Dr Avinash Mishra

Abstract: A simple and rapid method for micropropagation of succulent, salt accumulator and extreme halophyte Salicornia brachiata has been established for the first time using shoot tips and nodes. Individually, BA showed significant response compared to Kn and in combinations, improved shoot proliferation was observed with BA + NAA than BA + 2,4-D, however no significant response was observed with BA + IAA. Percentage of shoot response significantly increased with NaCl treatment in the combination of BA + NAA while BA + 2,4-D + NaCl combination showed reduced shoot proliferation followed by demises of most of cultures. Efficient shoot proliferation was observed with combinations BA (8.9 μM) + NAA (5.37μM) + NaCl (500 mM) and BA (13.3 μM) + NAA (5.37μM) + NaCl (250 mM) indicating that NaCl is required for the micropropagation. The developed method will facilitate functional analysis of novel salt responsive gene(s) isolated from S. brachiata and propagation of industrially important elite accessions.

Abstract: Salicornia brachiata is an extreme halophyte growing luxuriantly in the coastal marshes and frequently exposed to various abiotic stresses including heavy metals. A full length type 2 metallothionein (SbMT-2) gene was isolated using RACE and its copy number was confirmed by southern blot analysis. Transcript expression of SbMT-2 gene was analyzed by semi-quantitative Rt-PCR and real time quantitative (qRT) PCR. Expression of SbMT-2 gene was up-regulated concurrently with zinc, copper, salt, heat and drought stress, down regulated by cold stress while unaffected under cadmium stress. Heterologous expression of SbMT-2 gene enhances metal accumulation and tolerance in E. coli. Metal-binding characteristics of SbMT-2 protein show its possible role in homeostasis and/or detoxification of heavy metals. Significant tolerance was observed by E. coli cells expressing recombinant SbMT-2 for Zn(++), Cu(++) and Cd(++) compared to cells expressing GST only. Sequestration of zinc was 4-fold higher compared to copper and in contrast SbMT-2 inhibits the relative accumulation of cadmium by 1.23-fold compared to GST protein. Fusion protein SbMT-2 showed utmost affinity to zinc (approx. 2.5 fold to Cu(++) and Cd(++)) followed by copper and cadmium ions with same affinity. Halophyte S. brachiata has inherent resilience of varying abiotic tolerance therefore SbMT-2 gene could be a potential candidate to be used for enhanced metal tolerance and heavy metal phytoremediation.

Abstract: Production of biosurfactant from an alkaliphilic bacterium Cronobacter sakazakii (accession no. JN398668) was screened by haemolytic assay, emulsifying activity and surface tension measurement. Biosurfactant, comprised of total sugars (73.3%), reducing sugars (1.464%), protein (11.9%), uronic acid (15.98%) and sulfate (6.015%), showed low viscosity with pseudoplastic rheological behavior and exhibited significant emulsification activity with oils and hydrocarbons. A series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligosaccharides were detected in the positive ion reflector mode of MALDI TOF�TOF MS. GC�MS analysis revealed composition of monosaccharide moieties (w/w) viz. glucose (14%), mannose (24%), galactose (14%), xylose (20%) and arabinose (1.9%). 1H NMR, FT-IR and EDX analyses confirmed the characteristic various functional groups, bonds and elements respectively. Thermostability (up to 260 °C) and CI (0.456) were determined by TG and DSC analyses. Inherent properties of biosurfactant make it a potential candidate for bioremediation of oil and hydrocarbons.

Abstract: An alkaliphilic bacterium, Klebsiella sp. strain RJ-03, produced a biosurfactant, which showed low viscosity with pseudoplastic rheological behavior and exhibited emulsification activity with oils and hydrocarbons. The biosurfactant has excellent oil removing efficiency as compared to chemical surfactants. The isolated biosurfactant has compatibility with detergents and enhanced oil removing efficiency from soil and cotton cloths. It comprised of sugar, uronic acid, protein and sulfate. GC-MS analysis confirmed the presence of six monosaccharides (w/w), glucose (6.65%), galactose (23.98%), rhamnose (14.94%), mannose (17.54%), fucose (9.47%) and 6�O-Me-galactose (1.4%). It is a high molecular weight, thermostable biopolymer showing degradation above 300 °C. Positive ion reflector mode of MALDI TOF-TOF MS analysis revealed series of low and mid range mass peaks (m/z) corresponding to mono-, di-, tri- and oligo- saccharides content. The NMR, FT-IR, EDX-SEM, AFM and PSD analysis confirmed the presence of functional groups, bonds, elements and particle size respectively.

