Assessment of resistance to rice tungro disease in popular rice varieties in India by introgression of a transgene against Rice tungro bacilliform virus.

Rice crops in South and Southeast Asian countries suffer critical yield losses due to rice tungro disease caused by joint infection with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Previously, for generating RNA interference-based transgenic resistance against tungro viruses, RTBV ORF IV was used as a transgene to develop RTBV resistance in a popular high-yielding scented rice variety. The transgene from this line was then introgressed into five popular high-yielding but tungro-susceptible rice varieties by marker-assisted backcross breeding with a view to combine the resistant trait with the agronomic traits. The present work includes a resistance assay of the BC3F5 lines of these varieties under glasshouse conditions. Out of a total of 28 lines tested, each consisting of 12 individual plants, eight lines showed significant amelioration in height reduction and 100- to 1000-fold reduction in RTBV titers. The RNAi-mediated resistance was clearly manifested by the presence of virus-derived small RNA (vsRNA) specific for RTBV ORF IV in the transgenic backcrossed lines.

Whole Genome Characterization of a Few EMS-Induced Mutants of Upland Rice Variety Nagina 22 Reveals a Staggeringly High Frequency of SNPs Which Show High Phenotypic Plasticity Towards the Wild-Type.

The Indian initiative, in creating mutant resources for the functional genomics in rice, has been instrumental in the development of 87,000 ethylmethanesulfonate (EMS)-induced mutants, of which 7,000 are in advanced generations. The mutants have been created in the background of Nagina 22, a popular drought- and heat-tolerant upland cultivar. As it is a pregreen revolution cultivar, as many as 573 dwarf mutants identified from this resource could be useful as an alternate source of dwarfing. A total of 541 mutants, including the macromutants and the trait-specific ones, obtained after appropriate screening, are being maintained in the mutant garden. Here, we report on the detailed characterizations of the 541 mutants based on the distinctness, uniformity, and stability (DUS) descriptors at two different locations. About 90% of the mutants were found to be similar to the wild type (WT) with high similarity index (>0.6) at both the locations. All 541 mutants were characterized for chlorophyll and epicuticular wax contents, while a subset of 84 mutants were characterized for their ionomes, namely, phosphorous, silicon, and chloride contents. Genotyping of these mutants with 54 genomewide simple sequence repeat (SSR) markers revealed 93% of the mutants to be either completely identical to WT or nearly identical with just one polymorphic locus. Whole genome resequencing (WGS) of four mutants, which have minimal differences in the SSR fingerprint pattern and DUS characters from the WT, revealed a staggeringly high number of single nucleotide polymorphisms (SNPs) on an average (16,453 per mutant) in the genic sequences. Of these, nearly 50% of the SNPs led to non-synonymous codons, while 30% resulted in synonymous codons. The number of insertions and deletions (InDels) varied from 898 to 2,595, with more than 80% of them being 1-2 bp long. Such a high number of SNPs could pose a serious challenge in identifying gene(s) governing the mutant phenotype by next generation sequencing-based mapping approaches such as Mutmap. From the WGS data of the WT and the mutants, we developed a genic resource of the WT with a novel analysis pipeline. The entire information about this resource along with the panicle architecture of the 493 mutants is made available in a mutant database EMSgardeN22 (http://14.139.229.201/EMSgardeN22).

Residues of Diclofenac in Tissues of Vultures in India: A Post-ban Scenario.

Populations of three resident Gyps species (Indian white-backed vulture Gyps bengalensis, Indian vulture Gyps indicus, and Slender-billed vulture Gyps tenuirostris) in India have decreased by more than 90% since mid 1990s, and they continue to decline. Experimental studies showed the mortality of vultures to be due to renal failure caused by diclofenac, a nonsteroidal anti-inflammatory drug. India, Pakistan, and Nepal banned the veterinary usage of diclofenac in 2006 to prevent further decline in vulture population. This study was performed to know the current status of the impact of diclofenac on vultures in India. Between 2011 and 2014, 44 vultures comprising two species, namely Indian white-backed vulture (32) and Himalayan griffon Gyps himalayensis (12) were collected dead from Gujarat, Assam, and Tamil Nadu on an opportunistic basis. Kidney and liver tissues and gut content were analysed for diclofenac. Of the 32 dead white-backed vultures analysed, 68.75% of them had diclofenac ranging from 62.28 to 272.20 ng/g. Fourteen white-backed vultures had diclofenac in kidney in toxic range (70-908 ng/g). Of 12 Himalayan griffon studied, 75% of them had diclofenac in the range of 139.69 to 411.73 ng/g. Himalayan griffon had significantly higher levels of diclofenac in tissues than Indian white-backed vultures. It is possible that 14 of 29 white-backed vultures and 9 of 12 Himalayan griffon included in this study died due to diclofenac poisoning. Studies have indicated that diclofenac can continue to kill vultures even after its ban in India for veterinary use.

Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.).

BACKGROUND: Increased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, 'HTM-N22', exhibiting tolerance to a broad spectrum herbicide, 'Imazethapyr', and identified the mutations imparting tolerance to the herbicide. RESULTS: We identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2 plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies. CONCLUSIONS: A novel mutant tolerant to the herbicide "Imazethapyr" was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.

