The Efficiency of CBD Production Using Grafted Cannabis sativa L. Plants Is Highly Dependent on the Type of Rootstock: A Study

Abstract

1. Introduction

Medical cannabis and hemp (Cannabis sativa L.) contain many bioactive compounds with biological effects in the human body, and as a result, it is becoming important in the pharmaceutical industry and medicine [1]. Cannabinoids or phytocannabinoids represent largest group of secondary metabolites in cannabis plant. The dominant ones are tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinolic acid (CBNA), and cannabichromenic acid (CBCA) [2,3]. Cannabis is divided into four chemotypes based on the ratio of the neutral cannabinoids: chemotype I contains high concentrations of the cannabinoid tetrahydrocannabinol (THC) (>0.3%) and low concentrations of cannabidiol (CBD) (<0.5%) with THC/CBD > 1; chemotype II contains a lot of THC (>0.3%) and high CBD (>0.5%) with THC/CBD ≈ 1; chemotype III contains high CBD (>0.5%) and low THC (<0.3%) with THC/CBD < 1 [4,5,6,7]. Subsequently, in 1987, Fournier et al. identified chemotype IV, which contains over 0.3% cannabigerol (CBG) [8].

The goals of high-quality cannabis cultivation include a consistent and uniform yield of inflorescences and the production of these specific secondary metabolites. Therefore, professional medical cannabis producers are transitioning from greenhouses into more controlled and closed indoor growth chambers [9]. Indoor cannabis cultivation is one of the most energy-intensive industries to maintain [10], with the main energy consumption requirements of indoor production facilities being the intense lighting, dehumidification, space heating or cooling, drying, pre-heating of irrigation of water, generation of carbon dioxide, and 30 hourly air changes. This consumption results in power densities of 2000 W/m², which is comparable to modern computer data centers [11].

The bioactive substances of cannabis are mainly produced by unfertilized female plants, meaning only female plants are commercially cultivated for the production of secondary metabolites. Female plants can be obtained using feminized seeds [12], but due to heterogeneity of seedlings, selected female plants are usually vegetatively propagated to achieve uniform yields and easier production [2]. The rooting success rate in vegetative propagation by stem cuttings depends on many factors such as humidity [13], temperature [14], length and thickness of shoots [15], age and condition of mother plant [16], and genotype. The formation of adventitious roots is genotype-specific and determines the ability for successful vegetative propagation [17].

Grafting is an ancient, vegetative propagation technique. It involves the conjunction of at least two plant parts—root system (rootstock) and shoot (scion)—that form a grafted plant. Rootstocks can alter the biochemical and physiological processes of the scion and change plant tolerance to biotic and abiotic stresses [18]. It may serve as a source of endogenous plant hormones, thus leading to increased growth and yield of the scion in addition to disease control [19]. It has been shown that grafting exhibits excellent tolerance to serious soil-borne diseases. For instance, grafted watermelons show increased resistance to Fusarium L. and Verticillium N., bell peppers to Phytophthora B., and melons to Didymella bryoniae R., showing grafting’s role in tolerating diseases [20,21].

Choosing the right rootstock for a particular scion is crucial for the success of the grafted plants, as the rootstock has a direct influence on the survival rate, growth parameters, and the quality of the harvest. Rootstocks impact various growth parameters, including plant biomass, leaf area, and the length of generative development in different crops [22,23,24]. This underscores the critical importance of meticulous rootstock selection in grafting practices.

Plants grown from seeds usually have a single primary root (the taproot), while plants grown from cuttings have several primary roots. The total root mass of seedlings is higher than that of cuttings, resulting in differences in root mass and soil exploration ability [25,26]. Rootstocks can be dwarfing, in which they reduce vigour of the scion, or vigorous, which increases the vigour of the scion [27,28]. When grafting tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.), and bell pepper (Capsicum annuum L.) plants, seedlings are used as rootstocks and scions, and it is important that they are healthy and uniform in size at the time of grafting [29,30,31]. Studies have shown that the method of propagation influences root architecture, with stem cuttings developing shallower roots compared to seedlings [32,33,34].