Abstract: Salicornia brachiata is an extreme halophyte that grows in salty marshes and is considered a potential alternative crop for seawater agriculture. Salicornia seeds are rich in protein and its tender shoots are eaten as salad greens. Seed storage proteins were fractionated by sequential extraction using different solvents, including distilled water for albumins, NaCl (1.0 M) for globulins, NaOH (0.1 N) for glutelins and ethanol (70% v/v) for prolamins. Globulins accounted for 54.75% of the total seed storage proteins followed by albumins (34.30%) and glutelins (8.70%). The fractionated proteins were characterized using 2-D-diagonal-SDS-PAGE and Matrix-assisted laser desorption/ionization-Time of Flight (MALDI-TOF) mass spectrometry. The globulin fraction, composed of seven inter-molecular disulfide-linked polypeptide pairs of mol. wt. 63.5, 62.5, 54.7, 53.0, 43.2, 38.5 and 35.1 kDa, encompassed a basic and an acidic subunit. Two-dimensional gels revealed approximately 32 spots, with isoelectric points and molecular masses ranging from 4.93 to 11.6 and ~5.2 to ~109.4 kDa, respectively. Protein spots were identified by MALDI-TOF MS peptide mass fingerprint analysis and further classified. Homology analysis demonstrated that 19% of the proteins were involved in metabolism, 16% were involved in signaling and 15% were regulatory proteins. Peptide mass fingerprint analysis confirmed the presence of inter- and intra-molecular disulfide linkages in the globulin fraction. Sulfur-rich proteins are of high nutritional value and disulfides make S. brachiata a potential source of dietary supplementation.

Abstract: An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 μm, He pressure 1100 and 1350 psi with a target distance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5�7 mg L�1) of hygromycin. The integration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T0 transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function.

Abstract: Extracellular polymeric substances comprised of average molecule size 1264.354m, exhibited characteristic diffraction peaks at 6.025�, 9.675�, 22.775� and 28.475� with d-spacing 14.74755, 9.36297, 3.88747 and 3.11512A� , respectively. EDX confirms the presence of sulphate (2.7%) and 1H NMR reveals uronic acid, primary amine, aromatic-compounds, halides, aliphatic alkyl and sulfides. EPSs were thermostable upto 270 �C with CIxrd 0.12 and CIDSC 0.18. The dynamic viscosity is significantly high at pH 3.0 and decreases concomitantly with shear rate, confirming pseudoplastic rheological property. MALDI TOF�TOF represents a series of masses in linear mode corresponding to mass of pentose and hexose with ions. The positive ion reflector mode exhibited low mass peaks (m/z) corresponding to oligosaccharide and higher peaks for polysaccharide consist of different ratio of pentose and hexose associated with ions. EPSs allow further exploration of D. salina as potential EPSs producer and make it a promising candidate for biotechnological and industrial exploitation.

Abstract: Tau class glutathione transferases (GSTU) genes are plant specific, induced by different abiotic stress, and important for protecting plants against oxidative damage. GST gene was isolated using 50 RACE from an extreme halophyte Salicornia brachiata, cloned, sequenced and its protein structure was predicted. Transcript profiling of SbGST gene expression was studied under different abiotic stress conditions and plant growth regulator treatments, viz. salt, cold, drought, ABA and salicylic acid, with time period point and concentration point. The expression of SbGST gene was up-regulated in all stress conditions, except SA treatment. Seed germination percentage, GST enzyme assay, fresh weight and other growth parameters (root length, shoot length and leaf area) were studied and results indicate that over-expression of SbGST gene in transgenic tobacco leads to enhanced seed germination and growth under salt stress. Transgenic lines were evaluated for their performance under salt stress and tobacco plants over-expressing SbGST showed higher seed germination and survival compared to wild type. These results confirm that expression of SbGST gene is up-regulated by different stresses and over-expression of tau class SbGST gene in transgenic tobacco plays a vital role in abiotic stress tolerance. SbGST gene expressed conspicuously under salt stress leading to enhance seed germination and better growth. Furthermore, GST is a potential candidate gene to be used in genetic engineering for enhancing abiotic stress tolerance.

