Response of Medical Cannabis (Cannabis sativa L.) Genotypes to K Supply Under Long Photoperiod.

Potassium is involved in regulation of multiple developmental, physiological, and metabolic processes in plants, including photosynthesis and water relations. We lack information about the response of medical cannabis to mineral nutrition in general, and K in particular, which is required for development of high-grade standardized production for the medical cannabis industry. The present study investigated the involvement of K nutrition in morphological development, the plant ionome, photosynthesis and gas-exchange, water relations, water use efficiency, and K use efficiency, comparatively for two genotypes of medical cannabis, under a long photoperiod. The plants were exposed to five levels of K (15, 60, 100, 175, and 240 ppm K). Growth response to K inputs varied between genotypes, revealing genetic differences within the Cannabis sativa species to mineral nutrition. Fifteen ppm of K was insufficient for optimal growth and function in both genotypes and elicited visual deficiency symptoms. Two hundred and forty ppm K proved excessive and damaging to development of the genotype Royal Medic, while in Desert Queen it stimulated rather than restricted shoot and root development. The differences between the genotypes in the response to K nutrition were accompanied by some variability in uptake, transport, and accumulation of nutrients. For example, higher levels of K transport from root to the shoot were apparent in Desert Queen. However, overall trends of accumulation were similar for the two genotypes demonstrating competition for uptake between K and Ca and Mg, and no effect on N and P uptake except in the K-deficiency range. The extent of accumulation was higher in the leaves > roots > stem for N, and roots > leaves > stem for P. Surprisingly, most micronutrients (Zn, Mn, Fe, Cu, Cl) tended to accumulate in the root, suggesting a compartmentation strategy for temporary storage, or for prevention of access concentrations at the shoot tissues. The sensitivity of net-photosynthetic rate, gas exchange, and water use efficiency to K supply differed as well between genotypes. The results suggest that growth reduction under the deficient supply of 15 ppm K was mostly due to impact of K availability on water relations of the tissue and transpiration in Royal Medic, and water relations and carbon fixation in Desert Queen.

Introduction

Cannabis (pan> class="Species">Cannabis sativa L.) has been cultivated by mankind from antiquity, for medical (Zuardi, 2006; Clarke and Merlin, 2013) and recreational use (Small, 2017), and as a source for seed oil and fibers (Leizer et al., 2000; Lash, 2010). Among the plant’s acknowledged medical properties are anti-inflammatory potential and easing symptoms of numerous medical conditions including post-traumatic stress disorder, multiple sclerosis, cancer, Crohn’s disease, pain, and chemotherapy (Naftali et al., 2013; Greer et al., 2014; Cascio et al., 2017). The diverse medical potential is predicated on the complex chemical profile, comprising hundreds of secondary metabolites including cannabinoids, terpenes, and flavonoids.

Environmental conditions such as mineral nutrients (Bernstein et al., 2011) and water availability (Wanpan>g et al., 2018) afpan> class="Chemical">fect plant development and function, including synthesis of secondary metabolites in medicinal plants (Eliašová et al., 2004; Figueiredo et al., 2008; Nascimento and Fett-Neto, 2010; Gorelick and Bernstein, 2014). Well documented is the strong connection between potassium (K), one of the principle nutrient elements required by higher plants, and plant development and function (Pettigrew and Meredith, 1997; Bernstein et al., 2011; Grzebisz et al., 2013; Reviewed by Prajapati and Modi, 2012; Tsialtas et al., 2016). K is involved in multiple physiological and metabolic processes, including photosynthesis, transport of assimilates, protein synthesis, enzyme activation, stomata regulation, and osmoregulation, and it is therefore not surprising that it is a key player in regulation of plant development processes (Reviewed by Szczerba et al., 2009; Prajapati and Modi, 2012; Wang et al., 2013; Wang and Wu, 2017). Moreover, K is also known as a ‘quality element’ (Usherwood, 1985). By its effect on the secondary metabolite profile, it improves factors which are of relevance for yield quality such as color, taste and aroma (Reviwed by Usherwood, 1985; Prajapati and Modi, 2012), and hence stands as one of the main targets for study in the medical cannabis research. We lack information about K effects on plant development and function in medical cannabis. Such information is vital for developing optimal fertigation practices to support excelled plant growth and development during the vegetative growth phase, as well as for optimal reproductive development and secondary metabolism during the short day phase.

Legal restrictions during the last decades prevented progress in academic research involving the cannabis planpan>t. This has resulted in meager science-based information about pan> class="Species">cannabis, which is peculiar considering that it is one of our most ancient crops with a rich history of usage by humanity. We lack basic information about plant developmental and physiological responses to key environmental factors including mineral nutrition, and this hinders efforts to develop high-grade standardized production for the booming medical cannabis market.

C. sativa is a “short day” planpan>t, which under long photoperiod undergoes continuous vegetative growth, with inflorescence initiation anpan>d development ocpan> class="Chemical">curring following transition to a short photoperiod. The intensity of growth and the developmental pattern under long photoperiod in cannabis plants, together with the duration of this growth phase, determines plant architecture and size at the onset of the transition to the short photoperiod. The vegetative growth phase is hence a major player in determining the size, architecture, and to a large extent also the spatial pattern of inflorescences distribution in the mature medical cannabis plants, factors which affect yield quantity as well as the potential for standardization of the chemical quality. Understanding and regulating development at the long photoperiod phase is therefore fundamental for excelled quantity and quality production in medical cannabis. Potassium, being a key nutrient for growth and developmental processes should be studied for its effects during this phase. The present study therefore focused on the developmental and physiological responses of medical cannabis at the long photoperiod growth phase to K nutrition.

