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Wayne L.L.,Washington State University | Wayne L.L.,Dow AgroSciences | Wallis J.G.,Washington State University | Kumar R.,Washington State University | And 3 more authors.
Plant Cell | Year: 2013

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 heterozygotes segregated 1:1 heterozygous:wild type, indicating a gametophyte defect. Homozygous cbr1-2 seeds were deformed and required Suc for successful germination and seedling establishment. Vegetative growth of cbr1-2 plants was relatively normal, and they produced abundant flowers, but very few seeds. The pollen produced in cbr1-2 anthers was viable, but when germinated on cbr1-2 or wild-type stigmas, most of the resulting pollen tubes did not extend into the transmitting tract, resulting in a very low frequency of fertilization. These results indicate that cytochrome b5 reductase is not essential during vegetative growth but is required for correct pollen function and seed maturation. © American Society of Plant Biologists. All rights reserved.


Shamurailatpam A.,North - Eastern Hill University | Madhavan L.,National Bureau of Plant Genetic Resources | Yadav S.R.,Shivaji University | Bhat K.V.,National Research Center on Fingerprinting | Rao S.R.,North - Eastern Hill University
Nucleus (India) | Year: 2012

Wild species of Vigna are known to exhibit great range of phenotypic and genotypic diversity leading to development of several varieties and cultivars. However information regarding their chromosome biology is by and large lacking. The present study deals with karyotypic analysis in 10 taxa belonging to the genus Vigna. The details of chromosome morphology, karyotype formulae and symmetry index have been investigated. Heteromorphic chromosome pairs observed in two species viz. V. aconitifolia and V. radiata var. setulosa and NOR are also being reported in two taxa viz. V. mungo var. silvestris and V. sublobata. The karyomorphological studies confirmed that the asymmetry index of different taxa presently investigated had shown significant variation. The influence of karyotypic variation on genome evolution has been discussed in detail. © 2012 Archana Sharma Foundation of Calcutta.


Das A.,North Bengal Agricultural University | Das A.,ICAR Research Complex for NEH Region | Das S.,Tea Research Association | Mondal T.K.,North Bengal Agricultural University | Mondal T.K.,National Research Center on Fingerprinting
Plant Molecular Biology Reporter | Year: 2012

Drought is an important abiotic stress that limits the production of tea in different regions of the world. Young roots of tea are responsible for nutrient and water uptake; hence, they are the first tissues to perceive drought stress. In this study, a forward suppression subtractive hybridization library was constructed from the tender roots of drought-tolerant tea (Camellia sinensis (L.) O. Kuntze) cultivar (TV-23) subjected to 21 days of drought stress. A total of 572 quality expressed sequence tags were generated by sequencing of 1,052 random clones which have resulted to 246 unigenes comprising 54 contigs and 192 singlets. The unigenes were assigned to various functional categories, i. e. cellular components, biological processes and molecular functions as defined for the Arabidopsis proteome. There were 13. 04% of differentially regulated genes that have been associated to various stresses. A total of 123 putative drought-responsive genes were identified which include candidate genes of ubiquitin-proteasome, glutathione metabolism and sugar metabolism pathways and several transcription factors. In order to determine the possible expression, 10 genes associated to drought-responsive pathways were further analysed by reverse transcription polymerase chain reaction. This study provides a basis for studying the drought tolerance mechanism of this important commercial crop which will also be a valuable resource for the functional genomics study of woody plants in future. © 2012 Springer-Verlag.


Mondal N.,National Research Center on Fingerprinting | Bhat K.V.,National Research Center on Fingerprinting | Srivastava P.S.,Jamia Hamdard University
JAOCS, Journal of the American Oil Chemists' Society | Year: 2010

A germplasm collection of 33 entries comprising 22 sesame (Sesamum indicum L.) cultivars, 4 landraces of S. mulayanum and 7 other accessions of 4 wild species were analyzed for the fatty acid compositions of their seed oil. The entries varied widely in their fatty acid compositions. The percentage content of oleic, linoleic, palmitic and erucic acids ranged between 36.7-52.4, 30.4-51.6, 9.1-14.8 and 0.0-8.0, respectively. Linolenic and arachidonic acids were the minor constituents but varied widely in wild species. Oleic and linoleic were the major fatty acids with mean values of 45.9 and 40.5%, respectively and the mean of their combined values was 86.4%. The polyunsaturated fatty acid (PUFA) compositions ranged from 30.9 to 52.5% showing high variation in PUFA in the germplasm. Linoleic acid content was very high in one landrace (47.8) and one accession each of three wild species, S. mulayanum (49.3), S. malabaricum (48.2) and S. radiatum (51.6%). Use of fatty acid ratios to estimate the efficiency of biosynthetic pathways resulted in high oleic and low linoleic desaturation ratios and consequently high linoleic and very low linolenic acid contents in seed oil. The results of this study provided useful background information on the germplasm and also identified a few accessions having high linoleic acid which can be used for developing cultivars with desirable fatty acid compositions. © 2010 AOCS.