Evaluation of virus resistance and agronomic performance of rice cultivar ASD 16 after transfer of transgene against Rice tungro bacilliform virus by backcross breeding.

Severe losses of rice yield in south and southeast Asia are caused by Rice tungro disease (RTD) induced by mixed infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to develop transgene-based resistance against RTBV, one of its genes, ORF IV, was used to generate transgenic resistance based on RNA-interference in the easily transformed rice variety Pusa Basmati-1, and the transgene was subsequently introgressed to rice variety ASD 16, a variety popular in southern India, using transgene marker-assisted selection. Here, we report the evaluation of BC3F4 and BC3F5 generation rice plants for resistance to RTBV as well as for agronomic traits under glasshouse conditions. The BC3F4 and BC3F5 generation rice plants tested showed variable levels of resistance, which was manifested by an average of twofold amelioration in height reduction, 1.5-fold decrease in the reduction in chlorophyll content, and 100- to 10,000-fold reduction in the titers of RTBV, but no reduction of RTSV titers, in three backcrossed lines when compared with the ASD 16 parent. Agronomic traits of some of the backcrossed lines recorded substantial improvements when compared with the ASD 16 parental line after inoculation by RTBV and RTSV. This work represents an important step in transferring RTD resistance to a susceptible popular rice variety, hence enhancing its yield in areas threatened by the disease.

Removal of malachite green from aqueous solution by activated carbon prepared from the epicarp of Ricinus communis by adsorption.

The use of low-cost, locally available, highly efficient and eco-friendly adsorbents has been investigated as an ideal alternative to the current expensive methods of removing dyes from wastewater. This study investigates the potential use of activated carbon prepared from the epicarp of Ricinus communis for the removal of malachite green (MG) dye from simulated wastewater. The effects of different system variables, adsorbent dosage, initial dye concentration, pH and contact time were investigated and optimal experimental conditions were ascertained. The results showed that as the amount of the adsorbent increased, the percentage of dye removal increased accordingly. Optimum pH value for dye adsorption was 7.0. Maximum dye was sequestered within 50 min of the start of every experiment. The adsorption of malachite green followed the pseudo-second-order rate equation and fits the Langmuir, Freundlich, Dubinin-Radushkevich (D-R) and Tempkin equations well. The maximum removal of MG was obtained at pH 7 as 99.04% for adsorbent dose of 1 g 50 mL(-1) and 25 mg L(-1) initial dye concentration at room temperature. Activated carbon developed from R. communis can be an attractive option for dye removal from diluted industrial effluents since test reaction made on simulated dyeing wastewater showed better removal percentage of MG.

Removal of hexavalent chromium using distillery sludge.

Batch mode experiments were conducted to study the removal of hexavalent chromium from aqueous and industrial effluent using distillery sludge. Effects of pH, contact time, initial concentration and adsorbent dosage on the adsorption of Cr(VI) were studied. The data obeyed Langmuir and Freundlich adsorption isotherms. The Langmuir adsorption capacity was found to be 5.7 mg/g. Freundlich constants K(f) and n were 2.05 [mg/g(L/mg)(n)] and 3.9, respectively. Desorption studies indicated the removal of 82% of the hexavalent chromium. The efficiency of adsorbent towards the removal of chromium was also tested using chromium-plating wastewater.

Biochemical evaluation of the antiulcerogenic effect of Cauvery-100 (an ayurvedic formulation) in rats.

Due to the varied pathogenesis of ulcers, a number of pharmacologically different antiulcer drugs are now used in the treatment of the condition. The suitability of certain of these drugs is still to be established. Cauvery-100 is an ayurvedic formulation consisting of plant ingredients, and has been suggested to be useful in the treatment of gastrointestinal disorders. In the present study this drug was tested for its antiulcerogenic effect. The number of lesions on gastric mucosa decreased markedly after 15 days oral treatment with Cauvery-100 in rats with indomethacin-induced ulcers. The volume and total acidity of the gastric juice also decreased in the treated rats. The hexose, hexosamine and sialic acid levels of gastric contents, which were increased in untreated rats returned to near normal levels after 15 days of treatment. Protein in the gastric juice was elevated in untreated rats but returned to near normal levels after 15 days of treatment. Cauvery-100 acts both to decrease the acidity and to increase the mucosal defence in the gastric areas, thereby justifying its use as an antiulcerogenic agent.

Biochemical studies on the antidiarrhoeal effects of Cauvery-100, an ayurvedic formulation, in rats.

Diarrhoea is a common gastrointestinal disorder which is a state of fluid and ion loss from the gut. Cauvery-100, an Ayurvedic formulation has been used in this study for the treatment of diarrhoea. Diarrhoea was induced in experimental rats by oral administration of castor oil. The increased gastrointestinal motility in diarrhoea was brought back to near normal levels on the treatment of Cauvery-100. The activities of the enzymes alkaline phosphatase, total ATPase and Na+,K(+)-ATPase were decreased in the diarrhoeal group and was brought back to near normal levels in the treated group. The serum levels of sodium and potassium were decreased in the diarrhoeal group and brought back to normal levels in the treated group. Prior treatment of the drug Cauvery-100 did not induce diarrhoea on administration of castor oil, suggesting the protective influence of the drug on the gastrointestinal tract.