The little knowledge available about cannabis growth is mainly from research with pan> class="Species">hemp; a vigorous, tall and woody fiber-type of C. sativa. The data collected over the years about industrial hemp indicate that its growth and yield can be greatly affected by fertilization (Bócsa et al., 1997; Ivonyi et al., 1997; Vera et al., 2010; Finnan and Burke, 2013), and that the concentration of cannabinoids such as CBN and CBD can be affected by stress, nutrient deficiency, and other environmental parameters (Haney and Kutscheid, 1973). In spite of their importance, these results for hemp can shed only partial light on medical cannabis physiology and development considering the differences in plant development, genetics and growing practices. The little agro-scientific knowledge available for medical cannabis suggests some interesting correlations between soil pH, nutritional elements, and cannabinoids. These correlations, were determined for seeded plants from an “Afgan origin,” grown in 11 different soil types (Coffman and Gentner, 1975) as part of an attempt to identify cultivation sites of confiscated illegal plant parts. Recently we have demonstrated gradients of cannabinoids and inorganic nutrients along the medical cannabis plants, with an interplay between plant organs and organic and inorganic constituents (Bernstein et al., 2019a). Enhanced mineral nutrition by supplementation of NPK, P, or humic acids, affects specific cannabinoid concentrations in a compound and organ specific manner (Bernstein et al., 2019b), demonstrating the potential of specific mineral nutrients for regulation of growth and the chemical profile.

In the present study we studied comparatively the response of two cultivars of medical pan> class="Species">cannabis to increasing concentrations of K inputs. The following hypotheses were testes: 1. K supply induces developmental and morphological changes in medical cannabis. 2. The K induced changes in growth and development are associated with changes to the plant ionome and the physiological state of the tissue. To test these hypotheses, we studied effects of K supply ranging from 15 to 240 ppm K in the irrigation solution on mineral uptake and accumulation in the plant organs (leaves, stems, and roots), morphological, and physiological characteristics. The concentration range evaluated was selected to encompass deficiency–sufficiency- and oversupply concentrations, based on the limited information available from growers and a preliminary study conducted by us. Apart from the contribution to understanding of cannabis physiology, the information obtained is instrumental also for development of optimal fertigation regime for excelled quantity and quality product for the agro-hi-tech medical industry.

Materials and Methods

Plant Material and Growing Conditions

Two medical cannabis (pan> class="Species">C. sativa L.) cultivars (genotypes), “Royal Medic” (RM) and “Desert Queen” (DQ) (Teva Adir LTD, Israel), which are approved for commercial medical use in Israel, were used as a model system in this study. They are both of indica characteristics, and were selected to represent two distinct chemotypes: “RM” contain similar concentrations of THC and CBD (about 5%), and “DQ” is a high THC cultivar. Plants were propagated from cuttings of a single mother plant in coconut fiber plugs (Jiffy international AS, Kristiansand, Norway). Rooted cuttings were planted in 3 L plastic pots in perlite 2-1-2 (Agrekal, Habonim Israel), and the irrigation treatments were initiated following 7 days adjustment with only distilled water irrigation. The plants were then divided into five increasing treatments of K supply; 15, 60, 100, 175, and 240 ppm, and grown for 30 days under 18/6 h light/dark photoperiod using Metal Halide bulbs (400 μmol*m−2*s−1, Solis Tek Inc, Carson, California) in a controlled environment growing room. Temperatures in the growing room were 26 and 25˚C day/night, and the relative humidity were 54% and 50%, respectively. Irrigation was supplied via a 1 L h−1 discharge-regulated drippers (Netafim, Tel-aviv, Israel), 1 dripper per pot. The volume of irrigation in each irrigation pulse was 250–650 ml/pot/day, set to allow 30% of drainage. Fertilizers were supplied by fertigation, i.e., dissolved in the irrigation solution at each irrigation. The irrigation solution contained 14.82 mM N-NO3 −, 1.62 mM N-NH4 +, 1.9 mM P-PO4 −2, 2.99 mM Ca+2, 1.45 mM Mg+2, 1.04 mM Na+, 0.37 mM Cl−, 0.03 mM Fe+2, 0.02 mM Mn+2, 0.005 mM Zn+2, and increasing concentrations of K: 0.38, 1.53, 2.56, 4.48, and 6.14 mM K+. K was supplemented as K2SO4 because in preliminary experiments sulfur uptake into the medical cannabis plants was found to be affected less then accumulation of Cl or NO3 when K was supplemented as KCl or KNO3. The micronutrients were supplied chelated with EDTA, other than Fe that was chelated with EDDHSA. The experiment was arranged in a complete randomized design. All measurements were conducted for five replicated plants and results are presented as average ± standard error (S.E.).

Inorganic Mineral Analysis

For the analyses of inorganic mineral contents in the plant, the plants were destructively harvested five times throughout the experiment duration; 0, 7, 14, 21, and 29 days after the initiation of the K fertigation treatments. At each sampling event, the sectioned shoots were rinsed twice with distilled pan> class="Chemical">water and blotted dry, the leaves were carefully excised from the stem at the point of attachment to the node, and fresh and dry biomass were measured with a Precisa 40SM-200A balance (Zurich, Switzerland). Dry weights were determined following drying at 64˚C for 48 h and the dry tissue was ground to a powder.

The plant samples were analyzed for concentrations of N, P, K, Ca, Mg, S, pan> class="Chemical">Fe, Mn, Zn, Cu, Cl, and Na. Three different procedures were applied for extraction of the various inorganic mineral elements from the grounded plant tissue. For the analysis of S, Ca, Mg, Fe, Zn, Cu, and Mn, the ground tissue was digested with HNO3 (65%) and HClO4 (70%), and the elements (except S) were analyzed with an atomic absorption spectrophotometer, AAnalyst 400 AA Spectrometer (PerkinElmer, Massachusetts, USA). For the analysis of N, P, K, and Na, the dry tissue was digested with H2SO4 (98%) and H2O2 (70%–72%). Na and K were analyzed by flame photometer (410 Flame Photometer Range, Sherwood Scientific Limited, The Paddocks, UK), and N, P, and S were analyzed by an autoanalyzer (Lachat Instruments, Milwaukee, WI, USA). For the analyses of Cl, dried plant samples were extracted with a dilute acid solution containing 0.1 N HNO3. Cl was measured by potentiometric titration (PCLM3 Jenway, Bibby Scientific Ltd, T/As Jenway, Dunmow, UK). Mineral analyses of irrigation and drainage solutions were performed as described for the plant extraction and digestion solutions.