To this date, the only known rule for grafting cannabis is that both the rootstock and the scion must be clonally propagated from vegetative tissue [35]. This method is unique compared to other non-woody plants, where seedlings are usually used as rootstocks. Grafting with clonally propagated germplasm differs from conventional vegetative propagation with cuttings and has not yet been studied in detail. Unpublished experiments at our institution have shown that certain genotypes cannot be vegetatively propagated by stem cuttings easily. Grafting offers an alternative for the propagation of these problematic genotypes. However, as research has shown [36], the survival rates of grafted plants can also differ significantly between varieties and grafting can affect plant survival. One reason for researching cannabis grafting is the interest in gaining knowledge about this technique and optimising the vegetative propagation of cannabis.

Despite the growing interest in cannabis cultivation techniques, research on the application of grafting in cannabis production remains scarce, particularly in terms of effects on plant morphology, yield, and secondary metabolite concentrations. This gap underscores the urgent need for comprehensive studies investigating the efficacy of grafting in cannabis. Research on various plant species confirms that grafting can modify the secondary metabolism, leading to enhanced production of bioactive compounds [37,38,39,40]. The special physiological and biochemical requirements of cannabis call for targeted research. This should investigate how different combinations of rootstock and scion affect the production of important medicinal compounds, including CBD. Our study attempts to fill this knowledge gap and provide insights that could lead to significant advances in the field of medical cannabis.

To the best of our knowledge, the method for grafting cannabis using different rootstock types, including seedlings as rootstock, has not yet been described. By pioneering research into the use of seedlings as rootstock for cannabis grafting, this study lays the foundation for the development of cultivation strategies that could improve the sustainability and efficiency of cannabis production. The aims of our study were to (i) compare the effectiveness/success rate of obtaining grafted plants using different rootstock types (categorised as potentially vigorous, potentially dwarfing, and derived from seedlings) and (ii) evaluate the influence of these different rootstock types on the morphological and biochemical performance of grafted cannabis plants using two different scion genotypes in indoor experiment.

2. Results

3. Discussion

The percentage of successfully rooted cuttings depends on various factors. The genetic basis and the age of the plant material have the greatest influence on the formation of adventitious roots [42,43]. Additional factors include leaf area of the cuttings, the position of the cutting on the mother plant, the concentration of endogenous hormones, the light intensity, the rooting medium, the water content, and the nutrient consumption [44,45]. Regarding survival rates in our grafting experiment, the data showed variability depending on the rootstock used. For the potentially dwarfing rootstocks (PDR) and potentially vigorous rootstocks (PVR) obtained as clones from selected mother plants, it is interesting to note that in some cases a high success rate in rooting the rootstocks did not necessarily lead to a high survival rate after grafting. For example, in ‘Vigor-S26/ScionII’, 12 out of 13 rootstock plants were rooted (Table 1) but only 3 grafted plants survived. In addition, ‘Vigour-S26/ScionIII’ had 11 out of 12 rooted plants and only 3 grafted plants survive (Table 2). This indicates that successful rooting of rootstocks is not the only factor determining graft survival. In some combinations, rooting success was low, with only 3 out of 14 in the ‘Control/ScionIII’ combination. Apparently, the ‘ScionIII’ genotype itself has poor rooting ability and by using the grafting technique it is also possible to improve root development in genotypes with poor rooting ability [35]. Further research could focus on understanding the reasons for these discrepancies in survival rates and possibly improving grafting techniques to increase survival rates.

In contrast, seedling-as-rootstocks (SAR) means no loss of plant material during rootstock establishment and a higher efficiency in obtaining grafted plants compared to other combinations as well as to self-rooted (non-grafted) controls. At the same time, it must be considered that the use of SAR does not require special cultivation techniques. Mother plants require a consistent and controlled environment with optimal light, temperature, and humidity conditions to ensure healthy growth and propagation by stem cuttings [35], which is less stringent when using SAR. However, when seedlings are used for rootstocks, high germination rate of rootstock seeds, homogeneous emergence of rootstock seeds, and rootstock/scion compatibility are important factors [46,47].