Rana M.K.,National Research Center on Fingerprinting | Arora K.,National Research Center on Fingerprinting | Singh S.,National Research Center on Fingerprinting | Singh A.K.,National Research Center on Fingerprinting
Journal of Plant Biochemistry and Biotechnology | Year: 2013

Sequence-Related Amplified Polymorphism (SRAP) markers were used for genetic diversity assessment and cultivar identification among 31 cultivars of jute belonging to two cultivated species Corchours olitorius L. and C. capsularis L. Forty-three primer-pairs produced a total of 394 bands with an average of 9 bands per primer pair and 89% bands were polymorphic across the genotypes of two species. Average genetic diversity in the cultivars of C. olitorius and C. capsularis was 7. 2% (range 2. 8-12. 3%) and 7. 6% (range 2. 2-13. 1%), respectively. Jute cultivars JRC 698, JRC 7447, TJ 40, S19 and JRO 3690 were more diverse compared to rest of the cultivars. UPGMA cluster analysis grouped all cultivars into two clusters which were representative of C. olitorius and C. capsularis species. All the cultivars could be unequivocally differentiated from one another based on the pooled profile of 43 primer-pairs, however, 24 of 31 cultivars could be identified uniquely. The probability of chance identity of any two cultivars based on SRAP markers was very low and was 6.95 × 10-07 and 2.23 × 10-07 for cultivars of C. capsularis and C. olitorius, respectively. Primer-pairs EM1-ME5, EM4-ME1, EM8-ME1 and EM10-ME1 were found to be useful for genetic diversity and cultivar identification. Our results show that SRAP markers could be effectively used for genetic diversity analyses in jute. For poor genetic diversity and resulting narrow genetic base, these markers will prove to be highly useful for identifying elite germplasm in a jute breeding program. © 2012 Society for Plant Biochemistry and Biotechnology.


Randhawa G.J.,National Research Center on Fingerprinting | Chhabra R.,National Research Center on Fingerprinting
Methods in Molecular Biology | Year: 2013

India is one of the largest cotton-growing countries. Cotton is a fiber crop with varied applications from making tiny threads to fashionable clothing in the textile sector. In the near future, cotton crop will gain popularity as a multipurpose crop in India. The commercialization of Bt cotton in 2002 and consequently the fast adoption of Bt cotton hybrids by cotton farmers have enhanced the cotton production in India. Presently, genetically modified (GM) cotton has occupied 21.0 million hectares (mha) that comprise 14% of the global area under GM cultivation. In the coming years, improved cotton hybrids, with stacked and multiple gene events for improved fiber quality, insect resistance, drought tolerance, and herbicide tolerance, would further significantly improve the cotton production in India. With the dramatic increase in commercialization of GM crops, there is an urgent need to develop cost-effective and robust GM detection methods for effective risk assessment and management, post release monitoring, and to solve the legal disputes. DNA-based GM diagnostics are most robust assays due to their high sensitivity, specificity, and stability of DNA molecule. © 2013 Springer Science+Business Media New York.


Kumar R.,University of Missouri | Kumar R.,National Research Center on Fingerprinting | Tran L.-S.P.,University of Missouri | Tran L.-S.P.,RIKEN | And 3 more authors.
PLoS ONE | Year: 2012

Background: Synthesis of polyunsaturated fatty acids (PUFAs) in the endoplasmic reticulum of plants typically involves the fatty acid desaturases FAD2 and FAD3, which use cytochrome b 5 (Cb5) as an electron donor. Higher plants are reported to have multiple isoforms of Cb5, in contrast to a single Cb5 in mammals and yeast. Despite the wealth of information available on the roles of FAD2 and FAD3 in PUFA synthesis, information regarding the contributions of various Cb5 isoforms in desaturase-mediated reactions is limited. Results: The present functional characterization of Cb5 polypeptides revealed that all Arabidopsis Cb5 isoforms are not similarly efficient in ω-6 desaturation, as evidenced by significant variation in their product outcomes in yeast-based functional assays. On the other hand, characterization of Cb5 polypeptides of soybean (Glycine max) suggested that similar ω-6 desaturation efficiencies were shared by various isoforms. With regard to ω-3 desaturation, certain Cb5 genes of both Arabidopsis and soybean were shown to facilitate the accumulation of more desaturation products than others when co-expressed with their native FAD3. Additionally, similar trends of differential desaturation product accumulation were also observed with most Cb5 genes of both soybean and Arabidopsis even if co-expressed with non-native FAD3. Conclusions: The present study reports the first description of the differential nature of the Cb5 genes of higher plants in fatty acid desaturation and further suggests that ω-3/ω-6 desaturation product outcome is determined by the nature of both the Cb5 isoform and the fatty acid desaturases. © 2012 Kumar et al.