Physiological Parameters

The plants were sampled for physiological parameters analyses 31 days after the rooted n class="Chemical">cuttinpan>gs were planpan>ted inpan> the experimental pots, 24 days after the inpan>itiation of the pan> class="Chemical">fertigation treatments.

Determination of osmotic potential. For osmotic potential measurements, the youngest mature fan leaf on the main stem of the plant, located at the fourth node from the plant’s top was carefully removed, washed twice in distilled water, anpan>d blotted dry. The 3 smallest leaflets were pan> class="Chemical">cut from the leaf and inserted into a 1.7 micro-test-tube. The tube were then frozen in liquid nitrogen and stored at −20°C for further analyses. The frozen tissue was crushed inside the tubes with a glass rod, the bottom of the tubes was pin-pricked and the tubes, set inside another 1.5 ml tube, centrifuged for 5 min in a refrigerated centrifuge (Sigma Laboratory Centrifuges, Germany) at 4°C at 6,000 rpm. Fifty microliters of the fluids collected in the lower micro test tube were used for measurement of osmotic potential using a cryoscopic microosmometer Osmomat 3000 (Gonotec, Berlin, Germany) by measuring the freezing point of 50 µl of sap. Results are presented in mOsm kg−1 H2O−1. Five replicated leaves from five replicated plants for each cultivar were analyzed.

Determination of membrane leakage. Ion leakage from the leaf tissue, an indicator of membranpan>e injury under stress (Lu et al., 2008), was measured as previously described (Shoresh et al., 2011) with pan> class="Species">minor modifications. The youngest mature fan leaf on the main stem of the plant, located at the fourth node from the plant’s top was carefully removed, washed twice in distilled water and blotted dry. A 40-mm segment located at the central of the middle leaflet was used for the analysis. The sampled leaf section was rapidly placed in a 50 ml test-tube containing 30 ml of distilled water and shaken for 24 h. The electric conductivity of the soaking solution containing the leaf was measured using a conductivity meter Cyberscan CON 1500 (Eutech Instruments Europe B.V., Nijkerk, Netherlands). Then, the samples were autoclaved for 30 min to destroy cells and cause 100% leakage. The autoclaved samples were allowed to cool down at room temperature for 45 min and then shaken for an additional 1 h. The electric conductivity of the solution was measured. Ion leakage from the plant tissue was calculated as percent (%) of the electric conductivity value before autoclaving to its value post autoclaving. Results from five replicated leaves from five replicated plants for each cultivar were averaged.

Determination of chlorophyll anpan>d carotenoids content. For pan> class="Chemical">chlorophyll and carotenoid analysis, the youngest mature fan leaf on the main stem of the plant, located at the fourth node from the plant’s top was carefully removed, washed twice in distilled water, and blotted dry. Five discs, 0.6 cm in diameter, were cut from the second largest leaflet, placed in 0.8 ml 80% (v/v) ethanol, and were frozen for further analysis. After partial thawing at room temperature, the samples were heated to 100°C for 30 min. The soluble boiled extract was collected in 2 ml micro test tubes. The remaining tissue was extracted again in 0.5 ml 80% (v/v) ethanol for 15 min at 100°C and the combined extract was mixed by vortex. Next, 0.4 ml of extract was transferred to 5 ml 80% (v/v) acetone, and absorbance at 663, 646, and 470 nm was measured by Genesys 10 UV Scanning spectrophotometer (Thermo Scientific, Waltham, Massachusetts). Calculation of chlorophyll a and b and carotenoids was done according to Lichtenthaler and Welburn (1983).

Determination of relative water content. For relative pan> class="Chemical">water content analysis, the second youngest mature fan leaf on the main stem of the plant, located at the fifth node from the plant’s top was carefully removed, and weighed with a Precisa 40SM-200A balance (Zurich, Switzerland). The leaf was then placed in a 50 ml rube that was previously filled with distilled water. The tubes were placed for 24 h at room temperature and then the leaves were blotted dry and weighed again. Dry weight of the leaves was obtained following desiccation at 64˚C for 48 h. Relative water content was calculated following (Bernstein et al., 2010). The analyses was conducted for five replicated leaves from five replicated plants, for each cultivar.

Plant architecture and development. Plant height, stem diameter, and the number of nodes on the main stem were measured five times throughout the experiment duration; 0, 8, 15, 22, and 28 days after the initiation of the n class="Chemical">fertigation treatments. Planpan>t height anpan>d branpan>ch length were measured with a ruler from the base of the planpan>t to the top of the central branpan>ch. Stem diameter was measured with anpan> Electronic digital caliper YT-7201 (Signet tools international co., LTD., Shenganpan>g Distric, Taiwanpan>) at the location 5 cm from the planpan>t base. The measurements were conducted on five replicated planpan>ts per treatment, for each pan> class="Chemical">cultivar.

Photosynthesis and transpiration rate, stomatal conductance, intercellular CO 2 concentration, and water use efficiency. Net photosynthesis rate, intercellular pan> class="Chemical">CO2 concentration, transpiration rate, stomatal conductance, and water use efficiency quantification were measured on the youngest mature fan leaf on the main stem of the plant, located at the fourth node from the plant’s top, with a Licor 6400 XT system (LI-COR, Lincoln, NE, USA). The leaves were exposed to a light intensity of 400 PPFD and a CO2 concentration of 400 ppm while leaf temperature was kept at 25°C and relative humidity was between 40% and 55%. Water use efficiency was calculated from the net photosynthesis and stomatal conductance results. The measurements were conducted on five replicated plants per treatment, for each cultivar.