Taking all combinations together, the average survival rate of grafted plants was 38.1% for the scion ‘ScionIII’ and 29.5% for ‘ScionII’. In contrast to Purdy et al. (2022) [35], who used a one-step grafting method and reported a survival rate of up to 100%, a two-step grafting method was used in our experiment. They found that the older the rootstock, the lower the survival rate. In other words, the survival rate was highest when a fresh scion was grafted onto a fresh rootstock. It is possible that the two-step grafting method is less appropriate for cannabis grafting and one-step grafting is more suitable for obtaining a larger number of viable plants, which was also observed in our further (unpublished) experiments. However, SAR can only be used in the two-step grafting method.

The analysis of the biomass distribution revealed considerable differences depending on the combination of rootstock and scion. We expected that the different rootstock types would have the most pronounced and visible effects on the morphological characteristics of the grafted cannabis plants. Numerous studies have shown that certain rootstocks can increase both plant biomass weight and its fruit yields [48,49,50,51]. It is suggested that a more developed root system of vigorous rootstocks and root architecture can influence various physiological functions. These include improving water uptake, nutrient uptake, and subsequent transport to the scion, possibly leading to increased plant growth [48,52,53,54]. Turhan et al. (2011) [55] demonstrated that grafting tomatoes on suitable rootstocks significantly improves fruit yield, size, and number of fruits per truss compared to non-grafted plants. However, no significant differences were found between the different grafting combinations in terms of fruit characteristics. The improvement in growth and yield depends on the characteristics of the rootstock and its ability to modify the physiology of the scion [56,57]. However, as shown by Doñas-Uclés et al. (2015) [58], there is no clear correlation between yield and vigor conferred to the bell pepper (Capsicum annum L.) plant by the rootstock. In this context, it is crucial to understand the influence of rootstock on shoot growth, as rootstock can significantly influence hormone levels, which are closely linked to plant productivity and resource efficiency [59,60,61]. Albrecht et al. (2017) [25] showed that rootstocks, derived from seedlings, develop a well-defined primary root, and have a greater proportion of root mass and the lowest shoot-to-root ratio compared to clonally propagated rootstocks. Increased root biomass could lead to an increase in the aboveground biomass of the plant. For our study, this means that cannabis plants derived from seedling rootstocks (SAR), or potentially vigorous rootstocks (PVR), should perform better in terms of measured morphological traits (fresh weight and bud compactness index) than cannabis plants derived from potentially dwarfing rootstocks (PDR). This assumption was not confirmed, as the average UIN for both scion genotypes grafted on PVR gave the lowest values (118 for FW and the same BCI). However, our result is in line with the observation of Purdy et al. (2022) [35], who found that the size of the root system does not consistently correlate with cannabis yield. As for FW, the highest average UIN for both scion genotypes reached SAR (127), confirming the hypothesis of the dominance of the root system with a well-developed primary root.

Some authors suggest that the restricted passage through the grafting union, in specific grafting combinations, is directly related to anatomical changes in the tissue at the grafting union [62], which are thought to be partly due to the restriction of polar transport of auxin (IAA). Grafting prevents the passage of IAA into the root system, which could stimulate the synthesis of cytokinins and consequently increase the vigor of the scion [63]. That could also explain increased vigour using PDR in our case. On the other hand, it is assumed that a reduced IAA concentration in the root system reduces the ratio between xylem and phloem in some plants, which may result in dwarfing of the scion [64,65]. Reduced rootstock growth may also be related to the accumulation of IAA in the rootstock and reduced cytokinin synthesis. An excessively high concentration of IAA causes oxidative stress and triggers degenerative processes in the root system [66]. Rootstocks also differ in the concentrations of other hormones, such as ABA and its derivatives—gibberellins GA9, GA19, and GA53 [67]. Hormonal profiles often show no direct correlation with growth parameters. Certain distant scion signaling proteins such as SlCyp1 can influence root system and vascular development. All this suggests a complex signaling between different rootstocks and scions that needs to be further investigated in cannabis plants [68].

In our study, the BCI was statistically significant only for the scion ‘ScionII’, with all three genotypes of the PDR type having the highest BCI values (0.25–0.29 g/cm) and the control combination ‘Control/ScionII’ the lowest (0.12 g/cm). In contrast, the PDR type had the greatest negative effect on BCI for the ‘ScionIII’ scion genotype, demonstrating the variability in the effects of different rootstock types on the scion genotype. Increased BCI could be one of the desired morphological improvements of cannabis plants through grafting to increase yields in indoor cannabis cultivation.