Abstract: In the present study, EPS secreted by the endophytic bacterium Bacillus licheniformis was isolated and characterized. The molecular masses of the EPS were 1540 and 44565 kDa and 1H NMR, FT-IR and UV�Vis spectral analyses revealed prevalence of characteristic primary amine-group, aromatic-compound, halide and aliphatic alkyl-group in addition to Na, P, Ca, C, O, Cl and S as inferred from EDX analysis. XRD and DSC analysis confirmed the amorphous nature of EPS, showing an average particle size of 24.977 μm (d 0.5) with 191 nm average roughness. The positive ion reflector mode of MALDI TOF-TOF MS exhibited a series of low and high mass peaks corresponding to various oligosaccharides and polysaccharides respectively. Further, GC-MS analysis revealed its four monosaccharide constituents glucose, galactose, mannose and arabinose. The potential heterogeneous properties of EPS as revealed in the present study may be explored in various biotechnological and industrial applications.

Abstract: A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 , respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. 1HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CIxrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF-TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.

Abstract: Hydrogen peroxide content in Dunaliella salina cells increases concomitantly with salinity, and maximum content occurred at 5.0 m NaCl concentration. Activity of the antioxidant enzyme superoxide dismutase (SOD, EC 1.15.1.1) increased significantly by two- and three-fold in 1.0 and 2.0 m salt concentrations, respectively, thereafter declining significantly. Unlike SOD, catalase (EC 1.11.1.6) activity decreased in tandem with salinity; however, there was no significant decrease in catalase activity up to 2.0 m salt concentration (compared to 0.5 m salinity). There was no significant decrease in catalase activity from 2.0 m to 5.0 m salinity, whereas a significant decrease was observed in 4.0 and 5.0 m salinity (compared to 1.0 m salinity). Ascorbic acid peroxidase (EC 1.11.1.11) activity decreased concurrently with salt stress up to 2.0 m, thereafter no significant change was observed in cells grown in varying salt concentrations (0.5�5.0 m NaCl). Detailed study of hydrogen peroxide content and antioxidant enzymes in response to varying salinity (0.5�5.0 m NaCl) has not been reported previously in Dunaliella salina. Antioxidant enzymes and H2O2 scavenging systems have short-term adaptation mechanisms for protection against salt stress and are not important in imparting salt stress tolerance to Dunaliella salina at high salinity because of production of secondary antioxidant metabolites, like β-carotene, which protect the alga against salt induced oxidative damages.

Abstract: Differentially expressed genes in Dunaliella salina grown under super saturated salt stress (5.5 m) were isolated by subtractive hybridization, cloned and classified for further biotechnological and industrial exploration. Sequence homology analysis showed that 26% of the library represented salt responsive genes for metabolic enzymes, viz., fructose-1,6-diphosphate aldolase (AldP), glucose-6-phosphate dehydrogenase, carbonic anhydrase, phosphoglycerate kinase etc., while 6% of library clones contained hypothetical/unknown genes. Six clones (8% of the library) contained genes that differentially expressed under salt stress, however, 6�8% of the library represented genes over expressed under salt stress and involved in photosynthesis, defense, cell rescue (chaperon/protease inhibitor) and protein sorting. About 39% were other genes of which 6% and 14% were transcription factors and house keeping genes (putative enzymes/signaling) respectively, while the remaining 19% of genes had miscellaneous functions.

Abstract: The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2-3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2-3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA-based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.

Abstract: Sixty blackgram accessions, collected from diversity rich zones of India, were evaluated in field for nine quantitative characters as per IBPGR description, which classified accessions into eight non-overlapping clusters based on principal component and non-hierarchical euclidean cluster analysis. The maximum inter cluster distance (10.270) was present between cluster number VIII and III while minimum inter cluster distance (1.656) was observed between cluster I and V. The maximum numbers of accessions were grouped in cluster V, while minimum number of accession (1) was found in cluster VIII. SDS-PAGE for seed storage proteins of accessions classified them into eight groups. Total number of bands ranged from 17 to 28, spread over four zones (A B, C and D). Maximum (10) genotypes were grouped in 28 band group while minimum (2) genotypes were sorted in 17 band group. Based on euclidean distance and dissimilarity co-efficient obtained for SDS-PAGE of seed proteins, dendrogram was constructed which categorized genotypes into nine clusters. Present study resulted in the evaluation of germplasm and listing of genetic donors for various agronomic characters, suitable for future breeding and biotechnology programme which will facilitate the use of this germplasm for blackgram improvement.

Abstract: Pre-cultured cumin embryos were bombarded under 27 inches Hg vacuum, 25 mm distance from rupture disc to macrocarrier, 10 mm macrocarrier flight distance using 1100 psi rupture disc and 9 cm microprojectile travel distance. An average of 110 embryos was used per shot and 91% embryos showed transient GUS expression after 24 h. Shoot tips and roots of T0 plantlets exhibited GUS expression done after 3 months of bombardment. Transformation was confirmed with PCR amplification of 0.96 and 1.3 kb band of hptII and gus genes respectively from T0 transgenics and southern blot analysis using PCR amplified DIG labeled hptII gene as probe. It is the first successful attempt of transformation of cumin plant through direct gene transfer using particle gene gun and adequately exhibiting the possibility of stable transformation in cumin.

