Tissue culture techniques are essential in modern plant biotechnology, enabling controlled in vitro cultivation under sterile conditions. Among these, micropropagation stands out as a widely adopted method, facilitating the large-scale cloning of plants (Davey and Anthony, 2010). However, the lack of standardized protocols for Cannabis sativa L. micropropagation continues to be a significant limitation (Stephen et al., 2023).

While micropropagation focuses clonal propagation, protoplast-based technologies unlock versatile possibilities by removing the cell wall. These applications include cellular dedifferentiation, genetic transformation, genome editing, and expression system analysis, as well as the production of secondary metabolites (Jiang et al., 2013; Aoyagi, 2011; Hesami et al., 2021). Under optimal conditions, protoplasts can regenerate into whole plants, enabling advanced applications such as cloning, non-chimeric genetic modifications, ploidy modification, and interspecies somatic hybridization (Jiang et al., 2013; Adhikary et al., 2021; Hesami et al., 2021).

In recent years, there has been a notable increase in research activity focused on cannabis protoplasts, particularly in the optimization of isolation methods, their application for transient gene expression, and targeted genome editing. However, despite these advances, data on the de novo regeneration of cannabis from protoplasts remain lacking (Hesami et al., 2021).

The complexity of protoplast isolation lies in its reliance on multiple factors, including plant genotype, source material, cultivation, and enzymatic conditions, which require precise species-specific optimization to achieve high yield and viability (Evans and Bravo, 2013). The earliest documented attempt to isolate cannabis protoplasts was made as early as 1979. The study examined the effects of enzymes, solution osmolarity, and source material type, using young and old leaves and calluses for isolation. The highest reported concentration was 10⁵ cells/ml, achieved using pectinase and driselase. While the feasibility of isolation was demonstrated, essential details such as cell viability or the amount of input material were not published (Jones, 1979). It was not until 2007 that cannabis protoplasts were used in a study investigating cell death. This study described the composition of enzymatic solutions (cellulase, macerozyme, and pectolyase), but data on concentration and viability were not provided (Morimoto et al., 2007). Significant progress has been made over the past decade. Flaishman et al. (2019) investigated the isolation of cannabis protoplasts but did not disclose their methodology. Subsequently, Lazič (2020) optimized the isolation process from calluses, young leaves, and hypocotyls of in vitro germinated cannabis. The best results were achieved with etiolated hypocotyls, yielding a concentration of 1.8 · 10⁵ cells/ml with 25.7% viability. The year 2021 marked a turning point with two landmark studies. Matchett-Oates et al. (2021) focused on young leaves derived from in vitro cultivated apical explants, achieving yields of up to 7.8 · 10⁶ cells/g with 72% viability. A similar approach was applied by Beard et al. (2021) using an enzymatic solution developed for A. thaliana enriched with pectolyase, resulting in yields of 2.27 · 10⁶ cells/g and 82% viability. Further advances followed in 2022. Kim et al. (2022) optimized enzymatic treatments for fully developed greenhouse-grown leaves, yielding 9.7 · 10⁶ cells/g, although viability was not reported. Zhu et al. (2022) compared hypocotyl and cotyledon-derived protoplasts, with cotyledons providing the highest recorded yield of 1.15 · 10⁷ cells/g and 98.5% viability. The most recent study, published in 2024, focused on protoplast isolation from calluses derived from hypocotyls. Using a medium containing 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of lignin synthesis, significantly enhanced protoplastization efficiency by weakening the cell wall. The average yield was 8.8 · 10⁴ cells/ml, with a viability rate of 92.1% (Monthony and Jones, 2024). A comprehensive summary of available publications focusing on the isolation of cannabis protoplasts, along with detailed information on the isolation conditions, is provided in Table 1 .

De novo regeneration, a crucial attribute of protoplasts, enables transformative applications in genetic engineering and cultivar development. In cannabis, however, this regenerative potential remains largely unexplored, creating a critical bottleneck for advancements in protoplast-based biotechnologies (Li et al., 2022; Hesami et al., 2021; Monthony and Jones, 2024).

