The results from this study conducted on drug-type cannabis (marijuana) show that the response to tissue culture conditions is first and foremost influenced by the genetic background (genotype) of the plants tested. The findings also indicated that both meristems and nodal explants were responsive to the tissue culture conditions tested, and that measurements of shoot growth could be used to determine the response of genotypes and quantify effects of media amendments on growth. Lastly, the findings show that the recovery of rooted plantlets is influenced by the degree of internal contamination of nodal explants and the extent to which rooting and acclimatization of the plantlets could be achieved using different treatments. Knowledge of these variables can enhance the successful recovery of plantlets from tissue cultures of C. sativa, which was the main objective of this study. This study focused on stage 1 of the micropropagation process as described by Page et al. (2021). This phase is equivalent to an initiation phase (establishment of cultures) and did not involve repeated cycles of subcultures and multiplication of shoots as observed in stage 2, the multiplication phase that increases plant numbers through micropropagation (Monthony et al., 2021; Page et al., 2021). Research on stage 1 is valuable to establish the genotypic response of a range of cannabis strains to initial tissue culture conditions and to rapidly recover plantlets from meristems or nodal explants for a first cycle of propagation.

Many previous reports of tissue culture of Cannabis sativa L. have utilized hemp varieties because of legal restrictions placed on the cultivation of drug-type cannabis in most regions of the world (Adhikary et al., 2021; Monthony et al., 2021). However, Lata et al. (2009) and Page et al. (2021) researched some of the variables that can influence the response of drug-type C. sativa to tissue culture conditions. In these studies, nodal segments with axillary buds were used for direct regeneration of shoots in stage 1 micropropagation (Lata et al., 2009, 2016; Page et al., 2021). The differential response of various genotypes to tissue culture conditions, as reported in this study, was also noted by Monthony et al. (2021). Prior research has shown that the response to tissue culture conditions in other plant species is affected by genotype (Islam et al., 2005; Martínez et al., 2017). If cannabis micropropagation is to be successful, an assessment of the response of genotypes of interest to tissue culture conditions would first need to be established before a full-scale tissue culture method could be developed for commercial use. In this study, some of the cannabis genotypes, e.g., MBD, produced shoots over 4.5 cm in height and formed multiple shoots and buds, while the other genotypes (SPQ and CHQ) grew poorly. Strain MBD was selected for a further study on plantlet recovery. A higher frequency of shoots and buds can potentially give rise to more plantlets during stage 2 micropropagation and can enhance plantlet numbers.

Meristems and axillary buds have both been used as starting explant sources for shoot induction in tissue culture experiments of various plant species. For example, axillary buds are commonly used for the propagation of fruit and nut trees, while meristems have been used for sweet potato and strawberry propagation (Hussain et al., 2012). Growth of axillary buds in tissue culture has been studied in mint (Mentha species) (Rech and Pires, 1986), Cancer tree (Camptotheca acuminate) (Liu and Li, 2001), hops (Humulus lupus) (Roy et al., 2001), Andean blueberry (Vaccinium floribundum) (Cobo et al., 2018), and other woody plants (Sahoo and Chand, 1998). In order to obtain plants free from pathogens, particularly viruses, meristem tip culture is a preferred method. Successful eradication of viruses using tissue culture techniques alongside a secondary method, such as thermotherapy, has been demonstrated in sugarcane (Cheong et al., 2012), Lilium spp. (Chinestra et al., 2015), dahlias (Nerway et al., 2020), artichoke (Spanò et al., 2018), and many others. We compared meristem and nodal explant types in this study and successfully obtained plantlets from both, with the meristems showing significantly lower microbial contamination rates compared with the nodal explants bearing axillary buds. However, shoot production from the meristems required a longer time in culture (10 weeks) compared with the axillary buds (6 weeks).

