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INTRODUCTION
Cannabis sativa (L.) is a species native to Eurasia where it was domesticated (Small, 2018); it is used to obtain fiber, seeds and phytocannabinoids for medicinal and recreational purposes (Busta et al., 2022). In this sense, Ascrizzi et al. (2019), considered it with the greatest potential for use due to the cannabinoids present and also because of its low environmental impact, and its resilience to pests and diseases, making its organic production possible. This has allowed its distribution worldwide over the past two millennia, adapting to diverse environments and selecting diverse morphotypes in response to environmental conditions (Small, 2015; Naim-Feil et al., 2021). After being stigmatized by the UN as dangerous in 1961 (Janatová et al., 2018), this species has now been legalized in many countries due to legislative changes that have decriminalized its consumption and regulated the production of plant derivatives for therapeutic and, more restrictively, recreational purposes (Dufresnes et al., 2017).
The Colombian state through different laws and decrees promulgated since 2016, have supported the policy of using cannabis as a plant species for medicinal purposes, under the fulfillment of parameters established in the said regulations and with this, has ratified its willingness to place the medical cannabis sector in a priority and strategic way for the development of the country, within the framework of legality, entrepreneurship and equity. Since it is presumed that by 2024 the medical cannabis market will be worth 62.000 million dollars, of which Latin America will only take advantage of 9,1% (PINE, 2020); a lot of companies have become interested in venturing into this field and taking advantage of the market potential. However, the genetic improvement of this species was limited due to legal restrictions and today high-yield genotypes of phytocannabinoids and other secondary metabolites are required to enter the market.
The genus Cannabis, has a number of secondary metabolites, called cannabinoids, terpenoids, flavonoids, steroids, alkaloids, lignans, etc (Kojoma et al., 2006), which are accumulated in the form of carboxylic acid in the glandular trichomes of female flowers (Potter, 2014) such as delta-9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), which by decarboxylation are transformed into tetrahydrocannabinol (THC) and cannabidiol (CBD) according to Welling et al. (2016). The most well-known and specific chemical group of secondary cannabis metabolites includes cannabinoids, especially psychoactive Δ9-THC, for which Canada and the European Union countries have set a limit of 0.3 and 0.2%, respectively, and for their CBD modifier of 1.0% (Small, 2018). CBD is a non-psychoactive isomer with anti-inflammatory, anticonvulsant, analgesic, anticancer, and antioxidant properties (Hadener et al., 2019).
Piluzza et al. (2013) evaluated the genetic diversity of domesticated and wild cultivars from different countries, through the molecular analysis of variance, reporting the existence of genetic divergences between the accessions in 74 and 26% within the accessions, and grouped the accessions into eight groups. Similarly, García-Tejero et al. (2020) evaluated five varieties under macro tunnel conditions, reporting significant differences in biomass production, cannabidiol (CBD) and cannabigerol (CBG) content.
Through decree 631 of 2018 (Minsalud, 2018), the Colombian government created the legal figure of 'seed source' and determined before December 31, 2018, the registration and legalization before the Instituto Colombiano Agropecuario (ICA), of the variety of seeds that already existed in the country, intended for the planting of cannabis. For this reason, it is essential to conduct agronomic evaluation tests of cultivars in the different subregions where it is intended for planting, to then register in the National Registry of Cultivars (Registro Nacional de Cultivares), and be able to produce and market.
The objective of this work was to carry out the agronomic and phytocannabinoid content evaluation of cultivars under the conditions of northern Colombia.
MATERIALS AND METHODS
Location
The study was conducted at the La Esperanza farm, located in the municipality of Pueblo Bello-Cesar, in the Caribbean natural region of Colombia, 10°41' N, 73°52' W and height of 1,044 m a.s.l. The evaluation of the genotypes was made under greenhouse conditions with a polycarbonate cover and anti-aphid mesh, average temperature of 22.8°C, minimum of 16.1°C and maximum of 33.6°C; average relative humidity of 72%, minimum of 53% and maximum of 84%.
Genotypes
We evaluated 10 clones (Tab. 1) obtained from pre-existing seed source in the Colombian territory. From the mother plants of each clone, the 13 cm cuttings were extracted, necessary for the establishment of a population of 60 for each clone.