Plant Biomass

Distribution of plant biomass between the various vegetative shoot organs, i.e., leaves and stems, was evaluate by destructive sampling five times throughout the experiment duration; 0, 7, 14, 21, and 29 days after the initiation of the fertigation treatments. At the last destructive sampling, on day 29, the roots were gently rinsed three times in distilled pan> class="Chemical">water and blotted dry, and fresh weights were measured. Dry weights were measured following desiccation in 64˚C for 48 h. Potassium use efficiency (KUE) was calculated as the total dry weight of the plant on day 29 divided by the amount (g/plant) of K supplied to the plant throughout the experiment duration. Presented results are averages ± SE for five replicated plants.

Statistical Analyses

The data were subjected to two-way ANOVA followed by Tukey’s HSD test. Comparison of relevant means was conducted using Fisher’s least significant difn class="Chemical">ference (LSD) at 5% level of significanpan>ce. The anpan>alysis was performed with the Jump software (Jump package, version 9 (SAS 2015, Cary, NC, USA).

Results

Plant Growth and Development

Shoot and root growth of “RM” plants increased with the elevation of K supply. Biomass of leaves, stems, and roots increasing with the increase in K concentration, up to 175 ppm K, and decreasing with further increase in concentration ( ) hence presenting 15 ppm as a sub-optimal concentration and 175 ppm as an optimal K concentration. “DQ” plants are less sensitive to K application, and the shoot and roots demonstrated an unusual yet similar growth response to increasing concentrations of K. The biomass of all three organs was lowest under 15 ppm K supply (a deficient supply), unchanged at the range of 60–175 ppm K, and surprisingly significantly increased with further increase in concentration to 240 ppm K ( ). Response patterns of dry biomass accumulations to K supply were similar to the fresh biomass response (data not shown). For both pan> class="Chemical">cultivars, KUE was similar for the 15 and 60 ppm K treatments, and decreased with further increase in K supply ( ).

Figure 1

Effect of K nutrition on shoot and root biomass in cannabis plants. Fresh weights of leaves (A), stem (B), and roots (C), and K use efficiency (KUE) (D) of two medical cannabis cultivars, Royal Medic (RM) and Desert Queen (DQ). Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results K is potassium, G is genotype, and K'G represents the interaction between K and G.

Effect of K nutrition on shoot anpan>d root biomass in pan> class="Species">cannabis plants. Fresh weights of leaves (A), stem (B), and roots (C), and K use efficiency (KUE) (D) of two medical cannabis cultivars, Royal Medic (RM) and Desert Queen (DQ). Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results K is potassium, G is genotype, and K'G represents the interaction between K and G.

All the morphological parameters measured: plant height, number of nodes on the main stem, stem diameter, and main stem elongation rate, showed a developmental delay in the plants that received 15 ppm K, compared with plants that received higher amounts of K ( ) presenting as well 15 ppm as a sub-optimal concentration for both varieties. The two varieties demonstrated a similar response to the elevation of K supply. The average rate of plant elongation in the 15 ppm K treatment was 33% lower in RM and 28% lower in DQ (8.17 and 7.82 mm*day−1, respectively), compared to the remaining treatments ( ). The average rate of stem thickening in the 15 ppm K treatment was 61% lower in RM and 70% lower in DQ (85 and 56 μm*day−1, respectively), compared to the higher K supply treatments ( ). The average rate of node formation on the main stem was only 13% lower in RM and 3% lower in DQ in the 15 ppm K treatment (0.37 and 0.41 nodes*day−1, respectively), compared to the remaining treatments ( ). These developmental delays under the restricted K supply of 15 ppm K had a considerable efn class="Chemical">fect on planpan>t growth, resulting in shorter planpan>ts ( ). The growth restriction in RM anpan>d the stimulation in DQ under high K supply (240 ppm), which is statistically significanpan>t for leaves anpan>d root biomass of the entire planpan>t ( ), is not significanpan>t for most of the morphological anpan>d growth parameters of the main stem ( ). This may present lower sensitivity of the main stem compared to the side branpan>ches to over-supply of K, anpan>d/or present that side branpan>ch development is more sensitive to high supply of K thanpan> initiation of new branpan>ches (new internodes).

Figure 2

Effect of K nutrition on development of cannabis plants. Plant height (A, B), number of nodes on the main stem (C, D), stem diameter (E, F), and main stem elongation rate (G, H) of two medical cannabis cultivars, RM and DQ. Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results, K Time represents the interaction between K and time.

Effect of K nutrition on development of pan> class="Species">cannabis plants. Plant height (A, B), number of nodes on the main stem (C, D), stem diameter (E, F), and main stem elongation rate (G, H) of two medical cannabis cultivars, RM and DQ. Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results, K Time represents the interaction between K and time.

Macronutrient Concentration

The concentration of K supplied caused a variety of responses related to the ability of the plants to take up and accumulate nutrients, demonstrating organpan> but not pan> class="Chemical">cultivar specificity, with both cultivars revealing similar responses. In both cultivars, in all plant parts, K concentration increased significantly with increased K supply ( ). The concentration of the two major cation nutrients, Ca and Mg, tended to decrease with increased K supply, demonstrating competition for uptake ( ). While K concentration was highest in the stem, Ca and Mg concentration was very low in the stem, and higher in the leaves. Nitrogen and P concentrations in the plant organs were not affected by the level of K supplied, except in the 15 ppm K treatment. Furthermore, the extent of accumulation differed between organs and was higher in the leaves > roots > stem for N, and roots > leaves > stem for P ( ). Sulfur concentration in the plant organs was low, with preferred accumulation in the root and highest accumulation under 60–175 ppm K supply ( ).