Abstract: The antibacterial activity of Aloe barbadensis was tested on clinically isolated bacterial pathogens i.e. Enterococcus bovis, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Morganella morganii, and Klebsiella pneumoniae causing infection in human being. Ethanolic and aqueous extracts were used for the antibacterial effect, which was measured by the appearance of zone of inhibition. Relatively higher MIC concentrations were obtained for gram negative bacteria E. coli and K. pneumoniae, with ethanol extract; however, no inhibitory effect was noted for aqueous extract. Ethanolic extract possesses great inhibitory activity for gram positive bacteria, E. bovis followed by S. aureus. Among gram negative bacteria, highest inhibitory effect was observed with P. aeruginosa, followed by M. morganii, P. mirabilis, and P. vulgaris, which was significant (p�<�0.01) than E. coli and K. pneumoniae. Antimicrobial activity tests of crude extract of A. barbadensis were carried out to validate the use of traditional medicinal herbal and results of this study tend to give credence to the common use of A. barbadensis gel and leaf.

Abstract: Proteins are one of the targets for improving the nutritional quality, and attempts are being made through manipulation of its native gene(s). Pigeonpea (Cajanus cajan L.) is one of the nutritionally important legumes of tropical and subtropical regions of the world, and studies of the structure of seed storage proteins and their interactions have been limited by the difficulty of isolating single-protein subunits in large amounts from a complex mixture of the seed endosperm. One way to overcome this problem is the expression of seed storage protein-encoded gene(s) in heterologous systems that have additional advantages wherein specific gene modifications can be made and the new gene constructs can quickly be expressed. Legumin protein was extracted from pigeonpea seeds of different developmental stages (5th to 25th day after flowering [DAF]) and characterized. The legumin gene (leg) of size 1.482 kb was screened, using the deoxygenin-labeled legumin probe, from the complementary deoxyribonucleic acid (cDNA) library, constructed from 18-day-old (DAF) immature seeds of pigeonpea and sequenced (accession no. AF3555403). The legumin gene was further characterized by DNA blotting, and its probable secondary structure was predicted using online ExPASy server. Significant Protein Data Bank (PDB) alignment of the deduced legumin protein by BLASTP was observed with proglycinin of soybean. Comparative 3D structural homology was predicted by Cn3D software, and the legumin protein showed the 3D structure alignment and interaction homology with proglycinin chain 1FXZA (PDB no. 1FXZ). The legumin gene was subcloned in vector pET-24a driven by the bacterial promoter, and its expression was detected in Escherichia coli by immunoblotting using polyclonal antibodies, raised against the purified legumin protein.

Abstract: Twenty eight accessions of blackgram (Vigna mungo L. Hepper) were screened against salt stress under controlled conditions using five different parameters (germination percentage, plumule length, radicle length, plumule-radicle length ratio and dry matter weight) at four different salt concentrations (0.00 EC, 4.65 EC, 11.25 EC and 16.00 EC). Selected genotypes were further analyzed to assess salt stress-associated biochemical and RAPD markers. A band of peroxidase isoenzyme (Rm 0.38) was observed in tolerant genotypes at all four salt concentrations while in susceptible genotypes at high salt concentration (11.25 and 16.00 EC) only. A peroxidase band (Rm 0.50) was also observed in tolerant genotypes at 11.25 EC with faint intensity. Two bands of malate dehydrogenase (Rm 0.12 and 0.14) were observed under salt stress only. The RAPD banding pattern showed high polymorphism with several unique loci which can help in identification and discrimination of tolerant and susceptible genotypes. A 300 bp band, identified as a RAPD marker for discriminating tolerant from susceptible genotypes, was amplified by four primers (G 08, H02, H 06 and H 08) in the tolerant group only. The study results in categorization and listing of germplasm that can be explored while breeding for saline stress in blackgram.

