An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars

Agrobacterium-mediated transformation of indica rice varieties has been quite difficult as these are recalcitrant to in vitro responses. In the present study, we established a high-efficiency Agrobacterium tumefaciens-mediated transformation system of rice (Oryza sativa L. ssp. indica) cv. IR-64, Lalat, and IET-4786. Agrobacterium strain EHA-101 harboring binary vector pIG121-Hm, containing a gene encoding for β-glucuronidase (GUS) and hygromycin resistance, was used in the transformation experiments. Manipulation of different concentrations of acetosyringone, days of co-culture period, bacterial suspension of different optical densities (ODs), and the concentrations of l-cysteine in liquid followed by solid co-culture medium was done for establishing the protocol. Among the different co-culture periods, 5 days of co-culture with bacterial cells (OD_600 nm?=?0.5–0.8) promoted the highest frequency of transformation (83.04 %) in medium containing l-cysteine (400 mg l^?1). Putative transformed plants were analyzed for the presence of a transgene through genomic PCR and GUS histochemical analyses. Our results also suggest that different cultural conditions and the addition of l-cysteine in the co-culture medium improve the Agrobacterium-mediated transformation frequencies from an average of 12.82 % to 33.33 % in different indica rice cultivars.     

Protective and survival efficacies of Rv0160c protein in murine model of Mycobacterium tuberculosis

The proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) multi-gene families code for approximately 10 % of the Mycobacterium tuberculosis (Mtb) genome. These proteins are thought to be virulence factors that participate in impounding the host immune responses. While some members have been studied, the functions of most PE/PPE proteins are yet to be explored. The studies presented here have specifically characterized the roles of one of the PE proteins of Mtb, Rv0160c (PE4), in mycobacterial persistence and in prophylactic efficacy. We have expressed Rv0160c in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that the protein improves the survival of mycobacteria in macrophages and in mice. The protein has also shown its effect under physiological stress of bacteria, as evidenced by elevated expression in acidic and in hypoxic conditions. In mice, the level of Rv0160c was noticeably high during the chronic stage of tuberculosis. The seroreactivity of the protein against different categories of tuberculosis patients revealed a strong B-cell humoral response in freshly infected pulmonary tuberculosis patients. In mice, it exhibited increased IL-2, TNF, and IL-6 production. The antigenic properties of the protein directed towards the protective efficacy against the Mtb challenge. All together, our findings have identified Rv0160c as an in vivo expressed immunodominant antigen which plays a crucial role in the pathogenesis of mycobacterial disease and could prove to be a good preventive antigen for tuberculosis.     

Genetic variability, heritability and genetic advance of growth and yield components of Jatropha (Jatropha curcas Linn.) genotypes

A total of 113 Jatropha curcas clonal accessions collected from different regions of India were studied to quantify the magnitude of genetic variability present in the test population and to identify important yield-attributing characters useful for developing high-yielding Jatropha cultivars. High heritability was observed for fruits per plant, seeds per plant, 100-seed weight, seed/kernel (S/K) ratio and kernel oil percentage coupled with high genetic advance suggesting that the accessions can be considered improvement. The significant positive association of seed oil content (%) with 100-seed weight suggested the effectiveness of indirect selection for seed oil content through 100-seed weight. Accessions 76, 120, 29, 86 and 84 showed above average higher values for all yield attributes (viz. fruit and seed yield, 100-seed weight, S/K ratio and oil content) suggesting these as best out of the test accessions. Accessions showing higher values for one or the other yield attributes could be selected as parents for further improvement.     

Dilution of protein‐surfactant complexes: A fluorescence study

Dilution of protein–surfactant complexes is an integrated step in microfluidic protein sizing, where the contribution of free micelles to the overall fluorescence is reduced by dilution. This process can be further improved by establishing an optimum surfactant concentration and quantifying the amount of protein based on the fluorescence intensity. To this end, we study the interaction of proteins with anionic sodium dodecyl sulfate (SDS) and cationic hexadecyl trimethyl ammonium bromide (CTAB) using a hydrophobic fluorescent dye (sypro orange). We analyze these interactions fluourometrically with bovine serum albumin, carbonic anhydrase, and beta‐galactosidase as model proteins. The fluorescent signature of protein–surfactant complexes at various dilution points shows three distinct regions, surfactant dominant, breakdown, and protein dominant region. Based on the dilution behavior of protein–surfactant complexes, we propose a fluorescence model to explain the contribution of free and bound micelles to the overall fluorescence. Our results show that protein peak is observed at 3 mM SDS as the optimum dilution concentration. Furthermore, we study the effect of protein concentration on fluorescence intensity. In a single protein model with a constant dye quantum yield, the peak height increases with protein concentration. Finally, addition of CTAB to the protein–SDS complex at mole fractions above 0.1 shifts the protein peak from 3 mM to 4 mM SDS. The knowledge of protein–surfactant interactions obtained from these studies provides significant insights for novel detection and quantification techniques in microfluidics.     

A rapid,ideal, and eco-friendlier protocol for quantifying proline

Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λ _max of the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and H₃PO₄, indicating negative impact of H₃PO₄ on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and H₃PO₄ was achieved within 30 and 60 min, respectively. This revealed that H₃PO₄ has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.