De novo regeneration of Cannabis sativa L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability needed to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies, transformation and genome editing technologies, and opens the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplast have been isolated for transient gene expression studies, but attempts at protoplast-to-plant regeneration have not been reported. The present study aims to test the efficacy of using a callus culture system, as an abundant source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP) in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared to the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0×10^5 protoplasts per mL, division was observed, providing the first know report of cell division from cannabis protoplasts and setting the stage for future development of a protoplast-to-plant regeneration system.

A total of five protoclones were successfully cultured on PDA medium out of regenerated twenty two colonies of Termitomyces protoplast and further studied. Liquid MYG grown mycelial tissue is used for protoplast isolation by enzymatic digestionin a mixture containing Lysing enzyme 2% and Cellulase R10 2% in 0.6 M mannitol. The incubation conditions like temperature, shaking and time were standardized at 24ºC, 60 rpm and 10 hours, respectively for healthy protoplasts liberation. The purified protoplasts showed an average yield of 1.2 × 10⁷ cells/gm tissue with 31.60 ± 9.31% regeneration efficiency on specific medium and 77.12 ± 2.72% viability by FDA test. Four ISSR primers were used in this study resulting a total of 27 reproducible bands with mean value of 6.75. They showed similar banding pattern in all the lines with zero percent polymorphism ranged from 280 bp–2700 bp. The amplified rRNA-ITS gene showed ~600 bp size in gel and found a single restriction site for enzyme HaeIII in all the protoclones and parent with similar fragment size in all.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like Triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in Triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the Triticale ABA 8’-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in Triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in Triticale genes.

Plant cells are omnipotent and breeding of new varieties can be achieved by protoplast fusion. Such fusions can be achieved by treatment with poly(ethylene glycol) or by applying an electric field. Microfluidic devices allow for controlled conditions and targeted manipulation of small batches of cells down to single cell analysis. To provide controlled conditions for protoplast fusions and achieve high reproducibility, we developed and characterized a microfluidic device to reliably trap few of Arabidopsis thaliana protoplasts and induced a cell fusion by controlled addition of poly(ethylene glycol) (PEG, with a molecular weight of 6000). Experiments were conducted to determine the survival rate of isolated protoplasts in our microfluidic system. Afterwards, PEG induced fusion was studied. Our results indicate that the following fusion parameters had significant impact on the fusion efficiency and duration: PEG concentration, osmolarity of solution, and flow velocity. PEG concentration below 10% led to only partial fusion. Osmolarity of the PEG fusion solution was found to strongly impact the fusion process; complete fusion of two source cells sufficiently took part in slightly hyper osmotic solutions, whereas iso-osmotic solutions led to only partial fusion at 20% PEG concentration. We observed accelerated fusion for higher fluid velocities. Up to this study, it was common sense that fusion is one directional, i.e. once two cells were fused into one cell they stay fused. Here, we present for the first time reversible fusion of protoplasts. Our microfluidic device paves the way to a deeper understanding on the kinetics and processes of cell fusion.

Terrestrial algae are the group of photosynthetic organisms, which can survive in extreme conditions. pH is one of the most important factor, influencing on distribution of algae, both in aquatic and terrestrial ecosystems. The influence of different pH levels on cell volume and other morphological characters of authentic and reference strains of Chlorella vulgaris, Bracteacoccus minor, Pseudoccomyxa simplex, Chlorococcum infusionum, and Vischeria magna were studied. Chlorella vulgaris and Pseudoccomyxa simplex were the most resistant species, retaining its morphology in range of pH 4-11,5. The change of pH towards acidic and alkaline levels caused the increase of volume of Pseudoccomixa simplex and Vischeria magna cells according to polynomial regression model. Volume of Chlorella vulgaris cells increased from low to high pH according to linear regression model. Changes of pH levels did not have a significant impact on volume of Bracteacoccus minor and Chlorococcum infusionum cells. Low and high levels of pH caused thickening of cell walls of Chlorococcum infusionum and increase of oil containing substances in Vischeria magna and Bracteacoccus minor cells. Our study revealed a high resistance of studied species to extreme level of pH, which allow recommend these strains for broader use in biotechnology and natures protection.