Evaluation of a morphological marker selection and excision system to generate marker-free transgenic cassava plants

The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar “KU50” was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19–21% was observed. Among the total number of ESP explants, 32–38% regained normal extended shoot phenotype and 88–96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.     

Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes

Journal of Plant Research - The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion,...     

Crucial role of heme oxygenase-1 on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents

Cancer cells acquire drug resistance via various mechanisms including enhanced cellular cytoprotective and antioxidant activities. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation and drug resistance. We aimed to investigate roles of HO-1 in human cholangiocarcinoma (CCA) cells for cytoprotection against chemotherapeutic agents. KKU-100 and KKU-M214 CCA cell lines with high and low HO-1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents, gemcitabine (Gem) and doxorubicin. Inhibition of HO-1 by zinc protoporphyrin IX (ZnPP) sensitized both cell types to the cytotoxicity of chemotherapeutic agents. HO-1 gene silencing by siRNA validated the cytoprotective effect of HO-1 on CCA cells against Gem. Induction of HO-1 protein expression by stannous chloride enhanced the cytoprotection and suppression of apoptosis caused by anticancer agents. The sensitizing effect of ZnPP was associated with increased ROS formation and loss of mitochondrial transmembrane potential, while Gem alone did not show any effects. A ROS scavenger, Tempol, abolished the sensitizing effect of ZnPP on Gem. Combination of ZnPP and Gem enhanced the release of cytochrome c and increased p21 levels. The results show that HO-1 played a critical role in cytoprotection in CCA cells against chemotherapeutic agents. Targeted inhibition of HO-1 may be a strategy to overcome drug resistance in chemotherapy of bile duct cancer.     

Cellular adaptation mediated through Nrf2-induced glutamate cysteine ligase up-regulation against oxidative stress caused by iron overload in β-thalassemia/HbE patients

Oxidative stress caused as a result of iron overload is implicated in clinical manifestation of beta-thalassemia/haemoglobin E (β-Thal/HbE). In this study, we investigated the cellular adaptation against oxidative stress in β-Thal/HbE patients. Twenty-four paediatric β-Thal/HbE patients and 22 healthy controls were recruited in the study. Blood samples from patients exhibited iron overload, elevation of lipid peroxidation, and marked diminution in the reduced glutathione (GSH) level. However, expression of glutamate-cysteine ligase catalytic (GCLC) subunit, a key enzyme in GSH biosynthesis, was up-regulated when compared with that in controls. GCLC protein levels were correlated with serum iron. There was an enhanced binding activity of the oligonucleotide probe for Nrf2-driven antioxidant response element (ARE) to nuclear protein from blood mononuclear cells of thalassemia subjects. In conclusion, β-Thal/HbE patients exhibit elevated plasma levels of GCLC expression and Nrf2-ARE binding activity, which may account for their adaptive survival response to oxidative stress.