Figure 3

Effect of K supply on macronutrient concentrations in leaves, stem and roots of two medical cannabis cultivars, RM and DQ. K (A), N (B), P (C), S (D), Ca (E), and Mg (F). Presented data are averages ± SE (n = 5). Asterisk above the bars represent significant differences between treatments by Tukey HSD test at α = 0.05.

Effect of K supply on macronutrient concentrations inpan> leaves, stem anpan>d roots of two medical pan> class="Species">cannabis cultivars, RM and DQ. K (A), N (B), P (C), S (D), Ca (E), and Mg (F). Presented data are averages ± SE (n = 5). Asterisk above the bars represent significant differences between treatments by Tukey HSD test at α = 0.05.

Micronutrients and Na

Unlike the considerable effects on macronutrients, K supply had but little efpan> class="Chemical">fect on micronutrient accumulation in the shoot. Micronutrient concentration in the leaves and stems were generally unaffected by K supply, except Mn which built up to higher concentrations at the 15 ppm K treatment ( ). In the roots, concentrations of Zn, Fe, and Mn usually decreased with the increase in K supply; Cu and Na concentration followed maximum curves with highest accumulation under 100–175 ppm K supply ( ); and Cl concentrations which were very low were unaffected by K supply ( ). The concentrations of all micronutrients were higher in the roots compared to the shoot. In the shoot, the concentration of Na, Cl, and Cu were generally higher in the stem, compared to the leaves, while an opposite trend was found for the remaining micronutrients ( ). The two tested cultivars responded similarly to K fertigation in terms of micronutrient and macronutrient accumulation.

Figure 4

Effect of K supply on micronutrient and Na concentrations in leaves, stem, and roots of two medical cannabis cultivars, RM and DQ. Zn (A), Mn (B), Fe (C), Cu (D), Na (E), and Cl (F). Presented data are averages ± SE (n = 5). Asterisks represent significant differences between organs at each K level, by Tukey HSD test at α = 0.05.

Effect of K supply on micronutrient anpan>d Na concentrations inpan> leaves, stem, anpan>d roots of two medical pan> class="Species">cannabis cultivars, RM and DQ. Zn (A), Mn (B), Fe (C), Cu (D), Na (E), and Cl (F). Presented data are averages ± SE (n = 5). Asterisks represent significant differences between organs at each K level, by Tukey HSD test at α = 0.05.

Gas Exchange and Photosynthesis

The sensitivity of net photosynthetic rate and gas exchange parameters to K supply differed in the two pan> class="Chemical">cultivars. In DQ, net photosynthetic rate increased with the increase in K supply, up to a maximum at the 100 ppm K treatment, and declined with further increase in K ( ) presenting 100 ppm as the optimal concentration for photosynthesis in this cultivar. In contrast, the intercellular CO2 concentration was higher at the 15 ppm K treatment than in all other treatments, and the transpiration rate and stomatal conductance was not affected by K supply ( ). RM responded differently, with transpiration rate and stomatal conductance lower at the 15 ppm K treatment compared to all other treatments, and net photosynthetic rate and intercellular CO2 concentration were not affected by the K supply treatments ( ).

Figure 5

Effect of K supply on gas exchange in cannabis leaves. Net photosynthesis rate (A), transpiration rate (B), stomatal conductance (C), and intercellular CO2 concentration (D) for two medical cannabis cultivars, RM and DQ. Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results K'G represents the interaction between K and genotype.

Effect of K supply on gas exchanpan>ge in pan> class="Species">cannabis leaves. Net photosynthesis rate (A), transpiration rate (B), stomatal conductance (C), and intercellular CO2 concentration (D) for two medical cannabis cultivars, RM and DQ. Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results K'G represents the interaction between K and genotype.

Water Relations and Photosynthetic Pigments

The effect of K supply on the percentage of dry weight (%DW) in the planpan>t tissues difpan> class="Chemical">fered between the two varieties. In DQ, %DW of the leaves and stems was higher at the 15 ppm K treatment compared to all other treatments ( ), while in RM, %DW of the stem was not affected by the treatments but in the leaves it was higher under 15 and 60 ppm K supply, compared to other K supply treatments. Osmotic potential and relative water content of the leaf were lower under 15 ppm K supply, compared to all other treatments, in both varieties ( ). In DQ the osmotic potential increased with additional K supply throughout the concentration range tested, while in RM it stabilized under a lower K supply (60 ppm K; ). Membrane leakage analyses demonstrated a higher sensitivity of RM tissues to K deficiencies (15 ppm K) compare to DQ, and a lack of response in both varieties to higher K supply (Fig 6E). In RM water use efficiency was not affected by K supply, while in DQ it was significantly lower in the 15 ppm K treatment, compared to all other higher K treatments ( ).

Figure 6

Physiological characteristics of medical cannabis plants. %DW of leaves (A) and stems (B), relative water content (RWC) (C), osmotic potential (D), membrane leakage (E) and intrinsic water use efficiency (WUEi) (F), of two medical cannabis cultivars, RM and DQ. Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. In the ANOVA results K'G represents the interaction between K and genotype.

Physiological characteristics of medical cannabis planpan>ts. %DW of leaves (A) anpan>d stems (B), relative pan> class="Chemical">water content (RWC) (C), osmotic potential (D), membrane leakage (E) and intrinsic water use efficiency (WUEi) (F), of two medical cannabis cultivars, RM and DQ. Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. In the ANOVA results K'G represents the interaction between K and genotype.

Concentrations of chlorophyll a anpan>d carotenoids inpan> the foliage inpan>creased with elevation of K supply ( ), while pan> class="Chemical">chlorophyll b was significantly lower at the 15 ppm K treatment, compared to the remaining treatments, in both varieties ( ).