Table 1. Origin and name of the genotypes used in the research.
| Genotype | Origin | Name | Species |
|---|---|---|---|
| 1 | Magdalena | Mountain tradition | C. sativa |
| 2 | Magdalena | Old culture | C. sativa |
| 3 | Cundinamarca | Cundi Gold | C. sativa |
| 4 | Magdalena | Blondie Grl | C. sativa |
| 5 | Cauca | River cosmic1 | C. sativa |
| 6 | Cundinamarca | Ice Nilo | C. sativa |
| 7 | Antioquia | High Paisa | C. sativa |
| 8 | Cauca | Timbiquí Skunk | C. sativa |
| 9 | Magdalena | Algarrobo CBD | C. sativa |
| 10 | Atlántico | No High | C. sativa |
Experimental design
The randomized complete block design was used, with 10 treatments and three repetitions. Each experimental unit consisted of 20 plants per plot, with a density of 16 plants/m2 in the vegetative and reproductive phases, with distances between plants and rows of 14 cm.
The vegetative response variables considered in the study corresponded to: Number of leaflets (NL), height of plant in female flowering (FFPH), length of internodes of the main stem (ILMS), length of the petiole (LP), length of the central leaflet (CLL), width of the central leaflet (WCL), number of stems per plant (NSPP); the reproductive variables correspond to: Days to female flowering (DFL); days to harvest (DH); harvested stem height (HHS); weight of 1,000 seeds (W100S) and Yield of dry flowers (DFY). For the determination of tetrahydrocannabinol (THC) and cannabidiol (CBD), representative samples of one gram of flower were taken from each experimental unit. The data for the analysis of variance and Tukey's mean test for the response variables were performed using the GLM procedure of SAS version 9.2 (SAS, 2008).
Management of cuttings
The cuttings were planted in rooting trays of 72 alveoli each, properly labeled and separated by clone, treated with root-stimulating hormones, nitrogen, phosphorus, potassium, and calcium, under permanent lighting of 24 h for a period of 15 d.
Once rooted, the cuttings were transferred to the home mesh and transplanted into 6 L geotextile bags, until harvest. A substrate composed of perlite, coconut fiber, peat, composite forest material, pumice, worm casting, bat guano, soybean meal, alfalfa meal, fishbone meal, seaweed meal, green sand and beneficial mycorrhizal fungi was used: Funneliformis mosseae, Rhizophagus intraradices and Septoglomus desertícola.
Vegetative phase: An integrated management of irrigation and fertilizers, and of pests and diseases was implemented, with a photoperiod of 18 light hours, of which 12 h were of natural light and six of 200 W reflectors. The radiation intensity control was performed using 80% silver polyshade. Irrigation was done with self-compensating drippers of 4 L h-1, 1.5 L/plant, with contribution of nitrogen, phosphorus, potassium, sulfur, magnesium, boron, copper, manganese, zinc, and calcium.
Flowering phase: In this phase the photoperiod was 12:12 (light:dark), the control of radiation intensity, phytosanitary management, irrigation (2 L/plant) and fertilization were adjusted to the need of the plant. Harvesting was done by cutting one (1) inch from the neck of each plant; the total biomass of flowers was determined, and the relationships between variables were estimated. Subsequently, the proper drying and obtaining of the samples for cannabinoid analysis was carried out.
Obtaining phytocannabinoids
To obtain the THC and CBD contents, a sample of 10 plants for each experimental unit/clone were air-dried under dark conditions to a moisture content of 12%. Subsequently, they were ground to obtain a sample of 0.3 g for each experimental unit, and analyzed by gas chromatography according to the methodology of Poniatowska et al. (2022).
RESULTS AND DISCUSSION
The mean squares of the analysis of variance recorded in tables 2 and 3 highlight significant differences (P<0.01) for all the characteristics evaluated, similar results were reported by Piluzza et al. (2013) and Baldini et al. (2020), in Italy. The statistical significance of the vegetative and reproductive characteristics, show the genetic variation of the cultivars under study, whose coefficients of variation ranged between 0.78% for W100S and 14.79% in DFY, being lower than those reported by Petit et al. (2020).This indicates that the dispersion of the data of each variable around the mean was reduced and reflects a good precision and experimental reliability in the estimation of the genetic differences between the cultivars.
