Re-evaluation of the effects of growth regulators on callus induction and shoot regeneration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of lettuce

Lettuce (Lactuca sativa) transformation varies by genotype. Various culture parameters have been studied in order to improve the transformation efficiency of lettuce cultivars. However, no improved transformation procedure for recalcitrant lettuce cultivars has yet been established. Here, we demonstrate the effects of varying concentrations and distinct combinations of growth regulators on recalcitrant lettuce transformation efficiency. More precisely, we assessed differences in the effects of several growth regulator combinations, including N-6(2-isopentenyl)-adenine (2ip), on induction of callus and regeneration of shoots after co-cultivation with Agrobacterium. When two commercial recalcitrant cultivars, Red Romaine and Bibb, were cultured on a medium with 2ip 1 mg l⁻¹, IAA 0.1 mg l⁻¹, and subsequently transferred to a second medium with BA 0.4 mg l⁻¹, NAA 0.05 mg l⁻¹ for selection and shoot regeneration, transformation efficiencies reached 8 and 9%, respectively. Stable integration and transmission of the transgene in T1 generation plants were confirmed by molecular analysis. This procedure represents a simple, efficient, and general means of transforming various lettuce cultivars, including recalcitrant commercial cultivars.     

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.     

Comparison of vetiver root essential oils from cleansed (bacteria- and fungus-free) vs. non-cleansed (normal) vetiver plants

‘Karnataka’ and ‘Malaysia’ cultivars of vetiver (Vetiveria zizanioides (L.) Nash, =Chrysopogon zizanioides (L.) Roberty) were subjected to meristem tissue culture in order to produce plants that were bacteria- and fungi-free. Tissue cultured (“cleansed” or phytosanitary) vetiver was grown for five months in sterilized soil contained in pots, and the oil content of plants grown on the medium was compared to that of non-cleansed (normal) vetiver plants grown in unsterilized soil under the same conditions. Statistical analysis of 49 of the major oil components revealed numerous significant differences between tissue culture derived and natural plants for both genotypes.     

CHX14 is a plasma membrane K‐efflux transporter that regulates K+ redistribution in Arabidopsis thaliana

Potassium (K⁺) is essential for plant growth and development, yet the molecular identity of many K⁺ transporters remains elusive. Here we characterized cation/H⁺ exchanger (CHX) 14 as a plasma membrane K⁺ transporter. CHX14 expression was induced by elevated K⁺ and histochemical analysis of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K⁺ stress as compared with wild‐type seedlings. Roots of mutant seedlings displayed higher K⁺ uptake rates than wild‐type roots. CHX14 expression in yeast cells deficient in K⁺ uptake renders the mutant cells more sensitive to deficiencies of K⁺ in the medium. CHX14 mediates K⁺ efflux in yeast cells loaded with high K⁺. Uptake experiments using ⁸⁶Rb⁺ as a tracer for K⁺ with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low‐affinity K⁺ efflux. Functional green fluorescent protein (GFP)‐tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K⁺ efflux transporter involved in K⁺ homeostasis and K⁺ recirculation.