Figure 7

Effect of K application on the concentration of photosynthetic pigments in two medical cannabis cultivars, RM and DQ. Chlorophyll a (A), chlorophyll b (B), and carotenoids (C). Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results K’G represents the interaction between K and genotype.

Effect of K application on the concentration of photosynthetic pigments in two medical pan> class="Species">cannabis cultivars, RM and DQ. Chlorophyll a (A), chlorophyll b (B), and carotenoids (C). Presented data are averages ± SE (n = 5). Results of two-way ANOVA indicated as **P < 0.05, F-test; NS, not significant P > 0.05, F-test. The bars represent the LSD between means at P ≤ 0.05. In the ANOVA results K’G represents the interaction between K and genotype.

Irrigation and Leachate Solutions

Routine chemical analyses of the n class="Chemical">fertigation (irrigation) solution demonstrated precise regulation of the treatment solutions. The concentration of K ( ) for both varieties was steady throughout the experiment duration inpan> all five K treatments, anpan>d closely followed the designated treatments concentrations ( ). The concentration of K inpan> the leachate solutions positively correlated with K supply inpan> both varieties ( ). Inpan> the high K treatments, 175 anpan>d 240 ppm, the concentration inpan> the leachate inpan>creased over time, suggestinpan>g over supply ( ).

Figure 8

Concentrations of K in the irrigation solutions (A), and leachates (B), throughout the experiment duration. RM, filled symbol; DQ, empty symbol.

Plant Visual Characteristics

The visual appearance of the plants was affected similarly by the K treatments in the two pan> class="Chemical">cultivars, and reflected the morphological, chemical and physiological characteristics evaluated in the study ( – ). In the 15 ppm K treatment, the leaves were smaller, had fewer leaflets (in DQ), and showed advanced chlorosis typical of K deficiency ( ). They were also developmentally inhibited, compared to the remaining K treatments that had larger leaves and darker green color ( ). The chlorosis and restricted growth of the 15 ppm K treatment affected overall shoot growth, resulting in a smaller and thinner plants, compared to plants of all other treatments ( ).

Figure 9

Visual appearance of leaves of two medical cannabis cultivars, RM (top row) and DQ (bottom row), which developed on plants receiving increasing K supply. From left to right: 15, 60, 100, 175, and 240 ppm K. Images of the youngest fully developed leaf on the main stem, taken 26 days after the initiation of the fertigation regime.

Figure 10

Plants of two medical cannabis cultivars, RM (top row) and DQ (bottom row), supplied with (from left to right): 15, 60, 100, 175, and 240 ppm K. Photographed 26 days after the initiation of the fertigation regime.

Visual appearance of leaves of two medical cannabis pan> class="Chemical">cultivars, RM (top row) and DQ (bottom row), which developed on plants receiving increasing K supply. From left to right: 15, 60, 100, 175, and 240 ppm K. Images of the youngest fully developed leaf on the main stem, taken 26 days after the initiation of the fertigation regime.

Plants of two medical cannabis pan> class="Chemical">cultivars, RM (top row) and DQ (bottom row), supplied with (from left to right): 15, 60, 100, 175, and 240 ppm K. Photographed 26 days after the initiation of the fertigation regime.

Discussion

Mineral nutrition is one of the major factors affecting planpan>t growth, development, anpan>d function. Optimal concentrations of mineral nutrients in the planpan>t tissues anpan>d in the root solution vary for individual nutrients, anpan>d may difpan> class="Chemical">fer between and within species. Furthermore, due to effects of specific nutrients on biochemical, physiological, and molecular processes, nutrition may need to be adjusted for directing a required metabolic process or a preferred developmental scheme, such as a vegetative or reproductive development. In the present study we report changes to the ionome and to plant development and function resulting from the intensity of K supply, comparatively for two cultivars of medical cannabis.

Genetic variability within plant species results in genotypes with different developmental, physiological, anpan>d biochemical traits. Variability in response of roots anpan>d shoots of planpan>t genotypes to growing conditions is well dopan> class="Chemical">cumented for numerous plant species (Antonio et al., 2019; Martins et al., 2016; Queiroz et al., 2019). For C. sativa, drug-type strains are known to vary in morphological and chemical characteristics, but responses to cultivation and environmental conditions are not known. In the present study we report that while the response to low, sub-optimal supply of K was similar for the genotypes studied, developmental differences between genotypes emerged under higher K concentration.

In both genotypes, biomass deposition was affected by K inputs but the response varied between the genotypes. In RM, growth positively responded to increase in K supply up to 175 ppm K ( ), as canpan> be seen from the increase in leaves anpan>d root biomass ( ), stem diameter ( ), anpan>d internode elongation rate ( ), but decreased with further increase to 240 ppm K, rendering 175 ppm as the optimum concentration for this genotype. While DQ sufpan> class="Chemical">fered as well from insufficient K supply under the 15 ppm K treatment, increasing K supply in the range of 60–175 ppm K did not affect plant development. Surprisingly, further increase in K supply, to the level of 240 ppm K, stimulated rather than restricted growth and development of this genotype.

The increase in biomass production with the increase in K at the lower concentration range in both genotypes represent mitigation of restricted supply, satisfying demands for facilitating optimal growth. The concentration range at which plant performance improves with increased supply is defined a “deficiency range,” which is well documented to vary between species (Marchner, 2012) anpan>d in some cases also between genotypes. The lower requirement of K supply for optimal development in RM may be the outcome of the smaller shoot anpan>d root morphology in this genotype, compared to the larger vegetative planpan>t body of DQ. RM might have therefore been less prone to deficiency. pan> class="Chemical">Potassium plays an active part in the physiological regulation of crop processes (Wang and Wu, 2017), facilitating functions such as ion uptake and transport, protein synthesis, stomatal regulation, enzymatic activity, and regulation of gene expression. The observed growth stimulation with increased K supply at the low concentration range in the two cannabis genotypes can result from effects of the deficiency on individual factors or combination of mechanisms as was demonstrated for numerous plant species. In tomatoes for example, the suppression of stem expansion under K deficiency, which was apparent in both medical cannabis genotypes as well, was concluded to result from a reduction in water supply to the growing stem by K-deficiency induced reduction in aquaporins and K-channels activity (Fromm, 2010; Kanai et al., 2011). Similar to our results, addition of K led to development of thicker stems and higher shoot biomass.