Meristems have not been previously tested as an explant source in either hemp or cannabis, although shoot tips were used for direct regeneration of hemp (Wang et al., 2009), and stem tips were used for micropropagation and retipping studies on hemp (Lubell-Brand et al., 2021). Meristems are important starting material, as they contain a lower frequency of internally-borne microbes and viruses (Wang and Hu, 1980). Since C. sativa L. is reported to harbor fungi and bacteria internally as endophytes (Scott et al., 2018; Punja et al., 2019), consequently, explants taken from mother plants that naturally carry endophytes or pathogens have a higher risk of becoming contaminated after transfer to tissue culture media, as observed in this study. Monthony et al. (2021) circumvented this problem by first establishing in vitro plants in stage 1 that were subsequently used to provide an explant material for studies on micropropagation and callogenesis in stage 2. While this approach is advantageous to provide clean explants and maintain desired genotypes in vitro, the explants used in the present study were derived from donor plants grown under commercial greenhouse conditions, as they provided unlimited quantities of tissues, and the plants could be evaluated for commercially desired traits prior to introduction into the tissue culture environment. To avoid higher contamination rates from these tissues, we evaluated a range of decontamination methods. Following reports of the addition of fungicides to tissue culture media to reduce fungal contamination (Nagy et al., 2005; Panathula et al., 2014), we added captan at 0.02 g/L, but it did not show any effect. We also tested a widely used broad spectrum product, Plant Presrvative Mixture™ (PPM; Plant Cell Technology, Washington, DC, United States), which reduced initial contamination in tissue culture but not beyond 2 weeks. Similarly, nodes that had been surface-sterilized in 5% PPM for 4 h showed no difference in contamination levels compared with nodes sterilized with 10% bleach +0.1% Tween 20. Interestingly, the application of a systemic fungicide (Luna) to the indoor-grown donor plants, followed 3 weeks later by the removal of nodal explants, showed reduced contamination levels in tissue cultures of one strain by almost 3-fold.

The contaminating microbes in cannabis explants appear to reside within the central pith tissues, as shown in this study using scanning electron microscopy and reported elsewhere (Punja et al., 2019). This could explain the difficulty in eradicating them with surface-sterilization methods. Donor plants of some genotypes, e.g., CPH appeared to have a higher background level of contamination compared with other genotypes, e.g., PWE. Most of the contaminants emerged after several weeks in the tissue culture environment and originated from the central pith of the nodal explants (see Figure 5). Axillary buds may contain pathogens or endophytes living internally, which can easily be transferred into tissue culture (Wang and Hu, 1980). Internal contamination is less of a concern for meristems, as the vascular dome and first primordial leaves are generally free of bacteria, fungi, and viruses (Ramgareeb et al., 2010). Previous studies have demonstrated that an extensive array of fungal and bacterial endophytes can colonize hemp and cannabis plants (Kusari et al., 2013; Scott et al., 2018; Punja et al., 2019). Kusari et al. (2013) found 30 different species of fungal endophytes, of which Penicillium copticola was the most prevalent. Scott et al. (2018) found 134 bacterial and 54 fungal strains in three hemp cultivars. The most abundant fungal genera were Aureobasidium, Alternaria, and Cochliobolus, and the most abundant bacterial genera were Pseudomonas, Pantoea, and Bacillus. Punja et al. (2019) showed that endophytic and pathogenic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium, could colonize cannabis plants internally. Previous tissue culture studies on hemp and cannabis have not described problems with tissue culture contaminants. It is likely that the coco fiber used as a substrate for growing plants in this study harbored microbes that eventually colonized the internal tissues of the stems and became established (Punja et al., 2019). Other growing media, such as rockwool, may contain lower levels of contaminating microbes. Donor plants grown in a coco fiber substrate over prolonged periods of time in indoor environments, e.g., for up to a year, such as CPH, showed much higher background levels of contaminants. Recent microbiome studies have demonstrated that the bulk soil and rhizosphere of cannabis and hemp plants are the most influential in determining the subsequent composition of internal microbes in other regions of the plant (Barnett et al., 2020; Comeau et al., 2021). Therefore, attention should be given to the condition of donor plants with regard to the substrate they are grown in and their duration in the growing environment. Young plants grown in relatively sterile growth substrates should be selected for tissue culture studies.

A polymerase chain reaction-based assay showed conclusively that cannabis stem tissues contained a range of fungi. The method allowed the detection of 1 ng/ml of genomic DNA and could be used to screen donor plants to determine the background level of microbial contamination. Similar PCR-based methods have been used to screen mother plants and tissue-cultured plants such as strawberries, sweet potatoes, and roses to ensure they are free of bacteria and fungi (Moreno-Vázquez et al., 2014; University of California Davis, 2008). This approach can be applied to cannabis plants before they are deployed in tissue culture. In addition, if meristem culture of cannabis is used to obtain pathogen-free plantlets, it would have to be accompanied by a similar PCR-based assay to test for the absence of these pathogens. Nodal explant cultivation is unlikely to be free of pathogens given the high levels of internal contamination observed in this study. Therefore, shoots derived from nodal cultures should be avoided because of the potential for contaminants. Meristems represent the explant of choice to obtain pathogen-free plantlets from tissue cultures of C. sativa.

