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Patade V.Y.,Bhabha Atomic Research Center | Patade V.Y.,Defence Institute of Bio energy Research | Rai A.N.,Bhabha Atomic Research Center | Suprasanna P.,Bhabha Atomic Research Center
Protoplasma | Year: 2011

Identification of genes whose expression enables plants to adapt to any kind of stresses is integral to developing stress tolerance in crop plants. In this study, PCR-based cDNA suppression subtractive hybridization technique was used to construct sugarcane salt (NaCl) stress specific forward and reverse subtracted cDNA library. For this, mRNAs were pooled from the shoot and root tissues stressed with NaCl (200 mM) for various time intervals (0.5 to 18 h). Sequencing the clones from the forward subtracted cDNA library, we identified shaggy-like protein kinase (hereafter referred as sugarcane shaggy-like protein kinase, SuSK; NCBI GenBank EST database Acc: FG804674). The sequence analysis of the SuSK revealed homology to Arabidopsis thaliana shaggy-related protein kinase delta (E value, 1e -108), dzeta and iota. Alignment of the catalytic domain sequence of GSK-3/shaggy-like kinase with partial sequence of SuSK performed using ClustalW tool indicated kinase active-site signature sequence. Spatial and temporal transcript expression profiling of the SuSK gene based on Real-Time PCR revealed significant induction of transcript expression in response to short-term salt (NaCl 200 mM) or polyethylene glycol-8,000 (PEG; 20% w/v) induced osmotic stress in leaves and shoots of sugarcane plants. The transcript expression increased progressively under salt stress and reached to 1.5-fold of the control up to 8 h treatment. In response to PEG stress, the transcript expression increased by 1.5-fold over the control in 2-h treatment in leaf, whereas in shoots, the expression remained unchanged in response to the various treatments. Differences in growth parameters, relative water content, and membrane damage rate were statistically insignificant in the short-term salt or PEG-stressed plants as compared to the control, non-stressed plants. Expression analysis revealed the differential and temporal regulation of this gene under salt and PEG stress and that its early induction may indicate involvement in stress signaling. © 2010 Springer-Verlag.

Siddiqui M.A.,Kumaun University | Pande V.,Kumaun University | Arif M.,Defence Institute of Bio energy Research
Enzyme Research | Year: 2012

A thermophilic fungal strain producing polygalacturonase was isolated after primary screening of 40 different isolates. The fungus was identified as Rhizomucor pusilis by Microbial Type Culture Collection (MTCC), Chandigarh, India. An extracellular polygalacturonase (PGase) from R. pusilis was purified to homogeneity by two chromatographic steps using Sephadex G-200 and Sephacryl S-100. The purified enzyme was a monomer with a molecular weight of 32 kDa. The PGase was optimally active at 55°C and at pH 5.0. It was stable up to 50°C for 120 min of incubation and pH condition between 4.0 and 5.0. The stability of PGase decreases rapidly above 60°C and above pH 5.0. The apparent Km and Vmax values were 0.22 mg/mL and 4.34 U/mL, respectively. It was the first time that a polygalacturonase enzyme was purified in this species. It would be worthwhile to exploit this strain for polygalacturonase production. Polygalacturonase from this strain can be recommended for the commercial production because of its constitutive and less catabolically repressive nature, thermostability, wide range of pH, and lower Km properties. However, scale-up studies are needed for the better output for commercial production. © 2012 Mohd. Asif Siddiqui et al.

Kumari M.,Banaras Hindu University | Kumari M.,Defence Institute of Bio energy Research | Pudake R.N.,Sudan University of Science and Technology | Singh V.P.,Banaras Hindu University | Joshi A.K.,Banaras Hindu University
Euphytica | Year: 2013

The presence or absence of the staygreen trait was screened for 3 consecutive years in 963 wheat lines from various sources, including Indian and CIMMYT germplasm. Staygreen was assessed at the late dough stage by visual scoring (0-9 scale) and the leaf area under greenness (LAUG) measurement. Around 5. 5 % of the lines were staygreen, 10. 5 % were moderately staygreen, and the remaining lines showed little or no expression of the trait. One hundred lines showing diversity for the staygreen trait were sown under three different sowing dates (timely, late and very late) for 3 consecutive years in three replications to determine the association of staygreen with heat tolerance. There was a decline in yield, biomass, grain filling duration (GFD) and 1,000 grain weight (TGW) under late and very late sowing conditions owing to terminal stress at anthesis and later stages. However, the decline was relatively less in staygreen genotypes compared to the non-staygreen (NSG) ones. The correlation study showed that LAUG and canopy temperature depression (CTD) were strongly correlated. LAUG and CTD were also significantly associated with grain yield, GFD and biomass. To further confirm the association of the staygreen trait with terminal heat stress, individual F2-derived F7 progenies from the cross of the 'staygreen' lines with NSG were evaluated for yield and yield traits at the three sowing dates. In each cross, the staygreen progenies showed a significantly smaller decline in yield and TGW under heat stress than the NSG progenies. These results appear to suggest an association between the staygreen trait and terminal heat stress and, thereby, that the staygreen trait could be used as a morphological marker in wheat to screen for heat tolerance. © 2012 Springer Science+Business Media B.V.

Patade V.Y.,Bhabha Atomic Research Center | Patade V.Y.,University of Pune | Patade V.Y.,Defence Institute of Bio energy Research | Bhargava S.,University of Pune | Suprasanna P.,Bhabha Atomic Research Center
Plant Cell, Tissue and Organ Culture | Year: 2012

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (-0. 7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level. © 2011 Springer Science+Business Media B.V.

Grover A.,Defence Institute of Bio energy Research | Sharma P.C.,Guru Gobind Singh Indraprastha University
Critical Reviews in Biotechnology | Year: 2016

Molecular markers, due to their stability, cost-effectiveness and ease of use provide an immensely popular tool for a variety of applications including genome mapping, gene tagging, genetic diversity diversity, phylogenetic analysis and forensic investigations. In the last three decades, a number of molecular marker techniques have been developed and exploited worldwide in different systems. However, only a handful of these techniques, namely RFLPs, RAPDs, AFLPs, ISSRs, SSRs and SNPs have received global acceptance. A recent revolution in DNA sequencing techniques has taken the discovery and application of molecular markers to high-throughput and ultrahigh-throughput levels. Although, the choice of marker will obviously depend on the targeted use, microsatellites, SNPs and genotyping by sequencing (GBS) largely fulfill most of the user requirements. Further, modern transcriptomic and functional markers will lead the ventures onto high-density genetic map construction, identification of QTLs, breeding and conservation strategies in times to come in combination with other high throughput techniques. This review presents an overview of different marker technologies and their variants with a comparative account of their characteristic features and applications. © 2014 Informa Healthcare USA, Inc.

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