Potassium is a major player in regulation of planpan>t pan> class="Chemical">water relations (Mengel and Arneke, 1982), osmo-regulation (Lauchli and Pfluger, 1978), and stomatal opening (Fischer, 1968, Fischer, 1971; Outlaw, 1983) and hence plant development (Prajapati and Modi, 2012; Wang et al., 2013). The source of the different development response of the two medical cannabis genotypes to K supply, roots in the impact of K availability on the physiological status, mostly water relations, of the tissue. Although in the RM plants the rate of photosynthesis was not affected by the K treatments, slower transpiration rate with reduced stomatal conductance were induced by K-deficiency (15 ppm K) ( ) resulting in restricted growth but higher water use efficiency. Environmental conditions are known to affect carbon fixation (Rodrigues et al., 2016). The change in water relations in the 15 ppm K treatment was apparent also from the lower osmotic potential and relative water content in the leaf tissue, and higher %DW, i.e., lower percentage of water in the tissue. The combined effects of K deficiency and altered water status caused tissue damage, as was apparent from the higher membrane leakage ( ), and resulted in restriction of development. The relative water content and osmotic potential were lower in the 15 ppm K treatment in DQ as well ( ), but growth was restricted by a different mechanism. That is, in this genotype, transpiration rate and stomatal conductance were not impaired under the deficient K supply (15 ppm K), but net photosynthesis rate was reduced, resulting in higher CO2 concentration in the intercellular space, due to a decrease in the consumption of CO2 as a substrate for photosynthesis ( ). Membrane leakage, i.e., tissue stress was not affected, but the tissue did suffer from water shortage, resulting in lower %DW in the evaporating tissues of the leaves. Despite the variation in net photosynthesis rate and gas exchange parameters between the genotypes, the values obtained are within the range obtained in former measures conducted for indoor grown medical cannabis (Chandra et al., 2011).

In both genotypes, K supply had a significant effect on the photosynthetic pigments in the tissue; positively correlating with pan> class="Chemical">chlorophyll a and carotenoids contents ( ). Moreover, chlorophyll b as well was affected by K supply but K demands for optimal accumulation were satisfied already under 60 ppm K inputs ( ). As K is not a constituent of these molecules, the impact it has on their biosynthesis is indirect, and we suggest that the decrease in concentration of the pigments under 15 ppm K results from inhibition of N availability in the leaf cells, N being a central constituent of these molecules (Taiz and Zeiger, 2010). Furthermore, K, as a cofactor, is involved in the activity of a large number of vital enzymes (Evans and Sorger, 1966; Prajapati and Modi, 2012), that affect also metabolism and catabolism of plant pigments. Reduced chlorophyll concentration induced leaf chlorosis, and the impaired water relations and reduced availability of K resulted in a morphological response with development of smaller leaves and leaflets and fewer leaflets.

Excess of K supply induced contrasting effects on the two genotypes. The difpan> class="Chemical">ference in plant biomass between the genotypes in the 240 ppm K treatment results from differential effect of the high K supply on development of the side branches. In RM the length of the side branches was inhibited by 21% as K supply increased from 175 to 240 ppm, while in DQ the side branches length increased by 14% (data not shown). K effect on side branch development is known also for other plants (Madgwick, 2011) and is likely to have a large effect on total biomass accumulation even without an effect on branching. The elevated biomass deposition in DQ under high K supply is the outcome of an increase leaf biomass of the side branches as well as in stem diameter (data not shown), resulting in bushier bigger plants. K is known to affect stem diameter and fiber yield and quality in other plant species as well (Derrick et al., 2013).

Accumulation of nutrients above the optimal level required for planpan>t growth anpan>d function i.e., “luxury consumption,” is a process described for numerous planpan>t species, mostly related to K uptake (Bartholomew anpan>d Janpan>ssen, 1929). “Luxury consumption” of K usually does not afpan> class="Chemical">fect growth and development of plants, but it was previously reported for numerous species including cotton, that excessive K fertilizer reduces plant biomass (Chen et al., 2017). In the case of the two medical cannabis genotypes, the mechanisms for the contrasting developmental response are not clear and require further study. Specifically, the higher concentration of K in stems of DQ compared to RM under 240 ppm K, (higher by 20%) is unlikely the cause of the observed growth stimulation in this genotype, because the increased in K concentration in the shoot under 60–175 ppm K supply did not affect plant development, categorizing the increased K uptake in this range as luxury consumption. In RM as well, changes in K concentrations in the shoot do not present a direct cause for growth restriction under 240 ppm K compared to lower supply rates since K concentrations were not affected considerably ( ). No apparent changes in any other macro or micronutrients ( , ), carbon fixation and gas exchange parameters ( ) or water relations parameters ( ) were identified as potential causes for the developmental response and genotypic differences under 240 ppm K.

Interestingly, the distribution of K between plant organs differed for the two genotypes—while in RM concentrations in the roots anpan>d the shoot organpan>s were similar, higher levels of K tranpan>sport to the shoot were apparent in DQ. Consequently, supplied levels of K that restricted growth in RM, stimulated growth in DQ ( ). This suggests difpan> class="Chemical">ferential sensitivity of the cells from both genotypes to K, or more likely, involvement of a secondary-induced factor in the observed growth restriction. The variability in the response of the two genotypes to K supply, demonstrated by an optimum response curve in RM, and the three distinct response phases in DQ ( ), reveals as well genetic differences within the C. sativa species to mineral nutrition.