Table 2. Mean squares (CM) of the analysis of variance and means of vegetative characteristics of 10 cultivars of Cannabis sativa.
| FV | CM | ||||||
|---|---|---|---|---|---|---|---|
| NF | FFPH (cm) | ILMS (cm) | LP (cm) | CLL (cm) | WCL (cm) | NSPP | |
| Blocks | 0.15 | 1051.71 | 0.17 | 3.39 | 8.35 | 0.15 | 9.57 |
| Genotypes | 1.69 ** | 503.28** | 1.60** | 8.01** | 14.45** | 0.83** | 17.03** |
| Error | 0.097 | 53.78 | 0.064 | 0.55 | 1.44 | 0.049 | 1.75 |
| CV (%) | 7.85 | 5.26 | 5.6 | 11.67 | 9.03 | 7.45 | 9.74 |
| R 2 | 0.89 | 0.87 | 0.92 | 0.88 | 0.84 | 0.89 | 0.84 |
| Genotype | Mean | ||||||
| Mountain tradition | 3.26 d | 127.7 dc | 3.74 dc | 5.13 de | 11.48 b | 2.39 c | 15.43 ab |
| Old culture | 3.51 cd | 124.2 d | 3.53 d | 5.41 cde | 10.95 b | 2.50 c | 14.10 abc |
| Cundi gold | 3.93 bcd | 127.7 dc | 4.08 dc | 6.12 cde | 11.31 b | 2.83 bc | 14.11 abc |
| Blondie grl | 5.22 a | 160.0 a | 5.50 a | 8.41 ab | 17.21 a | 3.84 a | 9.43 d |
| River cosmic1 | 4.99 a | 153.6 ab | 5.06 ab | 7.38 abc | 14.61 ab | 3.42 ab | 10.37 cd |
| Ice nilo | 3.45 c | 124.4 dc | 5.24 a | 7.07 bcd | 12.65 b | 3.44 ab | 12.61 bcd |
| High paisa | 3.04 d | 138.8 bdc | 3.89 dc | 5.17 de | 12.23 b | 2.75 c | 17.21 a |
| Timbiqui skunk | 3.52 cd | 142.3 abcd | 5.40 a | 9.42 a | 16.94 a | 3.46 ab | 12.51 bcd |
| Algarrobo CBD | 4.43 ab | 147.5 abc | 4.30 c | 4.69 e | 11.95 b | 2.45 c | 14.29 ab |
| No high | 4.39 abc | 148.2 abc | 4.33 bc | 4.88 e | 12.55 b | 2.56 c | 15.54 ab |
| Media general | 3.97 | 139.35 | 4.51 | 6.37 | 13.29 | 2.96 | 13.56 |
**: P<0.01; *: P<0.05; means with the same letters do not differ significantly, according to Tukey's 5% test; NL: number of leaflets; FFPH: female flowering plant height; ILMS: internode length of the main stem; LP, length of the petiole; CLL, central leaflet length; WCL: width of the central leaflet; NSPP: number of stems per plant.
Table 3. Mean squares of the analysis of variance and means of reproductive characteristics and phytocannabinoids of 10 cultivars from Cannabis sativa.
