The scientific interest in cannabis plants’ beneficial properties has sparked certain interest in many areas recently ranging from recreation use, medical use, and textiles and apparel industry (Blake et al. 2017; Hill and Palastro 2017; Mahmud et al. 2021; Zimniewska 2022; Zimniewska et al. 2021). There is even further interest in cell-to-cell communication in these plants in the form of extracellular vesicles (EVs) (Jones et al. 2018), a class of vesicles that are known to be released by all forms of organisms. Isolation and functional characterization of plant-derived extracellular vesicles (PDEVs) have been used to demonstrate their contents to include biomolecules, lipids, proteins, metabolites, and small RNAs (sRNA) (Urzì et al. 2021; Cui et al. 2020). PDEVs play a crucial role in the cross-kingdom exchange of molecules ranging from plant-plant to plant host-microbes or vector trafficking molecules, especially during defense response. Establishing the most appropriate and efficient isolation procedure for PDEVs remains a challenge due to vast differences in the physio-structural characteristics of different plants within the same genera and species. For these experiments, we employed a crude but standard isolation procedure for the extraction of apoplastic wash fluid (AWF), which is known to contain the PDEVs (Huang et al. 2021).

In this study, we employed isolation of PDEVs from five (5) cultivars of cannabis plants: Citrus (C), Henola (HA), Bialobrezenski (BZ), Southern-Sunset (SS), and Southern Cat-Daddy (CAD) for these studies. The following cultivars were selected based on their usage in the production of fiber for textiles [HA, BZ] or cannabidiol (CBD) products [C, SS, CAD] products. Approximately, 75–150 leaves were collected from each plant cultivar. The plants ranged from 2 months in age to 10 months old. In brief, AWF was extracted via negative pressure permeabilization and infiltration of the plants using buffer containing [0.1 M NaCl, 30 mM (N-morpholino)ethanesulfonic acid, and 2 mM CaCl], followed by high-speed differential ultracentrifugation to collect PDEV pellets. Initially, we evaluated CAD (approximately 75 leaves) as our pilot cultivar (Fig. 1A). These PDEVs ranged in size from 10 to 1000 nm, with the average PDEVs size of all cultivars being of 121 to 175 nm (150 leaves). The mean particle size of PDEVs extracted from HA was significantly higher than that of CAD and BZ (Fig. 1B). The concentrations of PDEVs extracted from 150 leaves of each cultivar were also plentiful, ranging from 2.9 × 10⁷ to 2.3 × 10⁸ particle/mL. PDEVs were analyzed for DNA, RNA, and protein. SS PDEVs contained significantly higher DNA content in comparison to all other cultivars examined (150 leaves) (Fig. 1C and Table 1). However, HA had significantly higher sRNA content within HA-derived PDEVs isolated as compared to all other cultivars (Fig. 1C and Table 1). The PDEV isolation technique described in this study is similar to that previously described by Huang et al. (2021); however, the technique has been improved upon in this study given that we analyzed various strains of Cannabis sativa plant and found more tetraspanins (CD9 and CD81) including heat-shock proteins; we also employed the use of a wide range Zeta Particle Metrix analyzer for hydrodynamic particle sizes and concentration analysis. This finding is in line with HA-derived PDEVs having a large mean particle size (Fig. 1B). CAD-derived PDEVs had significantly higher protein levels in comparisons to all other cultivars examined (150 leaves) (Fig. 1C and Table 1). PDEVs from four CAD cultivars were examined for cluster of differentiations (CD) 81 and 9. CD9 was highly expressed in CAD PDEVs. All AWF from CAD plants were examined for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (75 and 150 leaves) (Fig. 1D). In addition, C, HA, BZ, SS, and CAD PDEVs all had low levels of expression of heat-shock proteins 60, 70, and 90. Rubisco protein was evaluated to rule out contamination from cytoplasmic molecules and cell debris in CAD cultivars.