To date, there are only two published reports on the regeneration of cannabis protoplasts. The first, by Flaishman et al. (2019), found that just 4% of protoplasts survived for 48 hours in liquid culture, with no plant regeneration achieved. More recently, Monthony and Jones (2024) provided the most comprehensive results to date, describing partial regeneration during the initial cultivation period. Cell division was observed after six days, and microcallus formation occurred after three weeks. However, cell viability declined significantly at this stage, preventing further development. The study also highlighted the critical role of culture density: no response was observed at 0,5 · 10⁵ cells/mL, limited division occurred at 1 · 10⁵ cells/mL, and the best results were achieved at 2 · 10⁵ cells/mL.

The initial study of cannabis protoplast isolation also involved interspecific somatic hybridization with tomato. However, no hybrid plant regeneration was achieved (Jones, 1979). Significant progress towards transient transformation techniques for cannabis protoplasts was initially reported by Matchett-Oates et al. (2021) and Beard et al. (2021). Both studies employed plasmid DNA carrying reporter expression cassettes, achieving transformation efficiencies of 23% and 31%, respectively. The following year, Kim et al. (2022) reported a transformation efficiency of 55.3% by using fusion genes involved in cannabinoid biosynthesis tagged with GFP, which allowed the visualization and localization of these enzymes in subcellular compartments. Later that year, Zhu et al. (2022) demonstrated a transformation efficiency of 75.4% using GFP to track the nuclear localization of the transcription factor CsMYC2. Transient transformation of protoplasts has emerged as a powerful tool for evaluating gRNA constructs for CRISPR/Cas9-based genome editing. Zhang et al. (2021) developed a system to validate gRNA specificity, enabling efficient mutagenesis of target gene regions. The phytoene desaturase marker gene was used as the editing target. This work culminated in the creation of the first stable genetically modified cannabis plant, albeit not derived from protoplasts.

Protoplastization enhances chromatin accessibility, initiating stochastic gene expression changes that drive dedifferentiation and foster regenerative potential. During this process, cells shift from their original somatic programming to a new state, enabling division and developmental plasticity (Zhao et al., 2001; Xu et al., 2021). However, successful dedifferentiation depends on external phytohormones, particularly auxins (Aux) and cytokinins, without which cells rapidly degenerate. Aux alone promote redifferentiation, while their combined action induces chromatin decondensation and re-entry into the cell cycle (Vissenberg et al., 2000; Zhao et al., 2001). As a marker of the activated Aux signaling pathway, the IAA-2 gene can be used (Abel et al., 1994, 1995; Yang et al., 2017; Gao et al., 2018; Král et al., 2022).

Proliferating cell nuclear antigen (PCNA) is a highly conserved protein found in all eukaryotes and archaea, playing a central role in DNA replication and various other nuclear processes. Its primary function is to coordinate the recruitment of replication-associated proteins, ensuring efficient replication progression. Additionally, PCNA is involved in translesion DNA synthesis, base and nucleotide excision repair, mismatch repair, recombination, chromatin remodeling, sister chromatid cohesion, and cell cycle regulation (Lee et al., 2019). For this reason, the PCNA gene is a widely used cell division marker and has been applied in protoplast cultures of various species, including tobacco and cucumber (Williams et al., 2003; Cápal and Ondřej, 2014), and was therefore selected for use in the present study.

Abscisic acid (ABA) is a pivotal phytohormone that orchestrates various physiological processes in plants. It plays a central role in managing responses to abiotic stresses and regulating developmental transitions from embryogenesis to senescence (Finkelstein et al., 2008). ABA regulates the expression of key gene families including protein phosphatases 2C (PP2C) and late embryogenesis abundant (LEA) genes. LEA proteins are critical protectants, safeguarding cells against dehydration and damage caused by extreme temperatures and salinity (Liu et al., 2019). In contrast, PP2C proteins function as negative regulators of ABA signaling, maintaining a balanced and appropriate adaptive response to environmental stresses (Park et al., 2009). To evaluate the extent of abiotic stress experienced by the cells in our system, we analyzed the expression of representative members of both LEA and PP2C gene families.