Abstract: Sixty blackgram accessions were evaluated and classified into different clusters to assess genetic diversity and traits using isoenzymes. Trait-specific expression was assessed, and isoenzyme bands were observed: a peroxidase band (Rm 0.60) associated with dwarfness and an esterase band (Rm 0.25) with tallness. Early maturing varieties were characterized by a specific esterase isoenzyme band of Rm 0.51. All yellow mosaic virus susceptible genotypes had two bands of esterase isoenzyme, Rm 0.42 and 0.70. Resistant genotypes showed three bands (0.32, 0.33, and 0.35) of alkaline phosphatase. Peroxidase isoenzyme was helpful to differentiate green-seeded from black-seeded varieties. Two bands (0.58 and 0.83) were observed in black-seeded accessions, and two different bands (0.74 and 0.76) were observed in green-seeded accessions. Clustering of germplasm and assessment of traits will facilitate the use of germplasm for the improvement of blackgram.

Abstract: Extracellular polymeric substances (EPSs), produced by Dunaliella salina strain, increase concomitantly with salt concentration and maximum (944mg/l) were obtained at 5M NaCl, whereas minimum (56mg/l) at 0.5M salinity. Emulsifying activity was measured in terms of strength to retain the emulsion and comparatively 85.76% retention was observed at 0.5M salinity thereafter it intends to decline. The FT-IR-spectra reveal characteristic functional groups NH stretching, asymmetrical CH stretching vibration of aliphatic CH(2)-group, CC stretching of aromatic, CN stretch of aliphatic amine, NH wag of primary amine and CX stretch of alkyl-halides with a stretching of COC, CO corresponding to the presence of carbohydrates. The FT-IR-spectra substantiated the presence of primary amine-group, aromatic-compound, halide-group, aliphatic alkyl-group and polysaccharides. Four monosaccharides (glucose, galactose, fructose and xylose) were also detected by HPLC analysis. Production of EPSs may allow further exploration of D. salina as potential EPSs producer and make it as a promising candidate for biotechnological and industrial exploitation.

Abstract: With the aim of grain sorghum improvement to enhance its economic value, HMW glutenin gene subunit(s) were isolated from wheat (T. aestivum var. PBW343) using allele specific primers (AS-PCR) designed on the basis of allelic data available in NCBI Gene Bank. Three glutenin genes viz. Ax1, Dx5 and Dy10 were isolated, cloned, sequenced and analyzed using on line bioinformatics tools. These sequences showed significant homology with the HMW glutenin gene sequences available in the NCBI gene bank. To facilitate transformation of these genes to sorghum, these genes were transferred to a pUC based vector under the regulation of kafirin promoter of sorghum which makes it specific for expression in the endosperm of sorghum. HMW glutenin gene cloned in pGEM vector was excised using restriction enzymes SacI and SacII and directional cloning was performed successfully to clone under the control of kafirin gene promoter of sorghum. Transfer of these gene(s) could be useful for improving the bread making quality of sorghum.

Abstract: A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11 degrees E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0-1.16 +/- 0.05 mug chl. a and 0.2-0.29 +/- 0.01 mug chl. b per 10(6) cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 +/- 0.12 mug chl. a and 0.84 +/- 0.4 mug chl. b per 10(6) cells) was observed which declined again at 5.5 M NaCl concentration (2.0 +/- 0.1 mug chl. a and 0.52 +/- 0.03 mug chl. b per 10(6) cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 +/- 0.07 mug), glycine betaine (5.7 +/- 0.28 mug), glycerol (3.7 +/- 0.18 ml) and total sugar (250 +/- 12.5 mug) per 10(5) cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 +/- 0.01 mug per 10(5) cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na(+) remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K(+) and Mg(++) was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.

Abstract: Study of the structure of high molecular weight (Mr) glutenin subunits and their interaction has been limited by the difficulty of isolating single subunits in large amounts from the complex mixture of gluten wheat endosperm. One way to overcome this problem is the expression of high Mr glutenin subunits in heterologous systems. These systems have the additional advantages that, specific gene modifications can be made and the new gene constructs can quickly be expressed. HMW glutenin gene subunits, associated with better dough quality, were isolated from wheat (T. aestivum var. PBW343) using allele specific primers (AS-PCR) designed on allelic gene sequences. Three glutenin genes 1Ax1, Dx5 and Dy10 of sizes 1.8, 2.0 and 1.47 kb were cloned and sequenced. Probable secondary structure of HMW glutenin protein was predicted using online ExPASy server. HMW glu Dx5 and Dy10 gave significant similarity (more than 90%) to the available database but, 1Ax1 didn't show significant similarity. Glutenin genes were further subcloned in expression vector pCAMBIA-1304 driven by CaMV35S promoter and their expression was observed in Escherichia coli. The transcription was confirmed by Reverse transcriptase PCR (Rt-PCR) and sequencing. The sequences of HMW glu genes showed 100% homology with cloned HMW glutenin genes. Although initial western blotting using polyclonal antibodies against glutenin protein has strongly indicated its expression at the translation level, it is needed to be further confirmed through purification and sequencing of proteins.