| FV | CM | ||||||
|---|---|---|---|---|---|---|---|
| DFL (d) | DH (d) | HHS (cm) | W100S (g) | DFY (g) | CBD | THC | |
| Blocks | 8.15 | 3.43 | 1138.69 | 0.0008 | 1729.54 | 0.158 | 0.042 |
| Genotype | 15.54** | 17.51** | 571.73** | 0.67** | 4763.48* | 103.570** | 158.623** |
| Error | 2.56 | 3.14 | 60.5 | 0.001 | 1896.92 | 0.154 | 0.925 |
| CV (%) | 3.17 | 2.21 | 5.88 | 0.78 | 14.79 | 7.91 | 10.6 |
| R 2 | 0.77 | 0.74 | 0.86 | 0.97 | 0.58 | 0.88 | 0.98 |
| Genotype | Mean | ||||||
| Mountain tradition | 49.27 ab | 76.67 c | 122.00 c | 12.08 d | 260.77 a | 6.71 b | 2.58 cd |
| Old culture | 50.43 ab | 77.00 bc | 117.23 c | 12.10 d | 265.27 a | 7.06 b | 3.38 cd |
| Cundi gold | 51.83 a | 77.33 bc | 119.37 c | 13.00 b | 251.83 a | 0.04 c | 13.59 b |
| Blondie grl | 52.07 a | 81.67 abc | 155.78 a | 13.50 a | 319.15 a | 0.04 c | 16.10 ab |
| River cosmic1 | 51.83 a | 80.00 abc | 148.37 ab | 13.08 b | 308.39 a | 0.04 c | 15.88 b |
| Ice nilo | 46.23 b | 82.00 ab | 119.07 c | 13.02 b | 341.10 a | 0.05 c | 18.82 a |
| High paisa | 50.90 ab | 79.00 abc | 129.30 bc | 12.50 c | 251.08 a | 6.95 b | 4.69 c |
| Timbiqui skunk | 46.93 b | 80.33 abc | 133.30 abc | 13.00 b | 263.16 a | 0.04 c | 14.42 b |
| Algarrobo CBD | 53.00 a | 86.67 a | 139.03 abc | 12.30 cd | 336.67 a | 14.79 a | 0.65 d |
| No high | 51.83 a | 82.00 ab | 139.70 abc | 12.80 b | 347.98 a | 13.99 a | 0.62 d |
| Media general | 50.43 | 79.97 | 132.32 | 12.74 | 294.54 | 4.97 | 9.07 |
**: P<0.01; *: P<0.05; means with the same letters do not differ significantly, according to Tukey's 5% test; DFL: days to female flowering; DH: days to harvest; HHS: height of harvested stem; W100S: weight of a thousand seeds; DFY: dried flower yield; CBD: cannabidiol; THC: tetrahydrocannabinol.
The highest NL was observed in the Blondie Grl genotype, without significant statistical differences with respect to ‘No High’, ‘Algarrobo CBD’ and ‘River cosmic1’. The CLL did not vary significantly in seven of the 10 genotypes. However, the Blondie Grl genotype presented longer CLL, without statistically differentiating from ‘Timbiquí Skunk’ and ‘River cosmic 1’. This indicates that this characteristic does not vary much between the genotypes studied. On the other hand, WCL and LP, were highly variable in the 10 genotypes, with the cultivars Blondie Grl, River cosmic1, Ice Nilo and Timbiquí Skunk having the highest WCL. Regarding LP, the highest were observed in ‘Timbiquí Skunk’, ‘Blondie Grl’ and ‘River cosmic1’. Both LP, CLL and WCL are allometric measures indicating that the leaf surface captures light for photosynthesis and therefore ‘Timbiquí Skunk’, ‘Blondie Grl’ and ‘River cosmic1’ have larger leaves, while ‘Algarrobo CBD’, ‘No High’, ‘High Paisa’, ‘Mountain tradition’, ‘Old culture’ and ‘Cundi Gold’ present smaller leaves, with lower magnitudes of LP, CLL and WCL (Tab. 2).
The highest FFPH was observed in the Blondie Grl, River cosmic1, No High and Algarrobo CBD genotypes, while those with the lowest height were ‘Old culture’, ‘Mountain tradition’ and ‘Cundi Gold’; the highest NSPP was expressed in the High Paisa, No High, Mountain tradition, Algarrobo CBD, Cundi Gold and Old culture genotypes, while the lowest NSPP was expressed in the Blondie Grl cultivar (Tab. 2). The FFPH records in the present study were lower than those of Schumann et al. (1999), who reported 173-254 cm. However, short plants have an agronomic advantage, since, having a shorter internode length, they allow a higher population density and with it a greater number of productive branches in a smaller area (Sarkar et al., 2017; Small, 2018). On the other hand, the High Paisa and Mountain tradition genotypes not only presented higher NSPP, but also lower ILMS (Tab. 2), so the size of the mother plant and the architecture of the stem are determinants of the cloning rate by cuttings. In this regard, the effects of light on the size of the internodes and the number of meristems has been studied, with results showing that under far-red LED lighting the length of internodes increases, while metal halide light statistically reduces their length. However, the greater length of internodes achieved with far-red LED light was industrially insignificant (Campbell et al., 2019).
