Arun Dev Sharma

DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.

Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses

The effect of various abiotic stresses on germination rate, growth and soluble sugar content in Sorghum bicolor (L.) Moench cv. CSH 6 seed embryos and endosperm during early germination was investigated. Under stress conditions germination, water potential and tissue water content decreased markedly. Subsequently, this reduction resulted in marked decreases in fresh weight both in embryos and endosperm. Conversely, a substantial increase in dry weight was observed. Furthermore, a considerable increase in the sugar contents in both embryo and endosperm was detected. The fructose level was always higher than glucose and sucrose in response to various stresses. However, as compared to the control the level of glucose and sucrose was higher in embryos and endosperm after stress treatments. Based upon these results a possible physiological role of sugars in the germination of sorghum seeds is discussed.

Effect of media supplements and culture conditions on inulinase production by an actinomycete strain

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.

RNA isolation from plant tissues rich in polysaccharides

Extraction of high-quality RNA is necessary formakingcDNAlibraries,isolatinggenesbyRT-PCR,orinvestigatinggeneexpressionprofiles.SeveralmethodsarecommonlyusedforisolationoftotalRNA[1–3]andare being developed because plants contain highamountsofmanydifferentsubstances;therefore,justonenucleicacidisolationmethodsuitableforallplantscanneverexist[4].Evenplantspeciesbelongingtothesamegenusorrelatedgeneracanexhibitenormousvariabilityinthecomplexityofpathwaysofdispensablefunctions.Thus,thebiochemicalcompositionsinplanttissuesofdifferentspeciesareexpectedtovaryconsid-erably.ThechemotypicheterogeneityamongspeciesmaynotallowoptimalRNAyieldfromoneisolationprotocoland,perhaps,evencloselyrelatedspeciesmayrequiredifferentisolationprotocols[5].Asouraimhasbeentostudydrought-induceddifferentialexpressionofgenesduringearlyandlateseeddevelopmentalstagesinsorghum,wemayneedaprotocolthatnotonlycangivethesamequalityandquantityofRNAateachstagebutalsoishighyieldinginordernottomisstherarelyex-pressedgenes.PreviousstandardplantRNAisolationproceduresfailedtoworkwhenappliedtotissuesrichinsecondaryproducts[3,6–8].Inthisreport,aproceduredevelopedbyLogemannetal.[3]wasmodifiedandappliedtosorghumseeds,whichareknowntocontainhighlevelsofpolysaccharides[9].Theextractionmethoddescribedinthisstudyissimple,notrequiringultra-centrifugationoradditionalprecipitationsteps,andal-lowsRNAextractionfromplantspeciesinwhichotherprocedureshavepreviouslybeenunsuccessful.Further,thequalityoftheisolatedRNAwasconsistentlyhighasindicatedbyspectrophotometricreadingsanditssepa-rationondenaturingagarosegels.TheyieldandqualityweresuitableforRT-PCRandNorthernblothybrid-ization.Seeds of sorghum (Sorghum bicolor cv. CSH-6)werepurchasedfromtheNationalSeedCorp.,(Pusa,NewDelhi,India)andseedsofchickpea(Cicerarieti-num),andsoybean(Glycinemax)werepurchasedfromPunjabAgriculturalUniversity(Ludhiana,India).Theseedsweresurfacesterilizedwith1%(w/v)mercuricchloride and 70% ethanol followed by rinsing withdeionizedwater.Seedswereimbibedfor6hindouble-distilledwaterat37 Candusedforfurtherstudies.Leavesofsoybeanandchickpeawerecollectedfrom6-day-oldgerminatedseedlingsgrowninaseedgermin-ater.Tissueswerestoredinliquidnitrogenforfurtheranalysis.Solutionsandreagentsusedwereasfollows:ho-mogenization solution (Solution I), NaCl (5M),Sarkosyl(2%);guanidinehydrochloridebuffer(pH7.0),8Mguanidinehydrochloride,20mMEDTA,20mMMes,

Effect of Water Stress on Expression of a 20 kD Cyclophilin-like Protein in Drought Susceptible and Tolerant Cultivars of Sorghum

Immunodetection studies revealed the presence of a 20 kD cyclophilin-like protein (designated as SorgCyp20) in leaves and seeds of sorghum (Sorghum bicolor L Moench). The expression of SorgCyp20 was temporally regulated in the leaves and after attaining maximum levels at either 60 or 70 days after sowing it declined after flowering. The effect of drought stress on SorgCyp20 levels in the leaves and seeds of sorghum was stage and cultivar dependent. The drought stress-induced enhancement in SorgCyp20 levels was many times higher in the leaves (3-fold increase at 30 days after sowing) and seeds (2.5-fold increase at 9 days post anthesis) of drought tolerant cv ICSV-272 than in the drought susceptible cv SPRU-94008B. The intercultivar differences in drought stress-induced changes in SorgCyp20 expression were not related to the difference in water potential thus suggesting differential regulation of SorgCyp20 in response to stress in the two sorghum cultivars.

VISUAL HAND GESTURES CLASSIFICATION USING WAVELET TRANSFORMS

This paper presents a novel technique for classifying human hand gestures based on stationary wavelet transform (SWT) and compares the results with classification based on Hu moments. The technique uses view-based approach for representation of hand actions, and artificial neural networks (ANN) for classification. This approach uses a cumulative image-difference technique where the time between the sequences of images is implicitly captured in the representation of action. This results in the construction of Motion History Images (MHI). These MHIs are decomposed into four sub-images using SWT. The average image (fll) is fed as the global image descriptors to the ANN for classification. The recognition criterion is established using backpropagation based multilayer perceptron (MLP). The preliminary experiments show that such a system can classify human hand gestures with a classification accuracy of 97%. The work is motivated by the previous research in appearance-based motion recognition of human hand actions. The overall goal of our research is to determine the reliability of using this wavelet based computationally inexpensive gesture classification technique that may be used for helping disabled or aged people interact with computers.

WAVELET DIRECTIONAL HISTOGRAMS OF THE SPATIO-TEMPORAL TEMPLATES OF HUMAN GESTURES

This paper evaluates the efficacy of directional information of wavelet multi-resolution decomposition to enhance histogram-based classification of human gestures. The gestures are represented by spatio-temporal templates. This template collapses spatial and temporal components of motion into a static gray scale image such that no explicit sequence matching or temporal analysis is required, and it reduces the dimensionality to represent motion. These templates are modified to be invariant to translation and scale. Two-dimensional, 3-level dyadic wavelet transforms have been applied on the template resulting in one lowpass sub-image and nine highpass directional sub-images. Histograms of wavelet coefficients at different scales are used for classification purposes. The experiments demonstrate that while the statistical properties of the template provide high level of classification accuracy, the global detail activity available in highpass decompositions significantly improve the classification accuracy.