Abstract: Tetraploid and hexaploid Indian wheat accessions were analyzed for genetic polymorphism using RAPD-PCR in order to study the gene rearrangements during polyploidization. Ten RAPD primers were employed to establish the evolutionary relationship amongst 10 tetraploid and 17 hexaploid wheat accessions. Tetraploid accession showed 75.7% polymorphism whereas hexaploid accessions showed 65.3% polymorphism. The Genetic Distance (GD) value of the tetraploid accessions ranged 0.400 to 0.966 was significantly higher than the GD values of the hexaploid accessions (0.630 to 0.952). RAPD primers clearly categorized tetraploid to hexaploid accessions in different groups according to similarity coefficient. Nearly all tetraploid accessions were grouped together. The same set of primers was also able enough to establish polymorphism amongst 20 Karnal bunt susceptible and resistant tetra and hexaploid wheat varieties. Like tetraploid accessions, susceptible varieties showed low genetic relationship. Some of the primers gave a distinct RAPD pattern for the discrimination between resistant and susceptible varieties but none of the primer was able to discriminate the resistant, susceptible and moderately susceptible wheat varieties. The phylogenetic polymorphism according to ploidy level and resistant status to KB is interpreted as the result of alteration in gene loci during polyploidization, genetic drift during inter and intra specific breeding and/or selection pressure for improved fertility.

Abstract: Gamma-kafirin gene of Sorghum bicolor L var. M35-1 was isolated using PCR based approach by designing specific primers. The primers gave highly reproducible amplification. The amplicons were then cloned and sequenced. Nucleotide sequences were subjected to the homology search and a comparative analysis was done with other prolamins. Amplified �-kafirin gene (accession no. AY 566298- 99) showed 99% homology with �-kafirin gene of Sorghum vulgare. As compared to Sorghum vulgare, only 3 extra bases were present in Sorghum bicolor at position 40 nucleotide bases downstream of transcription initiation site. These sequences were related with prolamins of other genera i.e. coix and maize with 84% sequence homology. Deduced protein sequence of �-kafirin gene of S. bicolor (accession no. AAS 73290) gave significant similarity of 99, 79 and 77 % with gamma-kafirin protein of S. vulgare, �-zein and �-coixin proteins, respectively. All cysteine residues of �-kafirin were highly conserved. Probable secondary structure of gamma-kafirin protein was predicted using online ExPASy server. The study of temporal expression of �-kafirin gene is needed for analysing the expression pattern of introgressed gene(s) driven by its promoter. Temporal expression of gamma-kafirin gene in endosperm studied by semi quantitative Rt-PCR with specifically designed primers, showed that �-kafirin expression started at 7 DAP (days after pollination). There is statistically non significant increase in expression upto 14 DAP there after expression increased significantly (at 5% level) and reached at maximum level on 21 DAP. Expression of �-kafirin started decreasing after 21 DAP and a very low expression was detected at 28 DAP.

Abstract: Modern biological tools of genetic engineering and biotechnology can allow transfer of gene(s) across crop species. The r-DNA technology has tremendous potential to transfer bread making character of bread wheat into sorghum by transferring glutenin gene(s), which can improve the visico-elastic property of the sorghum flour/dough. These genes in addition to improving quality can significantly contribute to improve the nutritional status by the addition of more protein fractions also. In the simplest approach, new HMW gluten loci may be created via transformation to bioengineer sorghum quality. For this, amplified γ-kafirin promoter (574 bp) was subcloned in pCAMBIA1304 by replacing CaMV35S promoter (ca. 800 bp) of the gus reporter gene resulting in vector pkaf-gus, where the expression of gus reporter gene is under the control of γ-kafirin promoter. In order to construct a gene cassette where HMW glu gene(s) will be under the control of γ-kafirin gene promoter, kafirin promoter was first cloned in pUC19 and then HMW gene(s) were excised from their respective vectors and cloned under the control of promoter. Finally, two gene cassettes were developed as pKaf-Dx5 and pKaf-Dy10 where expression of the HMW glu gene Dx5 (8.7 kb) and Dy10 (6.4 kb) was driven by the γ-kafirin gene promoter. Both gene cassettes are ready to clone in any vector to bioengineer sorghum by genetic transformation.