For their part, Bevan et al. (2021), maintained that the yield of the inflorescence is related to the fresh weight of the aerial part, the growth rate of the plant and the dry weight of the root, so a taller plant would have higher NF, CLL and WCL and consequently higher inflorescence yield and cannabinoid production according to Bernstein et al. (2019); while Trancoso et al. (2022), expressed that there is not always a connection with a greater amount of phytocannabinoids.
The DFL ranged between 46.23 and 53.00 (Tab. 3), which indicates that the genotypes evaluated are much earlier than those recorded by Baldini et al. (2020), who in Italy reported between 97 and 107 d. The earliest genotypes in bloom are ‘Ice Nilo’ and ‘Timbiquí Skunk’, and the later are ‘Algarrobo CBD’, ‘Blondie Grl’, ‘River cosmic1’, ‘Cundi Gold’ and ‘No High’ and this agrees with the positive and significant correlation between HHS with respect to DFL (Tab. 3), as previously reported by Faux et al. (2013), who also noted that this depends on the genetics of the cultivar, as well as variations in temperature and photoperiod.
The genotypes that needed more DH were ‘Algarrobo CBD’, ‘No High’ and ‘Ice Nilo’, while the earliest to harvest were ‘Mountain tradition’, ‘Old culture’ and ‘Cundi Gold’. It was observed that the DFL do not correlate with the DH (Tab. 3), that is, the plants that flower first are not necessarily also harvested first, because although these two characteristics correlate in genotypes such as ‘Algarrobo CBD’, ‘Mountain tradition’ and ‘No High’; in others like ‘Ice Nilo’ that flowered early, their harvest was later than expected. According to Faux et al. (2013) the duration from sowing to full flowering correlated better with stem and seed yield.
The highest HHS and W100S were observed in the Blondie Grl genotype (Tab. 3). Likewise, these variables presented positive and significant correlation with NF, FFPH, ILMS, CLL and DH (Tab. 4); in turn, DFY correlated positively with HHS, NL, FFPH and DH. This suggests that the genotypes that present female flowering and greater height at harvest are more yielding in flower biomass (Tab. 2 and 3), so these characteristics must be taken into consideration for the selection of cultivars with higher yields of dry flowers and heavier seeds.
Table 4. Phenotypic correlations between the vegetative and reproductive characteristics of Cannabis sativa L.
| Caract | NF | FFPH | ILMS | LP | CLL | WCL | NSPP | DFL | DH | HHS | W100S | DFY |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NF | 1 | 0.56** | 0.46* | 0.29ns | 0.47** | 0.39* | -0.51** | 0.47** | 0.31ns | 0.59** | 0.53** | 0.39* |
| FFPH | 1 | 0.49** | 0.15ns | 0.38* | 0.25ns | -0.21ns | 0.49** | 0.47** | 0.98** | 0.39* | 0.38* | |
| ILMS | 1 | 0.71** | 0.67** | 0.77** | -0.62** | -0.19ns | 0.51** | 0.49** | 0.82** | 0.33ns | ||
| LP | 1 | 0.88** | 0.87** | -0.58** | -0.39* | 0.13ns | 0.17ns | 0.67** | -0.04ns | |||
| CLL | 1 | 0.83** | -0.52** | -0.17ns | 0.22ns | 0.40* | 0.60** | 0.04ns | ||||
| WCL | 1 | -0.69** | -0.28ns | 0.27ns | 0.29ns | 0.81** | 0.09ns | |||||
| NSPP | 1 | 0.01ns | -0.28ns | -0.23ns | -0.58** | -0.04ns | ||||||
| DFL | 1 | 0.10ns | 0.45* | -0.05ns | -0.01ns | |||||||
| DH | 1 | 0.45* | 0.34ns | 0.66** | ||||||||
| HHS | 1 | 0.41* | 0.45* | |||||||||
| W100S | 1 | 0.25ns | ||||||||||
| DFY | 1 |
Caract: characteristic; NF: number of leaflets; FFPH: plant height in female flowering; ILMS: stem internode length major; LP: petiole length; CLL: length of the central leaflet; WCL: width of the central leaflet; NSPP: number of stems per plant; DFL: days to female flowering; DH: days to harvest; HHS: height of the harvested stem; W100S: thousand seed weight; DFY: dried flowers yield. **: P<0.01; *: P<0.05; ns: not significant.