Manipulation of the defense mechanism of cannabis plants is crucial as it can lead to changes in the content of secondary metabolites [69]. Grafting has been shown to influence biosynthetic pathways and their accumulation [39,70]. There are several transport and signaling mechanisms between the rootstock and the scion that promote shoot adaptation to stress factors through the transport of signaling molecules involved in the plant defence mechanism. Long-distance signaling molecules may be responsible for the induction of systemic resistance in grafted plants [71]. The biosynthetic pathways of cannabinoids and terpenoids share a common precursor, isopentenyl pyrophosphate (IPP). IPP is synthesized either via the mevalonic acid pathway in the cytosol (MVA) or the plastidial methylerythritol pathway (MEP) [72,73]. The MEP pathway is posited to play a crucial role in the synthesis of cannabinoid-terpenoid compounds [74]. IPP acts as a substrate for the synthesis of geranyl diphosphate for monoterpenes, farnesyl diphosphate for sesquiterpenes, and geranylgeranyl pyrophosphate (GGPP) for tetraterpenes. Notably, GGPP is the immediate precursor for carotenoids [75,76]. The selection of specific rootstocks has been demonstrated to significantly enhance both the qualitative and quantitative carotenoid profiles, evidenced by increased concentrations of β-carotene and lycopene [37,38,40]. Given the shared biosynthetic pathways between cannabinoids and terpenoids, it can be hypothesized that grafting may stimulate the production of desired secondary metabolites in cannabis. Our data showed an effect of rootstock on the biochemical composition of cannabis plants, especially on the content of tCBD and tTHC. The effect was more pronounced in the ‘ScionIII’ genotype, which belongs to chemotype III and has a genetic predisposition for a high CBD content. Potentially dwarfing rootstocks (PDR) caused the highest increase in tCBD content (27%), from 10.1% (non-grafted) to 12.8% (average content for ‘ScionIII’ on PDR). However, as the ratio of CBD to THC is fixed in cannabis [77], the tTHC content also increased at almost the same value (29%), so the ratio CBD:THC was not changed.

Terpenes are the second most important group of biochemicals in cannabis and their expression varies greatly depending on genotype and growing conditions [1]. We observed differences in terpene content between different rootstock types (Tables S2 and S3). However, the terpene content was low overall. To investigate the influence of rootstock type on terpene content in more detail, different genotypes (e.g., of medical cannabis) should be included in further studies.

The CBD yield (CBDY) is determined by the CBD content and the yield of the inflorescences (IY). Since cannabinoids are genetically determined and the time window for their manipulation is relatively narrow, CBDY is mainly influenced by IY. This study has shown that IY can be increased in grafted cannabis plants by the grafting technique. In the genotype ‘ScionIII’, which naturally grows faster and bushier, grafting led to an increase in IY of 20–22% and in the genotype ‘ScionII’, which grows more upwards and has fewer side shoots, up to 71% higher IY was observed. This means that less vigorous scions can significantly increase their IY potential when grafted onto a suitable rootstock.

An increase in IY led to an increase in CBDY in this study, with the highest CBDY per plant causing PDR (average 4.8 g CBD/plant) for the genotype ‘ScionIII’. This was 55% higher than the non-grafted control plants. This is not in line with Purdy et al. (2022) [35], who found no increase in CBDY in grafted plants with a yield of up to 2.4 g CBD/plant. On the contrary, they even found a decrease in CBDY in one graft, which they explained because of the lower biomass yield of this graft combination. For the ‘ScionII’ genotype, the relative comparison showed an even greater increase in CBDY (up to 83% for SAR). However, absolute values of CBDY per plant were much lower due to the characteristics of the ‘ScionII’ genotype (chemotype II), which belongs to industrial hemp. Given the prevailing market prices for CBD isolate ($952/kg–$1964/kg) [78], graft-induced CBDY enhancement could drastically improve the efficiency of cannabidiol production.

