Delivery of CRISPR/Cas Components into Higher Plant Cells for Genome Editing

Russian Journal of Plant Physiology - CRISPR/Cas genome editing of plants is realized in three basic variants, including knockout mutations as indels, insertion of alien DNA fragments, and base...     

Esteblishment and characterization of the line of Fagopyrum tataricum morphogenic callus tolerant to aminotriazole

It is shown that the inhibitor of catalase 3-amino-1,2,4-triazole (AT) at the concentration of 2 mM affected differently growth of tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.) callus lines differing in the morphogenecity. In some cases, AT induced the death of a great fraction of non-morphogenic callus cells; in other cases, it inhibited growth and reduced viability of morphogenic callus. The death of non-morphogenic callus cells may be related to the accumulation of hydrogen peroxide and the development of oxidative stress. After morphogenic callus treatment with AT, we obtained a modified line 1?C8 AT tolerant to AT and differing from the original line in morphology, cell sizes, proliferative activity, and some biochemical characteristics. In the 1?C8 AT line, catalase was sensitive to this inhibitor action. In this case, catalase inactivation with AT did not increase the content of hydrogen peroxide in the cells, which may indicate the compensatory functioning of another/others mechanism(s) destroying hydrogen peroxide.     

Morphological and Molecular Analysis of Isolated Cultures of Tobacco Adventitious Roots Obtained by the Methods of Biolistic Bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).     

Water-soluble polyol-methanofullerenes as mitochondria-targeted antioxidants: Mechanism of action

The mechanism of an antioxidant action of water-soluble polyol – methanofullerenes C₆₀[C₉H₁₀O₄(OH)₄]₆ and C₆₀[C₁₃H₁₈O₄(OH)₄]₆ as the mild uncouplers of an oxidative phosphorylation and respiration is postulated. According to this mechanism, hydroxyl group of methanofullerenols can be protonated under excess of protons in the intermembrane space of hyperpolarized mitochondria. Protonation of fullerene derivatives is confirmed by the decrease in their negative Zeta potential in the pH below 5.4. Heavily protonated methanofullerenols become positively charged and move into the mitochondrial matrix. As a consequence, the proton gradient is dissipated, which causes a decrease in mitochondrial transmembrane potential (ΔΨ_m) and reduction in ROS production.     

Expression of different liver cell markers during the early prenatal human development

The expression of the liver cell markers, vimentin, desmin, cytokeratins 7, 18, 19, and stem cell markers CD34 and Bcl-2 in the early stages of the human prenatal development was studied. Desmin was revealed in sinusoidal liver cells between 3.5 and 12 weeks of gestation; in mesenchymal cells of ventral mesentery and hepatoblasts it was detected at the 4–7th weeks of gestation. During the hepatic period of hemopoiesis, desmin-positive sinusoidal cells were located close to blood cells. So-called “cholangio-” cytokeratins 7 and 19 displayed different expression patterns. Cytokeratin 7 was found only in cholangiocytes, and cytokeratin 19 in hepatoblasts until 15–16 weeks of prenatal development. Mesenchymal cells of the ventral mesentery expressed cytokeratins 18 and 19 more than hepatoblasts at the 4–7th weeks of gestation. Bcl-2 was seen in the same period in most sinusoidal and mesenchymal cells of the ventral mesentery. CD34 positive cells were detected in liver sinusoids between the 4th and 9th weeks of gestation but probably they are not progenitors of hepatocytes during embryonic development. Ventral mesentery mesenchyma was negative for CD34. These results let us hypothesize that hepatocytes and cholangiocytes may arise from different embryonic sources: cholangyocytes derive only from duodenal epithelial cells, while hepatoblasts develop most likely with the participation of mesenchymal cells.     

The "direct mitogen" lead nitrate causes an intensification in lipid peroxidation and Ito cell activation in the rat liver]

We have used biochemical and immunohistochemical methods to study lipid peroxidation and activation of Ito cells in rat liver after a single administration of lead nitrate, a "direct mitogen". Lead nitrate was shown to injure hepatocytes through an increased lipid peroxidation. Response to the injury included increase in the proliferative activity of parenchymal and sinusoidal liver cells. In addition, activation of Ito cells has been noted, which manifested as increased expression of desmin and increased proliferation. However, no transformation of Ito cells into myofibroblasts has been observed. We discuss the possible role of Ito cell activation in creating conditions for the proliferation of liver parenchymal cells after the injury by lead nitrate.     

The Ito cells in ontogenesis and liver regeneration

This review regards the Ito cells, which constitute one of the four types of liver sinusoidal cells. These cells synthesize many cytokines and extracellular matrix proteins. The Ito cells make an important link in retinoid metabolism. The role played by the Ito cells in liver hemopoiesis and differentiation of liver epithelium in prenatal ontogenesis is emphasized. The importance of the Ito cells in reparative liver regeneration is also discussed.     

The first report on the induction of hairy roots in Trapa natans,a unique aquatic plant with photosynthesizing roots

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation.

     

Characterization of the lipids of psychrophilic bacteria <Emphasis Type="Italic">Shewanella frigidimarina</Emphasis> isolated from sea ice of the Sea of Japan

Lipids of ten Shewanella frigidimarina strains isolated from sea ice samples of coastal areas of the Sea of Japan and of the type strains of psychrophilic bacteria S. frigidimarina ACAM 591^T and S. hanedai JCM 20706^T were analyzed. Most of the new isolates contained isoprenoid quinones typical of the genus Shewanella (Q-7, Q-8, MK-7, and MMK-7), a high level of branched acids (i-13:0 and i-15:0), and polyunsaturated fatty acid (20:5 ω3). Phospholipid fractions of marine isolates and the type strain S. frigidimarina ACAM 591^T contained not only the main phospholipids (phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol), but also an unknown phosphoaminolipid, which is probably typical of this bacterial species. The isolates exhibited a high level of phylogenetic similarity but were phenotypically heterogeneous. Two strains distinguished by their phenotypic characteristics differed also in the composition of fatty acids, isoprenoid quinones, and phospholipids. The use of chemotaxonomic markers for primary species identification of psychrophilic bacteria of the genus Shewanella is discussed.     

Somatic Embryogenesis and Bud Formation on Cultured Fagopyrum esculentum Hypocotyls

An indirect somatic embryogenesis via the development of proembryogenic cell complexes (PECC) was observed in the in vitro cultured hypocotyl explants of 4–5-day-old buckwheat (Fagopyrum esculentum Moench.) seedlings. PECC development was shown to depend on culturing conditions, including 2,4-D concentration and the period of explant exposure to 2,4-D, sucrose concentration, and explant density. The culturing protocol was designed to ensure the development of buckwheat somatic embryos in two-month period. The cytogenetic analysis demonstrated that this protocol did not affect the chromosome numbers in the regenerated plants.