Sporadic endophytic contamination was observed in a few cultures following initiation and shoot proliferation. Bacterial endophytes, characterized by white slimy growth was observed from explants on a single Petri dish (Figure 4A). Such explants were immediately identified and discarded. Similar endophytic contamination was observed in shoot tip explants and cultures of C. sativa ‘TJ’ that were established from greenhouse-grown plants [32]. Microbial contamination in micropropagation cultures is commonly observed in several plant species [38]. Endophytes can negatively influence in vitro cultures by slowing growth rate of cultures, causing inconsistent shoot and root production, and in severe cases, result in total culture loss [39,40]. Some endophytes may also be pathogenic and have the potential to produce plant disease [41]. A diverse range of microbial bacterial and fungal endophytes have been observed in C. sativa [42]. Endophytic contamination in cultures is observed after a few weeks of culture establishment but in certain instances, endophytes can stay latent for several years in apparently clean cultures before being manifested in the growth medium [39]. In previous Cannabis micropropagation studies, explants were found to exhibit extremely high rates of endophytic contamination despite being subjected to rigorous surface-sterilization techniques [17,32]. This was attributed to the explant type and source plants from which they were obtained. The use of shoot apical meristems explants for initiating Cannabis cultures resulted in a significantly lower level of contamination compared to the use of nodal explants [17]. In our studies, all cultures were established after carefully excising the shoot apical meristems under a dissecting microscope (Figure 1C,D). This might be the reason for the low level of endophytic contamination observed in cultures and a high shoot proliferation rate. Meristem tip culture is the most widely used method for large-scale production of clean, disease-free plant materials. Micropropagation using meristem tip culture involve the careful excision of the shoot apical meristem under a dissecting stereomicroscope [43]. Such explants consist of the apical meristem along with a few leaf primordia and are typically between 0.5 and 1.0 mm in size. Apical meristems are the preferred choice of explants for elimination of endophytes due to the undifferentiated state of cells in the meristematic dome, which lack vascular differentiation and are rapidly dividing, which may result in exclusion of microbes from this region [16,43]. Successful excision of shoot apical meristems and their subsequent culture can result in rapid shoot proliferation and production of healthy, disease-free clonal plant material. Although micropropagation largely preserves clonal fidelity of regenerated plants, somaclonal variations may be introduced during various stages of culture [44] and are considered undesirable from a commercial point of view. The occurrence and extent of somaclonal variation is dependent on the explant type and developmental stage, culture duration, plant regeneration pathway and medium composition [45]. Genetic analysis of regenerated plants can assist in the identification of any somaclonal variations occurring during the culture process [24,25]. A large number of somaclonal variants were observed in micropropagation-derived Cannabis plants following prolonged maintenance under in vitro conditions [46]. Genetic analysis of regenerated plants using genotype by sequencing (GBS) revealed a high frequency of genetic variation that could not be detected/identified by utilizing single-sequence repeat (SSR) markers. A large number of identified variations did not exhibit any visible phenotypic differences compared to the mother plants.