Randhawa G.J.,National Research Center on Fingerprinting | Sharma R.,National Research Center on Fingerprinting | Singh M.,National Research Center on Fingerprinting
Journal of AOAC International | Year: 2012

Bt brinjal event EE-1 with cry1Ac gene, expressing insecticidal protein against fruit and shoot borer, is the first genetically modified food crop in the pipeline for commercialization in India. Qualitative polymerase chain reaction (PCR) along with event-specific conventional as well as real-time PCR methods to characterize the event EE-1 is reported. A multiplex (pentaplex) PCR system simultaneously amplifying cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos) terminator, aminoglycoside adenyltransferase (aadA) marker gene, and a taxonspecific β-fructosidase gene in event EE-1 has been developed. Furthermore, construct-specific PCR, targeting the approximate 1.8 kb region of inserted gene construct comprising the region of CaMV 35S promoter and cry1Ac gene has also been developed. The LOD of developed EE-1 specific conventional PCR assay is 0.01%. The method performance of the reported real-time PCR assay was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. The developed detection methods would not only facilitate effective regulatory compliance for identification of genetic traits, risk assessment, management, and postrelease monitoring, but also address consumer concerns and resolution of legal disputes.


Randhawa G.J.,National Research Center on Fingerprinting | Singh M.,National Research Center on Fingerprinting
Journal of AOAC International | Year: 2012

Qualitative and quantitative analytical methods based on PCR for Bacillus thuringiensis (Bt) rice hybrid, namely, MRP 5401 Bt expressing a modified version of the Bt cry1Ac gene, are reported here. Multiplex PCR assays were developed to target the cry1Ac transgene, Cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptII) marker gene, and an endogenous a-tubulin (TubA) gene in Bt rice. The 3.178 kb region of inserted gene construct comprising the region of the CaMV 35S promoter and cry1Ac gene was amplified, and the construct integrity was confirmed by the nested PCR. The LOD for cry1Ac gene-specific simplex PCR was 0.01%, as established using Bt rice DNA dilutions with 100, 10, 1.0, 0.1, 0.05, 0.01, and 0.001% genetically modified trait. A real-time PCR assay was also developed to quantify the cry1Ac gene. The method performance of the reported real-time PCR assay was in line with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with LOD and LOQ values of 0.05%. The reliable PCR assays prior to commercial release of Bt rice would facilitate efficient regulatory compliance for identification of genetic trait, labeling requirements, and effective risk assessment and management. They could also address consumers' concerns and legal disputes that may arise. © 2012 Publishing Technology.


Randhawa G.J.,National Research Center on Fingerprinting | Singh M.,National Research Center on Fingerprinting | Grover M.,National Research Center on Fingerprinting
Food and Chemical Toxicology | Year: 2011

The novel proteins introduced into the genetically modified (GM) crops need to be evaluated for the potential allergenicity before their introduction into the food chain to address the safety concerns of consumers. At present, there is no single definitive test that can be relied upon to predict allergic response in humans to a new protein; hence a composite approach to allergic response prediction is described in this study. The present study reports on the evaluation of the Cry proteins, encoded by cry1Ac, cry1Ab, cry2Ab, cry1Ca, cry1Fa/cry1Ca hybrid, being expressed in Bt food crops that are under field trials in India, for potential allergenic cross-reactivity using bioinformatics search tools. The sequence identity of amino acids was analyzed using FASTA3 of AllergenOnline version 10.0 and BLASTX of NCBI Entrez to identify any potential sequence matches to allergen proteins. As a step further in the detection of allergens, an independent database of domains in the allergens available in the AllergenOnline database was also developed. The results indicated no significant alignment and similarity of Cry proteins at domain level with any of the known allergens revealing that there is no potential risk of allergenic cross-reactivity. © 2010 Elsevier Ltd.

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