The tissue culture medium used for growth of plant tissues can influence the success in initiation and multiplication of shoot growth and elongation. Following shooting, a second medium with a higher concentration of auxin can be used to induce rooting (Lata et al., 2009; Wang et al., 2009; Chandra et al., 2017). We used the multiplication medium described by Lata et al. (2009) containing Murashige and Skoog (MS) basal salts supplemented with myo-inositol and activated charcoal. The growth regulators added were thidiazuron (TDZ, 1.0 μM) and naphthaleneacetic acid (NAA, 0.5 μM). On this medium, both explant types responded favorably, and shoots were produced in the initiation phase and could be transferred to subsequent media of the same composition for measurements to be made. However, continuous subcultures over extended time periods on MS salts medium tended to produce shoots that displayed hyperhydricity and developed signs of nutrient deficiency with low multiplication rates (authors, unpublished observations). These symptoms were also described by Page et al. (2021) on MS salts medium. The addition of activated charcoal appeared to improve growth on the MS medium; therefore, MS salts plus 1% activated charcoal was used in most of the experiments in this study. Activated charcoal, when added to tissue culture media, can absorb or bind some of the toxic waste compounds released from growing plants, in particular phenolic compounds, thereby improving in vitro plant tissue growth (Wang and Huang, 1976; Thomas, 2008; Chandra et al., 2017). This would be particularly useful in stage 1 micropropagation. Page et al. (2021) reported that a tissue culture medium based on DWK basal salts supported better canopy growth than MS basal salts from nodal explant segments that were intended for stage 2 micropropagation. They compared five cannabis genotypes and used two-node explants originating from micro-propagated plantlets grown on a DWK salts medium. Their results showed that explants grown on DKW produced a larger canopy area and had a higher multiplication rate than explants grown on MS. In this study, comparisons between the two basal salts media using two cannabis genotypes did not show consistent differences in shoot growth that could be attributed to the effect of medium composition during stage 1 micropropagation. Wang et al. (2009) used an MS basal salts medium with 30 g/L sucrose, 6.8 g/L phytagel, and 1 μM of TDZ to produce axillary buds from shoot tips of hemp during micropropagation. Lubell-Brand et al. (2021) used an MS salts medium described by Lata et al. (2016) in which TDZ was replaced with the growth regulator meta-topolin (mT). They reported that hyperhydricity was reduced by modifying the agar content of the medium, coupled with improved vessel ventilation and enhanced nitrogen levels. Therefore, both DKW and MS salts media can support short-term growth in tissue culture media during the initiation of cultures. However, continuous subcultures and multiplication on a DKW-based medium appear to yield healthier and more vigorous plants (Page et al., 2021) or on an MS medium supplemented with enhanced levels of nitrogen (Lubell-Brand et al., 2021).

Rooting is often the most challenging step in micropropagation (equivalent to Stage 3 micropropagation according to Page et al., 2021), especially in woody plant species (Ranaweera et al., 2013). IBA, a naturally occurring auxin, has previously been shown to induce rooting in cannabis plants at 5 μM (Lata et al., 2009), which was confirmed in this study. However, since the rooting frequency with IBA was not significantly different from that with MM, further rooting experiments with sodium metasilicate and silver nitrate were conducted on MM. KIN and 2,4-D were also tested for promotion of rooting. In callus cultures, these hormones induced rooting (Feeney and Punja, 2003). When added to MMC, neither KIN nor 2,4-D alone or in combination induced rooting in plantlets to the extent reported from callus. To promote root induction, sodium metasilicate (containing 22.9% silicon) was added to MM. Silicon was included because of its reported positive impact on rooting seen in other plant species (Zhuo, 1995; Soares et al., 2011). Previous research has also shown a positive effect of the addition of silicon to tissue culture media on leaf morphology of banana (Musa acuminata) (Luz et al., 2012). In this study, a significant increase in rooting and improved leaf morphology were observed when sodium metasilicate was added to MM at 6 mg/L. Sodium metasilicate has not been previously used in tissue culture of C. sativa. In this study, silver nitrate (AgNO₃) increased the number of rooted plants when added with IBA, but had no significant effect when added to MM. AgNO₃ could be combined with a lower concentration of IBA (5 instead of 37 μM) or with sodium metasilicate to determine the effects on plantlet recovery. However, the use of AgNO₃ may alter the sex expression toward male flower formation (Punja and Holmes, 2020).