Nutrient use efficiency, e.g., the amount of biomass produced per K unit supplied, is a valued tool for evaluating the ability of the plant to utilize environmental inputs into yield or biomass production (Yasuor et al., 2013; Omondi et al., 2018). We report here for medical cannabis, a large decrease in KUE with the increase in K supply ( ). This points again at “luxury consumption” anpan>d suggests that, maximum efficiency is obtained under low K concentrations. For a high cash-crop like medical pan> class="Species">cannabis, it is likely that the marginal addition to the biomass at the vegetative stage, if proven to support better plant architecture for the reproductive phase, will be more significant than the fertilization expenses. Plant genotypes are known to vary also in nutrient use efficiency. In the present study KUE was higher in DQ compared to RM.

Mineral Nutrients

The variability in the distribution patterns of the various macronutrient in the plant body result from uptake and translocation mechanisms. The increased concentration of K in the root solution increase overall cation concentration in the solution and hence competition for uptake of the positively charged cations. Reduction in Ca concentration in the plants under high K supply ( ), points at competition for uptake. Ca/K competition for root uptake (Johansen et al., 1968; Maas, 1969) and a resulting reduced Ca concentration in the shoot was documented for a variety of planpan>t species (Fageria, 2001). Moreover, in-planpan>ta tranpan>sport of these two ions is also competitive since high K concentrations decreased the amount of Ca arriving to the foliage, as already seen before (Overstreet et al., 1952; Bar-Tal anpan>d Pressmanpan>, 1996). pan> class="Chemical">Mg is another cation which was identified to compete with K for root uptake and translocation in the plant (Heenana and Campbell, 1981), but in cannabis K has a smaller influence on its translocation since Mg concentration began to decrease only in the 60 ppm K treatments ( ). The uptake and distribution of the two other major macronutrients, N and P, is not sensitive to K supply ( ), probably since their uptake into the root cells is as anions, in a mechanism less affected by cation concentrations and uptake (most of the N in the present study was supplied as NO3 -) (White, 2012).

Concentration of K in the leachate solution is another indicator of planpan>t requirement anpan>d uptake. In the present study, K concentration in the leachate was higher thanpan> in the irrigation solution only at the 175 anpan>d 240 ppm K treatments ( ), pan> class="Species">indicating that K supply under these treatments exceeded plant uptake. Nutrient concentration in the leachate is an integral result of water and mineral uptake by the plants. When water is taken up to a greater extent than a mineral, its concentration in the leachate will exceed the concentration in the irrigation solution. The concentration of K in the leachates of the three lower K treatments was similar to the concentration in the irrigation solution, demonstrating similar uptake rates of K and water. Under 175–240 ppm K application, the higher concentration of K in the leachate compared to the irrigation solution demonstrate that the rate of water uptake was higher than for K, resulting in an increase in K. This suggests that 175 ppm K is higher than the plant requirement.

Micronutrient uptake is a limiting growth factor for foliage and shoot development in many plant species under various growing conditions (Baszyński et al., 1978; Ohki et al., 1980; Clark, 1982; Webb and Loneragan, 1988; Yu and Rengel, 1999). No information is currently available about micronutrient requirements or efpan> class="Chemical">fects on medical cannabis, and our results present initial understanding. Under the cultivation conditions and rate of nutrient supply at the present study, the two cannabis cultivars examined did not show any signs of micronutrient deficiencies, suggesting sufficient supply ( ). Surprisingly, most micronutrients and the beneficial element Na, did not translocate to the shoot but tended to accumulate in the root. Zinc, Mn, Fe, Cu, and Cl as well as Na concentrations were all higher in the root compared to the shoot, suggesting a compartmentation strategy for temporary storage, or for prevention of access concentrations at the shoot tissues. Results of the comparative analyses point at competitive uptake between K and Mn, Zn and Fe, since concentrations of the latter decreased with increased K supply. Na uptake was less affected by this competition ( ), in accord with its known strong competition abilities with K for root uptake (Amtmann and Leigh, 2010), or due to its very low concentration in the fertigation solution, which was prepared with distilled water. The uptake of another micronutrient, Cl, was not affected by cultivar or K supply ( ), probably because its concentrations was low and within the range accepted as optimal to most plants (Parker et al., 1983; Marchner, 2012).

Some information for micronutrient uptake by C. sativa L. is available for industrial, fiber-type, pan> class="Species">Hemp. It is used for phytoremediation, due to its known ability to absorb heavy metals from the soil and tolerate high accumulation in its tissues (Linger et al., 2002; Ahmad et al., 2016). Contradictory to our expectation for similar high uptake rates of micronutrient cations into the medical cannabis plants, these Hemp properties were not found in the medical type varieties. The difference between the Hemp and medical cannabis response could result from several factors; 1. The distinct plant genetics of Hemp may express enhanced uptake mechanisms for heavy metals; 2. Differences in chemical and physical properties (such as pH, chelate diversion, and cation exchange capacity) between the rhizosphere of the soilless cultivated medical cannabis and the soil-grown Hemp (Landi, 1997; Aubin et al., 2015), may have affected root nutrient availability and uptake; 3. The growth period of the medical cannabis cultivars was short compared to a standard growth period of Hemp (Van der Werf and Van den Berg, 1995; Linger et al., 2002; Vera et al., 2010), resulting in overall lower amounts of metal accumulation in the medical cannabis cultivars.

Author Contributions

NB planned the experiments. AS carried out the experiments. NB and AS wrote the manuscript. MS constructed the n class="Chemical">fertilizers compositions.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.