Although the final yield of CBD depends on many factors during plant growth, determining the efficiency of CBD production begins with the efficiency of obtaining plants for flowering. When preparing grafted cannabis plants for CBD production, the critical step in evaluating the success of this new two-step grafting propagation method. This means how many rootstocks are needed at the beginning of the propagation process to obtain the desired number of plants for cultivation (CBD production). The number of rootstocks prepared is very important as these rootstocks require space and care, which incurs costs for electricity, growing space, and labour. Indoor design metrics such as electrical power, PPFD, efficiency, and photon conversion efficiency (g/mol) are the main drivers of electricity costs associated with CBD-rich biomass production and should be spent wisely [79]. Production Efficiency of cannabidiol index (PE_CBD) was established in this study to evaluate the efficiency of CBD production using different types of rootstocks. The PE_CBD index covers the entire production process, from rootstock preparation to CBD yield. The survival rate of the rootstock has the greatest influence on the calculation and follows the paradigm: the higher the survival rate of the rootstock, the higher the CBD yield at the end of the cultivation process.

The PE_CBD revealed that the seedlings in this study stood out as the most efficient rootstock for cannabis production with average UIN values of 252 and 216 for ‘ScionII’ and ‘ScionIII’, respectively. The reasons for these high values are: (i) the high germination rate of the seeds ensured 100% establishment of the rootstock to be used for grafting and (ii) the higher survival rate of SAR grafts (40–70%, Table 1 and Table 2), which means that more plants are available for CBD production, resulting in higher total CBD yields of a SAR rootstock type, notably affecting the PE_CBD value. ‘ScionIII’ proved to be more receptive to grafting compared to ‘ScionII’. The considerable indoor space required for large-scale production is a challenge, especially when traditional cloning takes up 20–25% of the production area [80]. Using the one-step tailor-made grafting methodology [35], this demand could increase greatly as both scions and rootstocks need to be vegetatively propagated. In this respect, the use of SAR seems even more promising, as it reduces costs (eliminating the maintenance of mother plants and the preparation of clones) and increases PE_CBD (a higher survival rate of SAR plants is expected). Although our results have shown that SAR has a high potential for practical application, some disadvantages (e.g., high variability among seedlings, risk of formation of unwanted rootstock shoots at flowering time) should also be considered and further researched.

4. Materials and Methods

5. Conclusions

This study showed that rootstock type influences the survival rate, morphological parameters, and biochemical composition of cannabis plants. A relative comparison between grafted and non-grafted plants showed better characteristics of the grafts in most cases, indicating the great potential of the grafting technique for cannabis production. Optimizing rootstock selection and grafting techniques, understanding the mechanisms behind the observed morphological changes, and a deeper exploration of the influence of rootstock on the biochemical composition and yield of cannabis plants are some of the topics for future research. Grafting is a new cultivation method in cannabis production and requires additional equipment, space, time and “experienced hands”. As the cannabis industry continues to expand, research in these areas will be of increasing importance in maximizing the potential of this plant. Further research on cannabis grafting is necessary, focusing on the physiological processes in the scion, changes in hormone balance, metabolic pathways, and genetic analyses, such as changes in gene expression in specific rootstock-scion combinations. In addition, priority should be given to breeding rootstocks that are resistant to biotic and abiotic factors to improve the success of grafting and the resistance of the plants.

Acknowledgments

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13081117/s1, Table S1: Results of the real measurement of the experiment with three types of rootstocks and two genotypes of scions; Table S2: Terpene content in combinations of rootstocks with the ‘ScionII’ genotype; Table S3: Terpene content in combinations of rootstocks with the ‘ScionIII’ genotype.

Funding Statement

This research was funded by the Slovenian Research Agency (SRA-ARRS): young researcher scholarship grant awarded Luka Bitežnik; program P4-0077 Genetics and Modern Technologies of Crops; and infrastructural center IC RRC-AG (IO-0022-0481-001). The funder had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Author Contributions

Conceptualization, L.B. and M.F.; methodology, L.B., R.Š. and M.F.; validation, L.B. and R.Š.; formal analysis, L.B., R.Š. and M.F.; investigation, L.B. and R.Š.; resources, R.Š. and M.F.; data curation, L.B., R.Š. and M.F.; writing—original draft preparation, L.B.; writing—review and editing, L.B., R.Š. and M.F.; visualization, L.B.; supervision, M.F.; project administration, M.F.; funding acquisition, M.F. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

All data from this study are included in this published article and as Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

References