The final step in tissue culture propagation is acclimatization of plantlets (Stage 4). At this stage, plantlets are removed from the jars/containers and acclimatized to external environmental conditions. When removing plantlets from the medium, roots should be carefully washed to avoid damage, and the agar should be washed off to prevent fungal growth from the residual sucrose. In this study, plantlets were transferred directly to rockwool, peat, and hydroponic substrates for a comparison of survival following acclimatization. Rockwool, peat, and coco fiber are the most common soilless growing media used worldwide for the production of fruits, vegetables, and cut flowers (Savaas and Gruda, 2018). In the present study, the plantlets generally grew better in rockwool during acclimatization, followed by peat and the hydroponic system, although the substrate response was not statistically different. The plantlets exhibiting a bushy morphology with long thin curled leaves acclimatized poorly. The addition of sodium metasilicate improved the morphology of the plants and was a contributing factor to improved acclimatization. Lata et al. (2016) acclimatized and hardened well-rooted cannabis plantlets with a 100% survival rate by 10-day pre-incubation on a coconatural medium before transfer into potting mix-fertilome. A mixed approach of using tissue culture medium with sterile rockwool cubes for multiplication and rooting of cannabis (Kodym and Leeb, 2019) may be a good option for improving acclimatization. Rooting can also be done ex vitro, i.e., outside the tissue culture environment. For example, a peat-based medium and high humidity conditions were used successfully for tea plants (Camellia sinensis L.) (Ranaweera et al., 2013). When compared with conventionally propagated tea plants using tissue culture, the ex vitro rooted micro shoots produced superior plants. Similarly, an in vitro–ex vitro micropropagation system was recently described for hemp (Lubell-Brand et al., 2021).

In addition to direct regeneration of shoots from axillary buds or meristems, efforts have been made toward indirect regeneration of shoots from callus cultures of hemp and cannabis. These have not been successful because of the recalcitrant nature of this species (Monthony et al., 2021). Differences in callus growth from petioles and leaves were attributed to different genetic backgrounds of the plants tested. Slusarkiewicz-Jarzina et al. (2005) and Wielgus et al. (2008) studied the effect of plant growth regulators on the development of callus and subsequent regeneration in five hemp genotypes. Their results showed that genotype was an important and determining factor for callus growth and regeneration. The hemp cultivar Fibrimon-24 produced the most calluses (83%), while a different cultivar, Silesia, had a regeneration rate of only 2.5%. In previous studies, callus formation has been induced from both hemp and cannabis explant tissues using combinations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), and the cytokinins kinetin (KIN) and thidiazuron (TDZ) (Braemer and Paris, 1987; Feeney and Punja, 2003; Lata et al., 2009; Wahby et al., 2013; Movahedi et al., 2015). Various explants have been studied for callus induction in hemp and cannabis, such as cotyledons and epicotyls (Wielgus et al., 2008; Movahedi et al., 2015), leaves (Mandolino and Ranalli, 1999; Page et al., 2021), and petioles (Slusarkiewicz-Jarzina et al., 2005). In this study and in that of Page et al. (2021), the genotype was shown to influence the extent of callus formation. Page et al. (2021) found that the addition of 2,4-dichlorophenoxyacetic acid to media was required for callus production, and that media containing DWK salts yielded healthier and faster-growing calluses than the MS medium. We did not test callus growth on the DWK salts medium. Interestingly, genotype SPQ, which responded poorly for shoot growth from meristems and nodal explants, responded well to callus production in this study. In contrast, genotype MDB, which responded very well to shoot growth, produced the least callus. The interest in deriving calluses from cannabis explants followed by regeneration of shoots is to allow genetic transformation studies to succeed (Feeney and Punja, 2003). In addition, there are numerous applications of tissue culture methods for cannabis and hemp improvement, and these have been described elsewhere (Adhikary et al., 2021). To date, however, there are few reports describing the successful utility of tissue culture approaches for C. sativa on a large and cost-effective scale.

The interest in micropropagation through tissue culture is to produce genetically identical, pathogen-free plants year-round in a limited amount of space (Yancheva and Kondakova, 2018; Lubell-Brand et al., 2021; Monthony et al., 2021). The results of this study, and those of previous investigators (Lata et al., 2009; Wang et al., 2009; Page et al., 2021) show that it is possible to obtain plantlets from tissue cultures of drug-type C. sativa, but the process requires a detailed assessment of the genotypic response, evaluation of the effect of basal salts medium, establishment of conditions promoting shoot growth and rooting frequency, and achievement of success in acclimatization. In addition, distinguishing between the requirements of stage 1 micropropagation (initiation of tissue cultures) verses stage 2 micropropagation (multiplication of shoots), as pointed out by Page et al. (2021), may result in differing protocols being developed. In contrast to the vegetative clonal propagation method that is widely used in the cannabis industry, tissue culture approaches will still play a minor role in commercial production until research efforts have resolved many of the remaining challenges and the cost-effectiveness of this approach is proven. This study has attempted to assess the variables that can affect success in plantlet recovery from stage 1 micropropagation using meristems and nodal stem explants in order to provide future directions in this area.