Abstract: Different cis acting elements of gamma kafirin gene from Sorghum bicolor var. M 35-1 were amplified and cloned using different combination of the primers. The amplified promoter was replaced with CaMV35S promoter of vector pCMBIA-1304 and resultant vector contained beta-glucuronidase (gus) gene under the control of amplified gamma-kafirin promoter. The resulting fusants were then transformed in to different explants of sorghum via particle bombardment. The regulation of uid gene expression was analyzed to find out the minimum required 5' regulatory sequence and cis acting elements for the efficient expression. However no gus expression was detected in leaves of micropropagated plants, scutellum and calli at any stage of growth. The expression of gus, with pKaf gus-P4 gene construct, was detected in immature embryos and endosperm 20 days after pollination (DAP). The result suggest that at least three motifs (two GCN4 and one prolamin box) besides TATA and CATC boxes are required for the efficient expression of the kafirin gene of sorghum. The study shows that PCR based isolation of different motifs and regions can be used as an alternate to deletion analysis for observing the role of various motifs and their importance in the gene expression and regulation.

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Abstract: Bioinformatics is a scientific discipline to understand biological functions through analysis of molecular sequence information from DNA, RNA or Proteins. It is the computer aided data handling approach to understand the life processes precisely. Biological problems require innovative database perspective for evolution and Phylogenetic studies, protein structure prediction, molecular sequence management and alignment, recognition of genes & regulatory elements and for gene expression. Bioinformatics is a strong tool to improve the crop . some common applications of bioinformatics in agriculture are hybrid machine model in rice against rice blast fungus, mapping of QTLs in plant breeding, in plant systematic etc and the best example is Sputnik- a database platform for comparative plant genomics.

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Abstract: Bioinformatics is a scientific discipline of discovering biological functions through analysis of molecular sequence information from DNA, RNA or proteins. It is the computer assisted data management discipline that enables one to understand life's processes precisely. It helps to discover better drug target after differentiating between healthy and diseased. Bioinformatics is being considered bedrock for the future of biotechnology and is being adopted worldwide by academicians, companies, national and international consortia. Statistical and computational analysis of the complex relationships between phenotypes and genotypes; animals and plants plays a crucial role in the future of biology.

Abstract: Polymerase chain reaction (PCR) based randomly amplified polymorphic DNA (RAPD) markers were used to study the genetic relationship and genetic diversity among 27 Indian wheat accessions (17 hexaploids and 10 tetraploids). The size of PCR amplified products ranged between 0.03 and 3.0 kb. Out of the 103 amplified total RAPD bands, 82 (79.6%) were polymorphic. Within hexaploids, of the total 98 amplified bands, 64 (65.3%) were polymorphic whereas within tetraploids, of the total 103 bands, 78 (75.7%) were polymorphic. The similarity coefficient between hexaploids and tetraploids ranged from 0.630 to 0.952 and 0.400 to 0.966, respectively. Primers UBC-535, UBC-552, UBC-600, UBC-534 and UBC-386 showed maximum number of polymorphic bands. The primers UBC-18, UBC-535, UBC-337, UBC-600, UBC-572 and UBC-534 were found to give distinguishable number of unique RAPD markers. These six primers were able to distinguish some of the genotypes like, IC-82233, IC-99785, IC-35161-D, IC-35177-D and IC-35720-D. As the similarity matrix value showed, the tetraploid genotypes had more wider genetic distance value than hexaploids.

Abstract: Polymerase chain reaction (PCR) based RAPD profiles, in conjunction with six primers, of Karnal bunt of wheat and rice bunt exhibiting distinct polymorphic DNA. A total of 84 RAPD loci were observed on polyacrylamide gel for both Tilletia sps. Out of 84, 16 loci were found monomorphic, while other 68 loci were unique. Usefulness of random primers was also checked with other seed borne fungal pathogens of wheat and rice. None of primers gave amplification with Magnaporthe grisea, a causative agent of rice blast. However, distinct RAPD profiles were obtained with Alternaria triticina, Fusarium monaliforme, Helminthosporium sativum and Rhizoctonia solani. These six arbitrary primers could distinguish T. indica, a quarantine fungal pathogen from a non-quarantine fungal pathogen, T. barclayana. The two Tilletia sps. could be discriminated not only on the basis of distinct RAPD profiles, but also by presence of few unique gene fragments amplified using all six primers.

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Abstract: Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. Specific gene silencing has been shown to be related to two ancient processes, co-suppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.