Protoplast viability is significantly affected by oxidative stress, caused by the accumulation of reactive oxygen species (ROS). While ROS are natural byproducts of cellular metabolism, their excessive generation during cell wall enzymatic digestion can disrupt the plasma membrane and exert toxic effects (Cassells and Curry, 2001; Zhang et al., 2022). Plants counteract ROS through various antioxidant mechanisms such as the ascorbate-glutathione cycle and catalases, which convert hydrogen peroxide into water via enzymatic activity of ascorbate peroxidases (APX) or catalases (CAT) (Caverzan et al., 2012; Ratanasanobon and Seaton, 2013). The success of protoplast cultivation depends on the timely activation of antioxidant systems, which typically peak within the first three days. This early cultivation phase is critical in determining whether protoplasts will successfully progress into the cell cycle (Kapur et al., 1993; Siminis et al., 1994; Ondřej et al., 2010; Moricová et al., 2013; Cápal and Ondřej, 2014). To assess the activation of antioxidant responses, we monitored transcript levels of CAT and APX genes by RT-qPCR throughout the cultivation period.

This study addresses key challenges associated with cannabis protoplast isolation and regeneration. By investigating expression changes of selected genes, we provide valuable insights into the physiological state and regenerative potential of cannabis protoplast cultures. Our findings help to establish a foundation for protoplast-to-plant regeneration methods in cannabis.

The donor material’s age, developmental stage, and cultivation conditions play a critical role in the success of protoplast isolation. Our study suggested that younger leaves are the most suitable, consistent with Evans and Bravo (2013) and Lazič (2020) findings. In contrast, attempts to isolate protoplasts from leaves of plants transferred to in vitro conditions, as well as from six-month-old cultures, were unsuccessful. This failure likely stems from physiological and morphological changes induced by stress during the transition to in vitro environments and suboptimal cultivation conditions (Evans and Bravo, 2013). Many samples exhibited senescence or vitrification, which rendered them unsuitable for protoplast isolation. These challenges may result from altered cell wall development, thinning of epidermal protective layers, or significant enlargement of vitrified cells (Pâques and Boxus, 1987; Rasco and Patena, 1997; Kemat et al., 2021). Efforts to improve protoplast isolation by shortening the enzymatic digestion period proved ineffective.

We tested various culture media, growth regulators, and container closure methods to optimize cultivation conditions. The findings confirm the beneficial effects of DKW medium, aligning with the results of Page et al. (2021). Among the tested growth regulators, 2iP yielded the most favorable outcomes, while TDZ produced the weakest response, consistent with the observations reported by Stephen et al. (2023). Reduced gas exchange under airtight conditions was identified as a critical limiting factor, as it decreases photosynthetic efficiency and forces greater reliance on nutrients from the culture medium. Elevated humidity further alters stomatal activity, increases concentrations of genotoxic H₂O₂, and amplifies overall cultivation stress. Under these conditions, the expression of stress and nutrition-related genes can be significantly affected, adversely impacting the cultivation process and leading to vitrification symptoms (Ševčíková et al., 2016; Xu et al., 2019; Abdalla et al., 2022; Král et al., 2022). The beneficial effects of more permeable closure observed in this study have also been demonstrated in other plant species (Majada et al., 1997; Tsay et al., 2006). Despite these optimizations, no enhancement in protoplastization efficiency was achieved.

The genetic background and genotype also significantly influence protoplast isolation outcomes, with variability observed even within plants of the same cultivar (Evans and Bravo, 2013). To ensure robust results, 9 cultivars were included in this study, representing both high and low CBD strains. Although previous studies have reported successful protoplast isolation from the ‘Finola’ cultivar (Lazič, 2020; Monthony and Jones, 2024), our findings revealed superior performance with the ‘USO 31’ cultivar. This study marks the first successful protoplast isolation from the leaves of this cultivar.

The enzymatic solution ER1a, developed by Matchett-Oates et al. (2021), proved to be the most suitable option for protoplast isolation in our study. Although the average yield was approximately 27% lower than previously reported, the maximum yield obtained in our experiments exceeded the values published by these authors. The reduced average yield may be attributed to genotypic differences between plant materials, variations in cultivation conditions, or a lower temperature used during enzymatic digestion, all known to influence protoplast isolation efficiency. Attempts to enhance isolation efficiency through the addition of pectolyase, as suggested by previous studies (Morimoto et al., 2007; Beard et al., 2021; Kim et al., 2022; Monthony and Jones, 2024), were unsuccessful. Specifically, lower pectolyase concentrations resulted in reduced yields, while higher concentrations led to complete isolation failure. These outcomes were likely caused by excessive enzymatic activity, resulting in oxidative stress and cellular damage (Ishii, 1987; 1988).

