Selection of suitable internal control gene for assaying gene expression in rice through qRT-PCR during sheath blight infection.

qRT-PCR is a globally accepted technique for assaying gene expression in relative terms which compares the difference between critical threshold (CT) values of a gene calculated form two independently isolated RNA samples. Independent RNA isolations, however, include error due to batch effect which must be normalized for error-free calculation of relative gene expression. Hence, CT values of internal control (IC) genes are used for normalization during the calculation of expression fold-change in gene expression analysis. The expression of ICs genes expected to be stable in all the experimental conditions. However, it is almost impossible to find such a gene which do not depict expression fluctuation in response to the changes in experimental conditions. Hence, it is necessary to identify suitable IC gene(s) for any given experimental condition before conducting any particular gene expression study. Here, we examined the suitability of eight candidate IC genes, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic elongation factor-1 (eEF-1α), 25 S rRNA (25 S), 18 S rRNA (18 S), ubiquitin C E2 ligase (UBC), Actin (Act), ubiquitin 5 (UBQ5) and ubiquitin 10 (UBQ10), for assaying gene expression in rice during sheath blight infection. Our analysis suggest that GAPDH might be the IC of choice when expression studies include contrasting genotypes differing in their tolerance to sheath blight pathogen as well as progressive infection time. While if expression analysis have to be performed only in one genotype but under progressive sheath blight infection, UBQ5 might be chosen as IC because of its high expression stability under the proposed experimental setup.

Radiographic Assessment of Anatomy of Nasopalatine Canal for Dental Implant Placement: A Cone Beam Computed Tomographic Study.

AIM: The aim of this study is to assess the morphology of nasopalatine canal (NPC) with cone beam computed tomography (CBCT). MATERIALS AND METHODS: A total of 460 subjects of both genders were subjected to CBCT with NewTom machine, and sagittal and coronal sections were used for evaluation of the shape of NPC and width of buccal cortical plate. Types of NPC were also assessed. RESULTS: Of 460 subjects, males were 210 and females were 250. The difference was nonsignificant (p = 0.1). Type III canals were mostly seen in both males and females, followed by types I and II. The mean length of NPC in males and females showed statistical significant difference (p < 0.05). Males showed significantly higher diameter of nasal opening, oral opening, and width of the buccal bone plate over the oral opening as compared with females. CONCLUSION: The exact location, morphology, and dimensions of NPC can be well visualized with CBCT. All findings were higher in males as compared with females. CLINICAL SIGNIFICANCE: The success of dental implant in maxillary anterior region may be affected by the approximation with NPC. The exact location and morphology play an important role for the correct placement of implant. Cone beam computed tomography is a useful tool providing three-dimensional images in all sections.

Status and Prospects of Next Generation Sequencing Technologies in Crop Plants.

The history of DNA sequencing dates back to 1970s. During this period the two first generation nucleotide sequencing techniques were developed. Subsequently the Sanger's dideoxy method of sequencing gained popularity over Maxam and Gilbert's chemical method of sequencing. However, in the last decade, we have observed revolutionary changes in DNA sequencing technologies leading to the emergence of next-generation sequencing (NGS) techniques. NGS technologies have enhanced the throughput and speed of sequencing combined with bringing down the overall cost of the process over a time. The major applications of NGS technologies being genome sequencing and resequencing, transcriptomics, metagenomics in relation to plant-microbe interactions, exon and genome capturing, development of molecular markers and evolutionary studies. In this review, we present a broader picture of evolution of NGS tools, its various applications in crop plants, and future prospects of the technology for crop improvement.

Host Delivered RNAi, an efficient approach to increase rice resistance to sheath blight pathogen (Rhizoctonia solani).

Rhizoctonia solani, the causal agent of rice sheath blight disease, causes significant losses worldwide as there are no cultivars providing absolute resistance to this fungal pathogen. We have used Host Delivered RNA Interference (HD-RNAi) technology to target two PATHOGENICITY MAP KINASE 1 (PMK1) homologues, RPMK1-1 and RPMK1-2, from R. solani using a hybrid RNAi construct. PMK1 homologues in other fungal pathogens are essential for the formation of appressorium, the fungal infection structures required for penetration of the plant cuticle, as well as invasive growth once inside the plant tissues and overall viability of the pathogen within the plant. Evaluation of transgenic rice lines revealed a significant decrease in fungal infection levels compared to non-transformed controls and the observed delay in disease symptoms was further confirmed through microscopic studies. Relative expression levels of the targeted genes, RPMK1-1 and RPMK1-2, were determined in R. solani infecting either transgenic or control lines with significantly lower levels observed in R. solani infecting transgenic lines carrying the HD-RNAi constructs. This is the first report demonstrating the effectiveness of HD-RNAi against sheath blight and offers new opportunities for durable control of the disease as it does not rely on resistance conferred by major resistance genes.