Among the two cultivars studied, ‘Purple’ produced a greater number of healthy shoots that exhibited normal shoot and leaf morphology (Figure 4B). Similarly, cultures exhibiting hyperhydricity were observed in cultures with higher mT concentrations (Figure 4C). Hyperhydric cultures appeared translucent and pale green in color, were characterized by water-soaked lesions and leaves appeared curled and brittle in contrast to healthy looking cultures (Figure 4C,D). Hyperhydricity can occur from an abnormal ratio of nitrate: ammonium ions in the medium, excessive levels of cytokinins or the production of ethylene during culture growth. High cytokinin concentration in the culture medium may result in hyperhydricity [47,48]. Ivanova and Van Staden [49] found cytokinins (TDZ, BA, and zeatin) to increase hyperhydricity in Aloe polyphylla in comparison to media without synthetic phytohormones. It was also found that an increase in the level of cytokinins (BA and zeatin) promoted hyperhydricity as well [49]. Liu et al. [50] found cytokinins to increase hyperhydricity in Allium sativum L. using BA and kinetin. Wróbel et al. [51] stated that the use of TDZ on industrial hemp shoots tip and nodal segments caused morphological issues such as hyperhydricity, leaf narrowing and rolling, and decreased shoot growth. Page et al. [26] performed a preliminary experiment with MS media and 0.5 μM of TDZ and observed several negative effects such as hyperhydricity, decreases in shoot multiplication, and callusing. In preliminary studies with medium optimization, we observed similar effects from the use of MS macroelements and microelements, and TDZ in the culture medium. In the current study, mT at higher concentrations may have inhibited plant growth by increasing hyperhydricity, which resulted in reduced shoot proliferation [47,48,50]. This may explain why more shoots were produced on control media. Cannabis is a fast-growing plant [52], which might be indicative of a high level of phytohormones that promote rapid growth and development. Thus, the addition of exogenous cytokinins may have caused an imbalance of the total levels in cultures, leading to hyperhydricity and poor shoot proliferation rate. Therefore, phytohormones may not be required for shoot proliferation in certain cultivars. Although, it is important to note that each cultivar reacts differently in vitro, which indicates the need for cultivar specific protocols to be developed when optimizing micropropagation protocols.

Cytokinins in the culture medium may influence ethylene biosynthesis [48,53]. Žd’árská et al. [48] found cytokinins to upregulate ethylene biosynthesis in Arabidopsis during root growth. In tissue culture, ethylene accumulates in culture vessels due to plant wounding, typically during transfer to fresh medium or as a stress response [1]. Ethylene production during the shoot proliferation stage can negatively influence culture growth [54]. Ethylene accumulation at high levels promotes hyperhydricity [55]. Hyperhydricity can cause a significant reduction in culture growth including shoot proliferation, poor rooting, and a low plant regeneration rate in tissue culture. Lack of chlorophyll is also a symptom of hyperhydricity [56]. A major cause of hyperhydricity is when the apoplast, which is the intercellular spaces within plants, is flooded with water instead of gas [56]. Van Den Dries et al. [56] found that air volume in the apoplast of hyperhydric Arabidopsis seedlings decreased significantly while water volume increased. When water in the culture vessel increases, due to high humidity or type of gelling agent, the apoplast can flood, resulting in hyperhydricity [56]. It was reported that an enzyme involved in ethylene biosynthesis is elevated when explants are hyperhydric [56]. High levels of auxin biosynthesis during rapid cell division and expansion also leads to excess water uptake and shoot deformation, resulting from hyperhydricity [54]. Culture hyperhydricity can be alleviated by increasing agar concentrations, using vented culture vessels to reduce humidity or release ethylene, altering the nitrate: ammonium ratio and/or decreasing the amount of cytokinin in culture media [30,57].

The incidence of in vitro flowering during culture proliferation was also observed. Male and female inflorescences were observed at various stages of culture growth and development (Figure 5). Normal floral morphology was observed in both male and female flowers, which was characterized by the presence of stamens and pistils (Figure 5B,D). Such cultures exhibited slow growth and eventual growth termination. Cannabis is a dioecious plant species where male and female flowers are borne on separate plants. In our studies, cultures were initiated and established from female stock plants with confirmed sex under field conditions. The production of male flowers in such cultures may be indicative of a stress response encountered during in vitro culture [58]. Since Cannabis is an annual species, in vitro flowering during micropropagation is undesirable, may result in a decline in shoot proliferation and eventually termination of cultures [59]. Ethylene biosynthesis and accumulation in culture vessels may also lead to in vitro flowering [1]. The use of culture vessels with vented caps may lead to a decrease in hyperhydricity, ethylene accumulation and significantly improve the quality of cultures and the response to micropropagation [18,30]. Alternatively, addition of chemicals such as silver nitrate and polyamines including spermidine and spermine to the culture medium can decrease the biosynthesis of ethylene and its subsequent negative effects such as in vitro flowering and hyperhydricity [47,60].