This study represents only the second report of cannabis protoplast proliferation initiation and microcallus formation, achieved earlier than previously reported (Monthony and Jones, 2024). For cultivation, we utilized a slightly modified regeneration medium originally developed for A. thaliana (Mathur and Koncz, 1998). The suitability of this medium for the initiation phase of cannabis protoplast cultures was demonstrated by expression analysis and successful microcallus formation.

Expression analysis of the proliferation marker PCNA revealed early activation, likely initiated during the isolation process, which is consistent with previous observations in other plant species (Cápal and Ondřej, 2014). This early upregulation, together with its continued increase during cultivation, supports the onset of the S-phase and indicates a strong proliferative potential of the isolated protoplasts (Williams et al., 2003; Cápal and Ondřej, 2014).; Importantly, although PCNA may also be expressed in non-proliferating cells (Van Diest et al., 1998), no such expression pattern was observed in our system, suggesting a close association with proliferative activity.

A significant increase in IAA-2 expression was detected after 24 hours of cultivation, indicating heightened auxin signaling activity upon exposure to phytohormones in the regeneration medium. The up-regulation of IAA-2 expression during successful protoplast cultivation has similarly been reported in A. thaliana (Pasternak et al., 2021).

PP2C-1 and LEA34 are associated with the abscisic acid (ABA) signaling pathway, which up-regulates their expression (Liu et al., 2019; Park et al., 2009). In our study, protoplast isolation resulted in a marked reduction in ABA signaling, which remained consistently low throughout the cultivation. Importantly, because the expression levels of these genes did not change significantly over time, it is likely that the protoplasts rapidly adapted to the in vitro culture conditions. These findings suggest that the cells were not exposed to substantial or increasing abiotic stress during cultivation, in contrast to the elevated stress levels observed in cannabis in vitro cultures (Král et al., 2022).

Oxidative stress, a critical factor affecting protoplast viability, was assessed through the expression of two key antioxidant genes, APX and CAT. These enzymes act within interconnected pathways linked by a shared substrate and display a dynamic balance during stress response (Caverzan et al., 2012; Ratanasanobon and Seaton, 2013). In our system, APX showed dominant upregulation during early cultivation, suggesting the primary role of the ascorbate–glutathione cycle in ROS detoxification. In contrast, CAT played a complementary role. Elevated expression levels of both genes correlated with reduced ROS levels, which is essential for chromatin reorganization and the initiation of dedifferentiation (Ondřej et al., 2008; 2010). The consistent expression trends of APX and PCNA genes further underscore the coordinated mechanisms driving protoplast proliferation. The functional relevance of PCNA, APX, and CAT expression was further supported by comparisons between protoplast cultures of differing viability. Cultures with high viability (>60%) maintained robust expression of all three genes, reflecting a strong capacity for proliferation and oxidative stress mitigation. In contrast, low-viability cultures (<15%) showed markedly reduced expression all three genes, consistent with impaired stress responses and limited proliferation potential (Ondřej et al., 2010).

This study successfully established a protocol for the Cannabis sativa L. protoplast isolation and cultivation using young leaves from in vitro-grown ‘USO 31’ seedlings. The application of a partially modified regeneration medium, originally designed for Arabidopsis thaliana, enabled the initiation of cannabis protoplast cultures. Transcriptomic analyses revealed that the protoplast cultures were viable and exhibited robust antioxidant responses, with stress levels lower than those of in vitro-germinated plants. Importantly, the protoplasts progressed into the S phase of the cell cycle, underscoring their potential for further developmental studies. By reporting only the second successful cultivation of cannabis protoplasts, this work lays the foundation for future research into cannabis protoplast biology and applications. The insights gained here could significantly advance research and development in the largely unexplored domain of cannabis protoplast cultures.