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

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 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, 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 cuttings were planted in the experimental pots, 24 days after the initiation of the 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, and blotted dry. The 3 smallest leaflets were 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 membrane injury under stress (Lu et al., 2008), was measured as previously described (Shoresh et al., 2011) with 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 and carotenoids content. For 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 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 fertigation treatments. Plant height and branch length were measured with a ruler from the base of the plant to the top of the central branch. Stem diameter was measured with an Electronic digital caliper YT-7201 (Signet tools international co., LTD., Shengang Distric, Taiwan) at the location 5 cm from the plant base. The measurements were conducted on five replicated plants per treatment, for each cultivar.

Photosynthesis and transpiration rate, stomatal conductance, intercellular CO ₂ concentration, and water use efficiency. Net photosynthesis rate, intercellular 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 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.

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 organ but not cultivar specificity, with both cultivars revealing similar responses. In both cultivars, in all plant parts, K concentration increased significantly with increased K supply (Figure 3A). The concentration of the two major cation nutrients, Ca and Mg, tended to decrease with increased K supply, demonstrating competition for uptake (Figures 3E, F). 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 (Figures 3B, C). Sulfur concentration in the plant organs was low, with preferred accumulation in the root and highest accumulation under 60–175 ppm K supply (Figure 3D).

Micronutrients and Na

Unlike the considerable effects on macronutrients, K supply had but little effect 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 (Figures 4A–F). 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 (Figures 4A–E); and Cl concentrations which were very low were unaffected by K supply (Figure 4F). 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 (Figures 4A–F). The two tested cultivars responded similarly to K fertigation in terms of micronutrient and macronutrient accumulation.

Gas Exchange and Photosynthesis

The sensitivity of net photosynthetic rate and gas exchange parameters to K supply differed in the two 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 (Figure 5A) 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 (Figures 5B–D). 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 (Figures 5A–D).

Water Relations and Photosynthetic Pigments

The effect of K supply on the percentage of dry weight (%DW) in the plant tissues differed 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 (Figures 6A, B), 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 (Figures 6C, D). 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; Figure 6D). 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 6F).

Concentrations of chlorophyll a and carotenoids in the foliage increased with elevation of K supply (Figures 7A, C), while chlorophyll b was significantly lower at the 15 ppm K treatment, compared to the remaining treatments, in both varieties (Figure 7B).

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.

Irrigation and Leachate Solutions

Routine chemical analyses of the fertigation (irrigation) solution demonstrated precise regulation of the treatment solutions. The concentration of K (Figure 8A) for both varieties was steady throughout the experiment duration in all five K treatments, and closely followed the designated treatments concentrations (Figure 8A). The concentration of K in the leachate solutions positively correlated with K supply in both varieties (Figures 8B). In the high K treatments, 175 and 240 ppm, the concentration in the leachate increased over time, suggesting over supply (Figure 8B).

Plant Growth and Development

Genetic variability within plant species results in genotypes with different developmental, physiological, and biochemical traits. Variability in response of roots and shoots of plant genotypes to growing conditions is well documented 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 (Figures 1A–C), as can be seen from the increase in leaves and root biomass (Figures 1A, C), stem diameter (Figure 2E), and internode elongation rate (Figures 2E, G), but decreased with further increase to 240 ppm K, rendering 175 ppm as the optimum concentration for this genotype. While DQ suffered 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) and 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 and root morphology in this genotype, compared to the larger vegetative plant body of DQ. RM might have therefore been less prone to deficiency. 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 plant 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) (Figure 5) 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 (Figure 6E), 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 (Figure 6), 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 (Figure 5). 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 chlorophyll a and carotenoids contents (Figures 7A, C). Moreover, chlorophyll b as well was affected by K supply but K demands for optimal accumulation were satisfied already under 60 ppm K inputs (Figure 7B). 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 difference 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 plant growth and function i.e., “luxury consumption,” is a process described for numerous plant species, mostly related to K uptake (Bartholomew and Janssen, 1929). “Luxury consumption” of K usually does not affect 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 (Figure 3). No apparent changes in any other macro or micronutrients (Figures 3, ​,4 ),4), carbon fixation and gas exchange parameters (Figure 5) or water relations parameters (Figure 6) 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 and the shoot organs were similar, higher levels of K transport to the shoot were apparent in DQ. Consequently, supplied levels of K that restricted growth in RM, stimulated growth in DQ (Figure 3A). This suggests differential 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 (Figures 1A–C), 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 (Figure 1D). This points again at “luxury consumption” and suggests that, maximum efficiency is obtained under low K concentrations. For a high cash-crop like medical 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.

The project was funded by the Chief Scientist Fund of the Ministry of Agriculture in Israel, Project No. 20-03-0018. We thank Bezalel Grushka for technical advice and assistance, and Geki Shoef and Ayana Neta for technical assistance. We are grateful for the cooperation of Gerry Kolin from Teva Adir, LTD, a certified commercial cultivation farm in Israel.