Incorporation of Magnetic Nanoparticles into Protoplasts of Microalgae Haematococcus pluvialis: A Tool for Biotechnological Applications

Intensive research on the use of magnetic nanoparticles for biotechnological applications of microalgae biomass guided the development of proper treatment to successfully incorporate them into these single-cell microorganisms. Protoplasts, as cells lacking a cell wall, are extensively used in plant/microalgae genetic manipulation as well as various biotechnological applications. In this work, a detailed study on the formation of protoplasts from Haematococcus pluvialis with the use of enzymatic and mechanical procedures was performed. The optimization of several parameters affecting the formation of protoplasmic cells and cell recovery was investigated. In the enzymatic treatment, a solution of cellulase was studied at different time points of incubation, whereas in the mechanical treatment, glass beads vortexing was used. Mechanical treatment gave better results in comparison to the enzymatic one. Concerning the cell recovery, after the protoplast formation, it was found to be similar in both methods used; cell viability was not investigated. To enhance the protoplast cell wall reconstruction, different “recovery media” with an organic source of carbon or nitrogen were used. Cell morphology during all treatments was evaluated by electron microscopy. The optimal conditions found for protoplast formation and cell reconstruction were successfully used to produce Haematococcus pluvialis cells with magnetic properties.

Optimization of Protoplast Formation, Regeneration, and Viability in Sorangium cellulosum

Sorangium cellulosum can product many secondary metabolites that is unique structural and makes these microorganisms highly attractive for drug development, especially epothilone, on cancer cells a cytotoxic macrolide which is naturally produced by Soxhlet cellulose that have the action of microtubule stabilization, is a promising anticancer drug. In this research, the factors affecting the regeneration and preparation of the protoplast of Sorangium cellulosum were discussed, those were regeneration media, enzymes and osmotic stabilizers. This study provide the distruction for improving the production of epothilone through genome shuffling, mutation, fusion and transformation.

Genome rearrangements and megaplasmid loss in the filamentous bacteriumKitasatospora viridifaciensare associated with protoplast formation and regeneration

Filamentous Actinobacteria are multicellular bacteria with linear replicons.Kitasatospora viridifaciensDSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium ofK. viridifaciensdrives morphological diversity. Characterization and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried lesions in the right arm of the chromosome. Strikingly, the lesion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.RepositoriesGenomic sequence data for strain B3.1 has been deposited in the NCBI SRA database under accession code SAMN11514356.

Pre-isolation, isolation and regeneration protoplasts from leaf mesophyll of in vivo Malus domestica ‘Anna’ cv.

To isolate protoplast, a pre-treatment was completed with the order to reduce and identify the phenolic contents round the year to encourage the isolation of protoplasts. Protoplasts from in vivo mesophyll leaves of apple cultivar “Anna” was isolated from 15 days old leaves by plasmolying in medium containing 90 g L-1 mannitol for half hour, then 130 g L-1 mannitol for half hour. Then using enzymatic mixture involving (1.5% cellulase + 0.5% pectianase + 1.5% Macrozyme) Prior to isolation. Anyhow, diverse factors, for example, Osmotic pressure, incubation period, sieve pore size, centrifugation period and hormonal balance was estimated using the techniques for isolation. The quantity of cells was computed as the quantity of cells per square on haemocytometer. A considerable higher yield of protoplast formation was noted in the CPW medium using a pore size of 25 µm with using incubation period for 20 hours. Moreover, the best protoplast regeneration with using of protoplast density of 2.0 x 105 in MS medium supplemented by 1.0 mg L-1NAA and 0.3 mg L-1BAP. We believed that our protocol might encourage the plant recovery using in apple somatic hybridization programs.