Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid

Gene therapy is a therapeutic strategy of delivering foreign genetic material (encoding for an important protein) into a patient’s target cell to replace a defective gene. Nucleic acids are embedded within the adeno-associated virus (AAVs) vectors; however, preexisting immunity to AAVs remains a significant concern that impairs their clinical application. Extracellular vesicles (EVs) hold great potential for therapeutic applications as vectors of nucleic acids due to their endogenous intercellular communication functions through their cargo delivery, including lipids and proteins. So far, small RNAs (siRNA and micro (mi)RNA) have been mainly loaded into EVs to treat several diseases, but the potential use of EVs to load and deliver exogenous plasmid DNA has not been thoroughly described. This review provides a comprehensive overview of the principal methodologies currently employed to load foreign genetic material into EVs, highlighting the need to find the most effective strategies for their successful clinical translation.

Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

TRANSFORMASI PLASMID PTRLI DENGAN TEKNIK ELEKTROPORASI PADA ASPERGILLUS TERREUS DAN UJI STABILITAS TRANSFORMAN

Aspergillus terreus is a Saprophyte fungus that produces several secondary metabolites as lovastatin (anti-cholesterol drug) and itaconic acid (a polymer material). Lovastatin is one of the statin class of drugs that have efficacy as antihypercholesterolemic. Plasmid transformation is the introduction and incorporation of exogenous plasmid into cells orprotoplast. In this study, pTRLI plasmid (pTRI inserts containing lovE gene as a regulator gene in the biosynthesis of lovastatin) will be transformed by electroporation transformation. The purpose of this research is transformation of pTRLI plasmid into protoplasts of Aspergillus terreus by electroporation and obtain stable transformants. The research was initiated by isolation of pTRLI plasmid. Then pTRLI plasmid was determined purity and concentration by nanodrop. Furthermore, Protoplasts of Aspergillus terreus were isolated enzymatically by adding an enzyme which can degrade the cell wall of Aspergillus terreus which contains chitin and cellulose. PTRLI plasmid were transformed into protoplasts of Aspergillus terreus by electroporation. These transformants were grown in Czapek-Dox medium containing pyrithiamine agar and the number of transformants mg-1 of pTRLI plasmid was calculated. Transformants were selected to grow in Czapek-Dox medium containing piritiamin 1 mg l-1. The number of transformants produced 187 transformants mg-1 of PTRLI plasmid. Transformants are stable up to five generations by growing the transformants in Czapek-Dox medium agar containing piritiamin 1 mg l-1. The success of the transformation indicated by ptrA gene in transformants that can be amplified by PCR. The size of fragment DNA is 801 bp.

Natural Competence and Recombination in the Plant Pathogen Xylella fastidiosa

ABSTRACTHomologous recombination is one of many forces contributing to the diversity, adaptation, and emergence of pathogens. For naturally competent bacteria, transformation is one possible route for the acquisition of novel genetic material. This study demonstrates thatXylella fastidiosa, a generalist bacterial plant pathogen responsible for many emerging plant diseases, is naturally competent and able to homologously recombine exogenous DNA into its genome. Several factors that affect transformation and recombination efficiencies, such as nutrient availability, growth stage, and methylation of transforming DNA, were identified. Recombination was observed in at least one out of every 106cells when exogenous plasmid DNA was supplied and one out of every 107cells when different strains were grown togetherin vitro. Based on previous genomic studies and experimental data presented here, there is mounting evidence that recombination can occur at relatively high rates and could play a large role in shaping the genetic diversity ofX. fastidiosa.