Wall Slip-Free Viscosity Determination of Filled Rubber Compounds Using Steady-State Shear Measurements.

The high-pressure capillary rheometer (HPCR) represents a state-of-the-art instrument for the determination of rheological properties for plastics and rubber compounds. Rubber compounds have an increased tendency to exhibit flow anomalies depending on the compound ingredients and the processing parameters. Combined with non-isothermal effects due to dissipative material heating, this causes rheological material measurements and the resulting material parameters derived from them to be affected by errors, since the fundamental analytical and numerical calculation approaches assume isothermal flow and wall adhesion. In this paper, the applicability of the empirical rheological transfer function of the Cox-Merz rule, which establishes a relationship between shear viscosity measured with a HPCR and complex viscosity measured with a closed cavity rheometer (CCR), is investigated. The Cox-Merz relation could not be verified for an unfilled EPDM raw polymer or for filled, practical rubber compounds. Using a closed cavity rheometer, a methodology based on ramp tests is then introduced to collect wall slip-free steady-state shear viscosity data under isothermal conditions. The generated data show high agreement with corrected viscosity data generated using the HPCR, while requiring less measurement effort.

Targeted knockout of a conserved plant mitochondrial gene by genome editing.

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

Targeted introduction of heritable point mutations into the plant mitochondrial genome.

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.