On the other hand, the analysis of variance for DFY showed significant differences (P=0.0465) between cultivars; however, Tukey's post hoc test did not estimate such differences because it is a conservative test, with a greater requirement to establish a significant difference between two means, which occurs when the p-value is very close to the type I error (α=0.05). However, the No High, Ice Nilo and Algarrobo CBD genotypes presented a difference of 103.7 g m-2 (35.2%) with respect to the rest. DFY varied between 251.8 g m-2 in High Paisa and 347.9 g m-2 in No High (Tab. 3).
The differences in DFY performance are supported by Vanhove et al.(2011), on three factors: light intensity, population density and cultivar genetics. In this study, the short plants were not the most efficient in flower production, but rather the tallest, as they produced greater flower biomass, due to a better use of light (photosynthesis) related to allometric characteristics such as NL, CLL and WCL.
Phytocannabinoid content
The analysis of variance with the mean squares of the sources of variation of the model, corresponding to the cannabinoids CBD and THC, in their basic form, and their mean values, are recorded in table 3. It was observed that the genotypes evaluated showed highly significant differences between them (P<0.01). Differences between cultivars have also been reported in works carried out by Glivar et al. (2020) and Busta et al. (2022), emphasizing that this is due to genetic differences, as a result of the differential expression of the genes responsible for the biosynthesis of cannabinoids, with uniformity within each clone. However, there is less variation in the CBD determinations, given its lower coefficient of variation.
With regard to the accumulation of tetrahydrocannabinol (THC) and cannabidiol (CBD), the enzymes THCA synthetase (THCAS) and CBDA synthetase (CBDAS) are necessary for the production of the acid forms THCA and CBDA, which are synthesized from cannabigerolic acid (Yamamuro et al., 2021), accumulate in the trichomes of the inflorescence and, when subjected to intense heat, undergo decarboxylation leading to the formation of CBD and THC (Welling et al., 2016; Wróbel et al., 2018; Cascini et al., 2019). According to the results, three groups are evident according to De Meijer et al. (2003) and Staginnus et al. (2014), the first, is made up of genotypes with the highest CBD content, which according to Cascini et al. (2019), correspond to the homozygous genetic combination B(D)/B(D) represented by the cultivars Algarrobo CBD and No High, this result is consistent with that reported by Vanhove et al. (2011).
The second group is represented by the heterozygous genotype of the codominant alleles B(T)/B(D), which includes the genotypes Old culture, High Paisa and Mountain tradition, with an approximate ratio 1:1 in the accumulation of THC and CBD, which can be variable, and is related to the efficiency in the transformation of CBGA by THCA and CBDA synthetase (De Meijer et al., 2003). The third group is represented by the homozygous B(T)/B(T) genotype, which groups five genotypes with the highest THC and lowest CBD levels, similar results were reported by Mark et al. (2009); and correspond to ‘Cundi Gold’, ‘Blondie Grl’, ‘River cosmic1’, ‘Timbiquí Skunk’ and ‘Ice Nilo’, which explains the genetic differences between them and their potential use for medicinal purposes, since CBD is believed to have a therapeutic effect on addiction to cocaine, opioids and psychostimulants, as well as on Alzheimer's (Evren and Umut, 2019), while THC is mainly used for recreational purposes (Cascini et al., 2019).
The precise characterization of the accumulation of CBD and THC in the cultivars is very important, since it allows discriminating the potential use to satisfy the needs of identifying parents in genetic improvement programs such as the pharmaceutical industry for the manufacture of cannabinoid-based products.
Considering the results obtained and given the importance of this species for both the primary and industrial sectors, this study allows for the selection of cultivars that respond to the agroecological conditions, needs, and demands of producers and the cannabis agroindustry in the country, which would promote the development and competitiveness of the cannabis agribusiness (Minsalud, 2018; PINE, 2020).
CONCLUSIONS
Genetic variability was identified both in the agronomic characteristics and in the cannabinoid content. Three groups of genotypes were identified based on the combinations of alleles coding for phytocannabinoid content: high THC, similar THC-CBD ratio and high CBD, which determines their potential use, mainly in medicine.
