium bicarbonate (AB)
-
Ninth leaf pair
-
Sixteenth leaf pair
-
Onset of flowering
0.5 g per plant
Table 3. F-tests for the effects of time (days since start), methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on plant height.
Table 3. F-tests for the effects of time (days since start), methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on plant height.
SourceSum of SquaresMean
Squares		Numerator
df		Denominator
df		Fp
Methyl jasmonate9.789.781300.400.53
Ammonium bicarbonate6.856.851300.28NS
Mother plant0.000.001300.000.99
Days since start65,743.0965,743.0913102674.77***
Days since start 216,516.7116,516.711310671.99***
Days since start 37040.937040.931310286.46***
Ammonium bicarbonate × days since start165.84165.8413106.75**
Ammonium bicarbonate × methyl jasmonate0.100.101300.00NS
Ammonium bicarbonate × mother plant4.374.371210.18NS
Days since start × methyl jasmonate9.399.3913100.38NS
Days since start × mother plant1719.741719.74131069.97***
Methyl jasmonate × mother plant2.872.871210.12NS
Ammonium bicarbonate × days since start × methyl jasmonate1191.471191.47131048.48***
Ammonium bicarbonate × methyl jasmonate × mother plant6.366.361210.26***
2,3 Superscripts represent the quadratic and cubic terms within the model for “days since start”, included to capture nonlinear growth patterns over time. NS, **, *** non-significant or significant at p ≤ 0.01, or 0.001, respectively.
Table 4. F-tests for the effects of methyl jasmonate, ammonium bicarbonate, and high-CBD cannabis mother plant on dry biomass.
Table 4. F-tests for the effects of methyl jasmonate, ammonium bicarbonate, and high-CBD cannabis mother plant on dry biomass.
SourceSum of SquaresMean SquaresNumerator dfDenominator dfFp
Methyl jasmonate 0.000.001240.73NS
Ammonium bicarbonate0.000.001240.01NS
Mother plant 0.020.0212411.05**
NS or ** non-significant or significant at p ≤ 0.01. The sum of squares and mean squares for ammonium bicarbonate and methyl jasmonate are <0.00, and exact values are, therefore, not shown.
Table 5. F-tests for the effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on trichome density.
Table 5. F-tests for the effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on trichome density.
SourceSum of SquaresMean SquaresNumerator dfDenominator dfFp
Methyl jasmonate228.94228.9412720.14***
Ammonium bicarbonate6.896.891270.61NS
Mother plant15.8115.811271.39NS
NS or *** non-significant or significant at p ≤ 0.001.
Table 6. F-tests for the effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on total CBD concentration.
Table 6. F-tests for the effects of methyl jasmonate (MeJA), ammonium bicarbonate (AB), and mother plant identity on total CBD concentration.
SourceSum of SquaresMean SquaresNumerator dfDenominator dfFp
Methyl jasmonate0.500.501230.92NS
Ammonium bicarbonate4.614.611238.43**
Mother plant5.285.281239.66**
Ammonium bicarbonate × methyl jasmonate2.402.401234.39*
NS, *, **, non-significant or significant at p ≤ 0.05 or 0.01, respectively.
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Da Cunha Leme Filho, J.F.; Schuchman, S.; Shikanai, A.; Sharma, S.; Alberti, T.; Diatta, A.A.; Walters, A.; Gage, K.L. Methyl Jasmonate and Ammonium Bicarbonate: Distinct and Synergistic Impacts on Indoor Cannabis Production Dynamics. Int. J. Plant Biol. 2025, 16, 78. https://doi.org/10.3390/ijpb16030078

AMA Style

Da Cunha Leme Filho JF, Schuchman S, Shikanai A, Sharma S, Alberti T, Diatta AA, Walters A, Gage KL. Methyl Jasmonate and Ammonium Bicarbonate: Distinct and Synergistic Impacts on Indoor Cannabis Production Dynamics. International Journal of Plant Biology. 2025; 16(3):78. https://doi.org/10.3390/ijpb16030078

Chicago/Turabian Style

Da Cunha Leme Filho, Jose F., Spencer Schuchman, Avery Shikanai, Shiksha Sharma, Thais Alberti, Andre A. Diatta, Alan Walters, and Karla L. Gage. 2025. "Methyl Jasmonate and Ammonium Bicarbonate: Distinct and Synergistic Impacts on Indoor Cannabis Production Dynamics" International Journal of Plant Biology 16, no. 3: 78. https://doi.org/10.3390/ijpb16030078

APA Style

Da Cunha Leme Filho, J. F., Schuchman, S., Shikanai, A., Sharma, S., Alberti, T., Diatta, A. A., Walters, A., & Gage, K. L. (2025). Methyl Jasmonate and Ammonium Bicarbonate: Distinct and Synergistic Impacts on Indoor Cannabis Production Dynamics. International Journal of Plant Biology, 16(3), 78. https://doi.org/10.3390/ijpb16030078

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