Novel Chitinase Gene LOC_Os11g47510 from Indica Rice Tetep Provides Enhanced Resistance against Sheath Blight Pathogen Rhizoctonia solani in Rice.

Sheath blight disease (ShB), caused by the fungus Rhizoctonia solani Kühn, is one of the most destructive diseases of rice (Oryza sativa L.), causing substantial yield loss in rice. In the present study, a novel rice chitinase gene, LOC_Os11g47510 was cloned from QTL region of R. solani tolerant rice line Tetep and used for functional validation by genetic transformation of ShB susceptible japonica rice line Taipei 309 (TP309). The transformants were characterized using molecular and functional approaches. Molecular analysis by PCR using a set of primers specific to CaMv 35S promoter, chitinase and HptII genes confirmed the presence of transgene in transgenic plants which was further validated by Southern hybridization. Further, qRT-PCR analysis of transgenic plants showed good correlation between transgene expression and the level of sheath blight resistance among transformants. Functional complementation assays confirmed the effectiveness of the chitinase mediated resistance in all the transgenic TP309 plants with varying levels of enhanced resistance against R. solani. Therefore, the novel chitinase gene cloned and characterized in the present study from the QTL region of rice will be of significant use in molecular plant breeding program for developing sheath blight resistance in rice.

Functional Characterization of Novel Chitinase Genes Present in the Sheath Blight Resistance QTL: qSBR11-1 in Rice Line Tetep.

Rice sheath blight disease caused by Rhizoctonia solani is one of the most devastating diseases in rice leading to heavy yield losses. Due to the polygenic nature of resistance, no major resistance gene with complete host resistance against R. solani has been reported. In this study, we have performed molecular and functional analysis of the genes associated with the major R. solani-resistance QTL qSBR11-1 in the indica rice line Tetep. Sequence analysis revealed the presence of a set of 11 tandem repeats containing genes with a high degree of homology to class III chitinase defense response genes. Real-time quantitative PCR analysis showed that all the genes are strongly induced 36 h after R. solani infection. Comparison between the resistant Tetep and the susceptible HP2216 lines shows that the induction of the chitinase genes is much higher in the Tetep line. Recombinant protein produced in vitro for six of the eleven genes showed chitinolytic activity in gel assays but we did not detect any xylanase inhibitory activity. All the six in vitro expressed proteins show antifungal activity with a clear inhibitory effect on the growth of the R. solani mycelium. The characterized chitinase genes can provide an important resource for the genetic improvement of R. solani susceptible rice lines for sheath blight resistance breeding.

Chemical Treatment as a Pre- requisite for Effective Disposal of Tri Nitro Toluene (TNT).

Present methods for the disposal of old and rejected ammunition carry high risk and are not environment-friendly. Various processes such as wet air oxidation, molten salt oxidation, hydro thermal oxidation, incineration, electrochemical reduction, biodegradation and other methods have limited use for decontamination and are not suitable for disposal of large quantities of explosives. Thus there is dire need to develop alternate method for safe disposal of rejected explosives which will be eco-friendly. In this paper we have attempted to combine two methods i.e. chemical treatment followed by biological/microbiological treatment. For this purpose we have selected Tri Nitro Toluene (TNT) as a model compound, which is used extensively in many types of ammunition. As reported previously from our laboratory the presence of nitro group from TNT was toxic to bacterial growth. By chemical treatment, nitro groups from TNT were converted into amine and mixed in soil for biodegradation. Our results suggest that after converting 'nitro groups' to 'amine groups' were much preferred by bacteria and faster mineralization is observed. Thus combined treatment to TNT as discussed in this study, showed much less phyto-toxicity and may have great potential to scale up the process for large quantities of explosive such as TNT.

Waste explosives and other hazardous materials--hazard potential and remedial measures: an overview.

A large amount of energetic materials including propellants, high explosives, pyrotechnics are subjected to disposal either due to expiry of their useful life or rejection in the manufacturing process. The environmental regulations do not allow the hazardous materials for open burning / detonation in view of the health hazard involved in these operations. The present paper describes the hazard potential of energetic materials and associated hazardous chemicals. It also deals with global technological status for remedial measures of hazardous chemicals along with their merits and demerits.