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Abstract: Metallothioneins (MTs) are cysteine-rich polypeptides that are involved in metal detoxification andhomeostasis in both prokaryotes and eukaryotes. MT genewas isolated through 5' RACE from EST database generatedat CSMCRI Bhavnagar. In silico analysis reveals that SbMT gene belongs to the class II, type-2 metallothionein-likegenes. The SbMT2 gene is 421 bp long, and contains 237 bpORF region encoding 78 amino acids protein with anestimated molecular mass of 8.5 KDa. For expressionanalysis, SbMT2 gene was cloned into bacterial expressionvector PGEX6p-3. The SbMT2 protein was purified,analysed on SDS-PAGE and further confirmed by MALDI-TOF-TOF. BL21(DE3) cells transformed with SbMT2 gene showed tolerance to 0.5 mM ZnSO4 and 1.0 mM CuSO4 but not significant to 0.5 mM CdSO4 as compared to PGEX6p-3 vector only.

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Abstract: Among the major cereals, grain sorghum ranks third in area (11.7 Mha) and production (10.5 mt). Almost half of it is grown during rabi season primarily in Maharashtra, Karnataka and Andhra Pradesh. In these states along with Tamil Nadu, Gujarat and Rajasthan, it is the staple food mainly among the poor. It contains similar levels of starch (68 -73 %) and protein (9-14 %) as in other cereals, which are considered nutritious. However, some factors like poor dough making quality, presence of tannins and low lysine content make it less palatable. Poor shelf life and inability to make soft fluffy, palatable and easily digestible roti limits its role in economic empowerment of sorghum producers and end users. Unlike sorghum, wheat possesses good dough making quality due to presence of certain proteins having stretchable properties. The unique elastic and cohesive properties of wheat are mainly due to its water insoluble (gluten) proteins. It was thought that value addition in sorghum, in terms of roti making quality could be enhanced through introgression of HMW- gs genes for dough strength. Thus expression of HMW- gs in sorghum will improve it quality in two ways a) Improvement in chapatti making characters. b) Expression of gene in sorghum protein bodies may enhance its digestive properties. With the advent of genetic engineering, it is now possible to create new HMW glutenin loci through heterologous gene transfer to bioengineer sorghum quality. For expression of appropriate HMW glutenin subunit of wheat in the endosperm of sorghum grain. Seed specific promoters are inevitable. Proper designing gene construct of HMW-gs gene under the influence of strong seed specific promoters from sorghum in one of novel and simplest approach of genetic engineering of sorghum for quality improvement. The improvement of sorghum cultivars is a major incentive to poor and marginal farmers, since sorghum can sustain yield in unpredictable environmental conditions of arid and rain-fed areas of our country. With the aim of improvement of grain sorghum to enhance its marginal value for poor consumers and producers, the present study was done as a part of NATP project. In present investigation, putative cDNA library of wheat (var. PBW 343) was prepared for HMW glutenin gene, γ- kafirin promoter of sorghum (cv. M-35-1) was isolated and its strength was analyzed in the terms of the specificity by biolistic bombardment. Different HMW glu gene(s) were procured from the abroad and HMW glu gene was fused with the γ-kafirin promoter through subcloning. Presently glu gene Dx5 and Dy10 under the control of γ-kafirin promoter is cloned in pUC 19 and ready to subclone in any binary vector for the transformation to sorghum.

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Abstract: Important Accessions: 1. Mishra Avinash and Jha B. 2008. Cloning of Na+/H+ antiporter (NHX-1) gene promoter from Salicornia brachiata and its application in abiotic stress. NCBI Accession No.: EU6286802. 2. Mishra Avinash and Jha B. 2009. Differentially expressed salt induced cDNAs from Dunaliella salina under super saturated salt stress using subtractive hybridization. NCBI EST database: GT032423- GT032494. 3. Mishra Avinash, Mandoli A and Jha B. 2008. Dunaliella salina 18S ribosomal RNA gene. NCBI Accession No.: EF195157.

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Abstract: This invention relates to a Gamma kafirin gene promoter of sorghum bicolor Var. M35-1 comprising minimum and essential cis acting elements containing three essential motif, viz one prolamin box, two GLM (GCN-4 like motif) and a method for expressing it for the temporal and spatial expression of genes comprising operably linking the nucleotide sequence to a promoter comprising SEQ IDNO1 and operably linking the nucleotide sequence encoding signal peptide with a promoter comprising SEQ IDN02, wherein different cis acting elements were amplified, cloned using different combinations of the primer as described herein and the amplified promoter is fused with (3-glucuronidase encoding gene from PCAMB1A-1304 vector at the place of CaMV35S promoter to produce an expression cassette and generating a transgenic plant comprising the expression cassette, the transformation method being a biolistic or Agrobacterium mediated one.

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