Browsing by Author "Govind Kumar Rai"

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Assessment of molecular diversity in chickpea (Cicer arietinum L.) rhizobia and structural analysis of 16S rDNA sequences from Mesorhizobium ciceri
(Polish Society of Microbiologists, 2013) Akhilesh Yadav; Asha Lata Singh; Govind Kumar Rai; Major Singh
Molecular diversity studies of 19 rhizobia isolates from chickpea were conducted using simple sequence repeats (SSR) and 16S rDNA-RFLP markers. Phenotypic characterization with special reference to salinity and pH tolerance was performed. These isolates were identified as different strains of Mesorhizobium, Rhizobium, Bradyrhizobium, and Agrobacterium. Twenty SSR loci of Mesorhizobium ciceri, distributed across the other rhizobial genome, clearly differentiated 19 rhizobial isolates. Analogous clustering supported the results of 16S rDNA sequence-based phylogeny. Analysis of the 16S rDNA sequences from M. ciceri strains revealed that nucleotide variables (signature sites) were located at 20 different positions; most of them were present in the first 820 bp region from 5' terminal. Interestingly, 14 signature sites were located in two main regions, the variable region V1 (nt 527-584), and variable region V2 (nt 754-813). The secondary structure and minimal free energy were determined in these two regions. These results will be useful in characterizing the micro-evolutionary mechanisms of species formation and increase understanding of the symbiotic relationship.
Bacterial Community Structure in the Rhizosphere of a Cry1Ac Bt-Brinjal Crop and Comparison to Its Non-transgenic Counterpart in the Tropical Soil
(2013) Amit Kishore Singh; Govind Kumar Rai; Major Singh; Suresh Kumar Dubey
To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4 +-N, NO3 --N, and PO4 --P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes. © 2013 Springer Science+Business Media New York.
Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato
(2012) Govind Kumar Rai; Neha Prakash Rai; Sanjeev Kumar; Akhilesh Yadav; Sushma Rathaur; Major Singh
An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog's (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l -1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l -1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T 1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars. © 2012 The Society for In Vitro Biology.
Expression of rd29A:: AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants
(2013) Govind Kumar Rai; Neha Prakash Rai; Sushma Rathaur; Sanjeev Kumar; Major Singh
Transgenic tomato lines (cv. Kashi Vishesh) over-expressing AtDREB1A/. CBF3 driven by stress-inducible rd29A promoter showed significantly higher activities of key antioxidant enzymes when exposed to water-deficit for 7, 14, and 21 days. Transgenic tomato plants exposed to water-deficit recorded lower levels of hydrogen peroxide and superoxide anion formation compared to the non-transgenic plants, suggesting alleviation of reactive oxygen species (ROS). A significant increase in activities of enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) was observed in response to the different durations of water-deficit conditions. In contrast, enzyme guaiacol peroxidase (POD) activity was lower in the transgenic lines and showed a negative correlation with ROS, ascorbic acid (AsA), and glutathione levels. The concentrations of AsA, glutathione and their reduced forms were higher in the transgenic plants and increased with ROS levels. These results indicate that AtDREB1A transgenic tomato lines are better adapted to water-deficit as they showed lower drought-induced oxidative stress due to activation of the antioxidant response. © 2013 Elsevier Masson SAS.
Monitoring the genetic fidelity of micropropagated plantlets of Spondias mangifera Willd. using RAPD marker assays
(Headley Brothers Ltd, 2012) Madhu Tripathi; Nishi Kumari; Neha Prakash Rai; Govind Kumar Rai; Major Singh
Random amplified polymorphic DNA (RAPD) markers were used to screen for clonal fidelity in in vitro-propagated plantlets of Spondias mangifera produced through direct organogenesis. One micropropagated plantlet was selected at random after each sub-cultural passage (six sub-cultures), along with the donor plant, for RAPD marker analysis. Twenty-five RAPD primers were used to study genetic similarities or dissimilarities with the mother plant as well as among the regenerated plants. Individual primers showed that the same pattern of RAPD markers was shared by all in vitro-propagated plantlets and the mother plant. No variation was observed among the micropropagated progenies. Thus, in vitro-regenerated plantlets of S. mangifera were clonally uniform and genetically stable.
Shoot and fruit borer resistant transgenic eggplant (Solanum melongena L.) expressing cry1Aa3 gene: Development and bioassay
(2013) Neha Prakash Rai; Govind Kumar Rai; Sanjeev Kumar; Nishi Kumari; Major Singh
Eggplant shoot and fruit borer (ESFB), Leucinodes orbonalis Guenée, is a major insect pest of eggplant, Solanum melongena L. In this study, cry1Aa3 gene was introduced into eggplant cv. Kashi Taru through Agrobacterium-tumefaciens mediated transformation. Hypocotyl explants from 7 day-old seedlings were pre-cultured for two days and inoculated with Agrobacterium strain EHA105 harboring a synthetic cry1Aa3 gene with full codon-modification in a binary vector. PCR and Southern blot analyses confirmed the presence of single copy insertions of the cry1Aa3 gene in plants of four independent transformation events. In addition, single copy gene insertions were confirmed by segregation analysis of T1 seedlings. Expression of the cry1Aa3 transcripts was confirmed by qRT-PCR; and ELISA revealed presence of Cry1Aa3 protein in fresh leaves (30.9-44.3ngg-1) and fruits (20.5-35.7ngg-1). Expression of the Cry1Aa3 protein resulted in high ESFB larval mortality in the shoot as well as fruit tissues of the transformed eggplant lines. Such transgenic eggplant lines possessing resistance to the ESFB could reduce dependency on pesticide use for ESFB control, leading to a safer environment and eggplant fruits. © 2013 Elsevier Ltd.
The Southeastern Asian house mouse (Mus musculus castaneus Linn.) as a new passenger host for Cryptococcus neoformans var. grubii molecular type VNI
(Oxford University Press, 2017) Karuna Singh; Jyoti Rani; Neelabh; Govind Kumar Rai; Major Singh
We describe Mus musculus castaneus as a new mammalian host for Cryptococcus neoformans var. grubii (VNI). Eighteen apparently healthy adults and pups of the rodent were collected from human dwellings in Varanasi, a city of India. Both clinical and behavioral examinations of the rodents did not reveal any sign of the disease. Among visceral organs, histological examination of only liver exhibited the presence of single celled, encapsulated, Southgate's mucicarmine positive fungal structures consistent with C. neoformans. Nevertheless, culture of tissue homogenates of brain, lungs, liver, and kidneys yielded white colonies on Sabouraud's dextrose agar and brown mucoid colonies of C. neoformans on Staib's and Tobacco agar media. The pathogen was isolated from habitat soil as well as fresh faeces of the animals. All isolates were urease positive, nitrate and canavanine-glycine bromothymol blue negative, exhibited phenoloxidase activity and grew at 37°C. The isolates were identified as C. neoformans var. grubii with ITS primers and unique marker (GACA)4. The pathogen when inoculated in immunosuppressed mice showed low pathogenicity. To our knowledge, we for the first time report case cluster of Mus musculus castaneus as new passenger host for C. neoformans var. grubii (VNI). © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved.