Department of Biosciences and Bioengineering

Browse

Browsing Department of Biosciences and Bioengineering by Issue Date

Filter results by year or month
Now showing 1 - 20 of 263
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    Exploring the structure and dynamics fo proteins in non-native states using fluorescence spectroscopy
    (2008) Kumar, Satish
    In the present thesis, structure and dynamics of non-native states in proteins are discussed under different experimental conditions. Owing to their conformation heterogeneity their involvement in protein folding pathway including their role in different protein misfolding diseases, non-native forms of protein are important both for basic and applied areas of biological science. Local structures in protein under extreme denaturing conditions are known as residual structures and are believed to act as nucleation site for protein folding events. Locating these structures are important to reveal protein folding/unfolding events. I used fluorescence from trp(s) as a simple and sensitive tool to hunt for these structures in different proteins, namely, barstar, subtilisin carlsberg (SC), human serum albumin (HSA), melittin, myelin basic protein (MBP), glucagon, Ribonuclease T1 (RNase T1), Trp-Met-Asp-Phe, bovine serum albumin (BSA) and hen egg white lysozyme (HEWL) after nightlong (~12 hours) incubation in 6 M GdnCl at room temperature. Except BSA and HEWL all other proteins used here contain single trp per polypeptide chain..
  • No Thumbnail Available
    Item
    Prodrug Gene Therrapy Vectors in combination Therapies
    (2008) Gopinath, P.
    Prodrig gene therapy , commonly known as suicide gene therapy, provides a selective approach to eradicate tumer cells. Development of efficient suicide gene therapy vectors to improve the therapeutic efficacy is the main theme of this thesis. ...
  • No Thumbnail Available
    Item
    Genetic Engineering of COWPEA For Storage Pest Resistance
    (2008) Solleti, Siva Kumar
    Cowpea (Vigna unguiculata L. Walp) is an important grain legume widely consumed by 120 million people. Cowpea seeds and fresh peas are a rich source of protein, certain minerals and vitamins. However, cowpea seeds are highly susceptible to storage pests, bruchid species causing massive damage to the stored seeds and seriously limiting its yield potential. Success through conventional breeding methods for developing resistant varieties is limited due to the natural genetic barriers in cowpea germplasm. Consequently, the transfer of insect pest resistance genes by genetic engineering could potentially aid plant breeders in overcoming these constraints. A critical step in the development of robust Agrobacterium tumefaciens-mediated transformation system in recalcitrant grain legume is the establishment of optimal conditions for efficient T-DNA delivery into target tissue and recovery of transgenic plants. We report a dramatic increase in efficiency of T-DNA delivery by constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404. A geneticin based selection system permitted rapid and efficient identification of transgenic shoots without interfering with their regeneration, and eliminated the bulk of escapes. Supplementation of 0.5 µM kinetin to medium containing 5.0 µM benzyl aminopurine after 1week of culture followed by 3 weeks of culture were found critical for optimal multiplication and elongation of transformed shoots from cotyledonary node explants. Combining these three developments, we recovered fertile transgenic plants at a frequency of 1.64%, significantly higher than previous reports. The presence, integration, expression and inheritance of transgenes were confirmed by molecular analysis. We employed the developed Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants at a frequency of 1.67%. The presence, integration, expression and inheritance of αAI-1gene was confirmed by molecular analysis. Expression of αAI-1 gene under bean phytohemagglutinin promoter resulted in accumulation of αAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine α-amylase in vitro. Transgenic cowpeas expressing αAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    Computationalinvestigation of HIV-1 protease dynamics by comparing the effects of mutation, force fields and pressure
    (2009) Meher, Biswa Ranjan
    AIDS, one of the major epidemics of the human society has been the constant worry. UNAIDS projection shows the existence of millions of AIDS patients at the end of 2008 with high mortality rate and fresh infections in coming days. All the FDA approved drugs are getting resistant against the clever HIV because of its frequent mutations. Hence there is an urgent need of developing new drugs with greater potential. AIDS is mainly due to the infection of retrovirus HIV. It infects the CD4+ cells of immune system thereby reducing its number to reach an immune deficiency condition. The viral life cycle is controlled by the activities of its essential proteins like gp41, gp120, HIV-RT, HIV-IN, and HIV-pr. Each protein has substantial role on the viral life cycle to be continued. The present thesis focuses on the protein HIV-pr, which is important for the cleavage of Gag and Gag-Pol polyproteins to form the smaller structural and functional proteins. Structurally, the homodimeric aspartyl protease has 198 residues in both chains with the catalytic aspartate in the dimer interface. Glycine rich flexible flaps cap the active site of HIV-pr, which controls the accession of ligands and size of the active site. The conformation of the protein plays a pivotal role in ligand binding and the catalytic process, which is affected by the rapid point mutations and various physiological parameters. In the present thesis, the conformational dynamics of HIV-pr is being studied in an atomistic detail by MD simulations. Both biological and technical aspects of the protein conformation and dynamics have been explored. With regards to the biological aspect, initially the effect of I47V mutation on the dynamics of HIV-pr and its effect on ligand (JE-2147) binding were investigated and was observed to have both direct and indirect effects on drug resistance. Also it gave the clues for increase ddynamics under high pressure compared to normal condition. It was observed that protein under high pressure validate the general decrease in the proteinDs structural degrees of freedom and pressure plays a crucial role in reducing the structural variability in proteins. Coming to the technical aspects, we studied the effects of polarization and the difference in force fields on the protein conformation. It was found that, polarizations of force fields influence both the global and specific local motions of protein and solvent thereby increasing the rigidity in proteins. Also the water movements around different types of residues are marked to be different and are high for charged residues. Comparative study of the HIV-pr dynamics by multiple force fields shed light on the difficulty in modeling dynamics of proteins with flexible binding site and in silico drug design against flexible receptors. The complex dynamics of HIV-pr can be sensitive enough to the force field difference. Hence a careful examination with different simulation parameters is required to conclude regarding the biological functions drawn from MD simulation studies. Altogether, these studies indicate that conformational dynamics of HIV-pr is sensitive enough to the simulation setups and force fields difference. Also mutation in some specific region has both direct and indirect effects on the conformation and dynamics of HIV-pr. The outcome of the thesis has noteworthy applicat...
  • No Thumbnail Available
    Item
    Characteristics and application potential of an alchhol oxidase from the hydrocarbondegrading fungus aspergillus terrrus
    (2009) Kumar, Adepu Kiran
    A hydrocarbon-degrading Aspergillus terreus was isolated from the oil contaminated soil. The organism could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39 D 4 g lD1 (wet weight) was observed when n-hexadecane was used as the sole source of carbon and energy. Using SEM the morphological change of the hyphae during growth of the fungi on hydrocarbon substrates was demonstrated. In glucosegrown cells the hyphae were smooth with a thick cell wall, whereas, the cell wall of the n-hexadecane cells was uneven and thin. The adaptation of the cells for up taking the hydrophobic hydrocarbon substrates through sorption mechanism and accumulation of high lipid content in the cells, as evident from the nearly seven-fold more lipid production (4.4 g%) in the n-hexadecane-grown cells than the glucosegrown cells (0.62 g%), were attributed to the observed morphological change. Analyses of the fatty acid profile by ESI/MS in the isolated lipid showed that nine different fatty acids induced in the hexadecane-grown cells were void in the glucosegrown cells. The oleic acid and palmitic acid were the fatty acids with highest peak intensity in the spectral profile corresponding to the glucose-grown cells and hexadecane-grown cells, respectively. It was revealed by analyzing the percentage distribution of different fatty acids in each cell type that palmitic and stearic acids were the predominant fatty acids in the hexadecane-grown cells; whereas, oleic and linoleic acids were the predominant fatty acids observed in the glucose-grown cells. Unlike glucose-grown cells, considerably high amount of fatty acids with chain length C32 and C33 were present in n-hexadecane-grown cells and each accounted nearly 9% (based on peak intensity) of the total fatty acid content in the cells. A microsomal membrane bound alcohol oxidase enzyme was isolated from this fungus during its growth on n-hexadecane substrate. This oxidase could oxidize short chain alcohol-, iii long chain alcohol-, secondary alcohol-, and aryl alcohol- substrates and was localized in the microsomes of the cell. The optimal pH and temperature of the enzyme were found to be 8.1 D 0.5 and 27-33 oC, respectively. The stability of the alcohol oxidase was drastically decreased beyond 30 oC. The enzyme showed 33% enantiomeric excess for the R(-)2-octanol over S(+)2-octanol, which may be correlated with the lower Km values of the enzyme for the R(-)2-octanol than the S(+)2-octanol. The fluorescence emission spectrum of the protein (at 443 nm excitation) was similar to that obtained with authentic FAD. In this flavoenzyme, flavin was non-covalently but avidly associated. The native protein molecular mass was 269 D 5kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13- kDa. The enzyme showed highest affinity for n-heptanol (Km = 0.498 mM, Kcat = 2.7 x 102 s-1). The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. The high aggregating property of the alcohol oxidase may be correlated with the observed lipoidic nat...
  • No Thumbnail Available
    Item
    Molecular detection and Characterization of antagonistic and in vitor adhesion property of laction acid bacteria
    (2009) Singh, Atul Kumar
    The objective of the research work carried out in this part of thesis was to develop a convenient and reliable method of generating template DNA for routine PCR-based detection and molecular fingerprinting of lactic acid bacteria (LAB). Template DNA extracted from Lactobacillus, Lactococcus, Pediococcus and Leuconostoc using a combination of urea, SDS and NaOH yielded amplicons of expected size in PCR with genus-specific primers. Apart from LAB, the proposed method could also be adopted to generate PCR-compatible template DNA from a number of Gram-positive and Gram-negative bacterial strains. DNA template prepared by the proposed method from various standard strains of Lactobacillus sp. also generated discriminating fingerprints with BOXA1R primer in rep-PCR. A significant finding of the investigation was that a comparable banding profile of LAB strains was obtained in rep-PCR using template DNA prepared by urea-SDS-NaOH method and a commercially available DNA isolation kit. This was further evidenced by high Dice coefficient values obtained in the range of 81.8-96.7 when cluster analysis was performed by UPGAMA method. The application potential of this DNA extraction method for PCR-based direct detection of LAB in fermented food samples such as dahi, idli batter and salt-fermented cucumber was validated by detecting specific amplicons of LAB genera in the fermented samples. The applicability of the proposed template DNA extraction method was further substantiated when 29 bacteriocinogenic LAB strains (Bac+) previously detected in salt-fermented cucumber by PCR generated differentiating fingerprints in BOX element based rep-PCR and formed clusters with reference LAB strains...
  • No Thumbnail Available
    Item
    Studies on Biodegradation of Pyrene by Mycobacterium Frederiksbergense
    (2009) Mahanty, Biswanath
    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants produced via natural and anthropogenic sources, mainly generated during the incomplete combustion of solid and liquid fuels or derived from industrial activities. Due to high hydrophobicity and recalcitrant nature, they tend to contaminate soil and water and pose serious threat to receiving environment because of their mutagenicity and carcinogenicity. Of the various treatment methods for PAHs, biodegradation have been shown to be more successful, eco-friendly and cost effective one. In this study, Mycobacterium frederiksbergense, preliminarily known to degrade pyrene, was investigated of its potential to degrade this model PAH compound, in single and mixed substrate condition, employing three different biodegradation systems. In the first slurry phase degradation system containing pyrene as the sole substrate, the Mycobacterium showed a lag of 48 h in pyrene degradation in batch shake flask for an initial concentration of 50 mg l-1. Immediately following this lag period, pyrene was actively degraded with an observed rate of 19.86 mg l-1 d-1. However, in fermenter, pyrene was degraded without any lag phase with an over all rate of 6 mg l-1 d-1. To study biodegradation of pyrene in a ternary mixture along with naphthalene and anthracene in this slurry phase system, a 23 full factorial design of experiments was employed. Statistical analyses of the PAHs degradation rates by the culture performed in the form of analysis of variance (ANOVA) and studentDs t-test gave interpretation of main and interaction effects of the substrates.In surfactant aided system for biodegradation of pyrene, initially five synthetic chemical surfactants viz. TritonX-100, Tween 80, Tween 20, sodium dodecyl sulphate (SDS) and cetyl trimethyl ammonium bromide (CTAB) were evaluated of their molar solubilisation ratio for pyrene, which inferred superiority of Tween 80 over others. Also, a biosurfactant produced by an indigenous microbial culture isolated from the soil of a gasoline filling station, was comparatively evaluated of its efficiency to solubilise pyrene. The partially purified biosurfactant also showed emulsification activity, stability at different temperature, pH towards a range of solvents. However, the culture failed to biodegrade pyrene. Tween 80 aided pyrene biodegradation in batch shake flask indicated a prolonged lag phase, which was however absent in fermenter experiments with a maximum degradation rate at 25 mg l-1 initial pyrene concentration. Results of Tween 80 aided mixed PAHs biodegradation studies with pyrene, naphthalene and anthracene were also analysed in terms of their main and interaction effects on their biodegradation. To further evaluate the potential of the mycobacterium in biodegradation of pyrene, two liquid phase partitioning bioreactor (TPPB) system was developed. After an initial screening procedure, silicone oil was chosen as the non-aqueous phase liquid in the TPPB system. Further, results of hydrodynamic study in this TPPB system indicated the best operating conditions to be 1.5 vvm of aeration rate, 600 rpm agitation rate, with silicone oil fraction of 0.2. At this optimized sets of operating conditions in the TPPB system, biodegradation study carried out at different initial pyrene concentration in silicone oil (200-1000 mg l-1) gave very high pyrene degradat.
  • No Thumbnail Available
    Item
    Development of Polymeric nanocarriers for Drug Delivery to Cancer Cells
    (2009) Sahu, Abhishek
    Designing of nanosystems that are able to deliver therapeutic molecules to the right place, at a desired dose, at the appropriate time in a controlled manner is currently an important area of research. Pharmaceutical nanocarriers have evolved as one of the major means for drug delivery in cancer chemotherapy due to their unique advantages over conventional dosage. The present research work deals with the development of polymeric nanocarriers for delivery of curcumin, a potential anticancer molecule. Curcumin has showed great promises in the last decade for cancer therapy but the clinical progress of this compound has been slow due to its poor water solubility and low bioavailability. Nanocarrier based delivery of curcumin could provide an opportunity to expand the clinical repertoire of this efficacious agent by enabling aqueous dispersion. Based on these considerations, the objectives of the present research work were fixed as follows: 1. Development of a synthetic nanocarrier and its application in curcumin delivery. i. Synthesis and characterization of synthetic mPEG-PA nanocarrier. ii. Evaluation of the nanocarrier for drug encapsulation. iii. Delivery of the nanocarrier encapsulated drug to in vitro cultured cancer cells. 2. Formulation of curcumin in Pluronic nanocarriers. i. Encapsulation of curcumin in Pluronic nanocarriers by self assembly. ii. Study of drug release and stability of the Pluronic based formulations. iii. Delivery of Pluronic encapsulated curcumin to in vitro cultured cancer cells. 3. Natural food protein based nanoparticle for curcumin delivery. i. Purification and characterization of casein micellar nanoparticles from milk. ii. Fluorescence study of curcumin binding to casein micelle nanoparticles. iii. Effect of curcumin complexed casein micelles on in vitro cultured cancer cells. Chapter One starts with a brief historical development of the concept of drug delivery and moves onto describe the fundamental understanding of drug delivery systems with an emphasis on nanosystems. A review of different types of nanocarriers used in drug delivery and their advantages has been discussed with a focus on cancer therapy. Detailed discussions on the use of inorganic, lipid and polymer based nanocarrier systems are presented along with information about the various commercialized drug delivery products based on nanosystems. The chapter also discusses about the chemistry and multi-therapeutic potential of curcumin. The clinical progress of curcumin, recent advances in the area of its delivery and its promising future as a chemotherapeutic drug against cancer are also reviewed. Chapter Two describes the synthesis of a novel synthetic nanocarrier for curcumin delivery. An amphiphilic polymer, mPEG-PA was synthesized by conjugating mPEG with palmitate by an ester linkage in a single step reaction. The conjugate forms micelle based nanocarriers by self-assembly in aqueous solution. mPEG-PA was characterized through 1HNMR and FT-IR spectroscopy. The size and morphology of the micelle nanostructures were characterized by DLS and AFM. The mPEG-PA based micelle nanocarriers were able to encapsulate curcumin in its hydrophobic core and also efficiently delivers it to the cancer cells. Chapter Three deals with use of Pluronics for curcumin delivery. Pluronics are commercially available FDA approved tri-block copolymers for pharmaceutical appl...
  • No Thumbnail Available
    Item
    Some Investigation on protiein Structure, Function and Dynamics in Disordered states and Non-ideal conditions
    (2010) Kumar, M. Venkata Satish
    The proteins we observe in nature have evolved through selective pressure to do certain specific functions such as catalyzing and regulating biochemical reactions, transporting molecules that permit cells to grow and reproduce. The three dimensional structure of protein is fundamentally related and tied to its biological function. So understanding the structure of protein is important to obtain insights on its function. The details of ordered secondary structures like helices and sheets in proteins are extensively discussed in the literature, but there are few reports on the remaining secondary structures comprising of turns, bends, bulges and other irregular structures that are commonly grouped and referred to as loops. Loops are essentially regions of non-repetitive conformation connecting regular secondary structures. These regions are important parts of protein structures as they have been shown in some cases to be the nucleation sites for protein folding, the sites of catalytic activity in enzymes or interfacial regions in the interaction between other proteins, ligands and nucleic acids. Thus loops play an important role in protein function. The structural-genomics initiative is expected to boost the population of high resolution protein 3D structures. Consequently, it has been argued that manual analysis of protein structure must be replaced by automated approaches of protein structure analysis. Loops play a major role in determining protein fold, hence methods that can facilitate automated analysis of loops in proteins are desirable. We have devised a method to identify and investigate functionally active loops and unstructured regions in protein structures using the MSRP parameter. This method (a) provides a unique classification tool for loops and folds among proteins, (b) permits automated identification of functional loops in protein structures, (c) provides clues on the diversity of conformations sampled by a disordered region during a molecular dynamics simulation. Traditionally acknowledged concept of protein function was the structurefunction paradigm, represented as Amino acid sequence-> 3D structure->Function. The 3D structure of protein in the folded state is related to its function and thus the native protein structure is the ordered 3D structure. However, recently it has emerged that the actual functional state for many proteins and protein domains are intrinsically unstructured. These unstructured proteins are also called intrinsically disordered proteins or natively unfolded proteins. They comprise a large fraction of eukaryotic...
  • No Thumbnail Available
    Item
    Studies on heme-Based redox enzymes from Aspergillus terreus MTCC 6324 and their potential applications for bioelectroni
    (2010) Vatsyayan, Preety
    The cytochrome P450 monoxgenase(CYO) activity in the cells of Aspergillus terreus MTCC 6324 was localized in the cytosol of n-hexadecane grown cells, white, it was apparently distributed in light mitochondrial and microsomal fractions of glucose grown cells. The Substrate ......
  • No Thumbnail Available
    Item
    Production purification and characterization of glutaminase-free L-asparaginase from pectobacterium carotovorum MTCC 1428
    (2010) Kumar, Sanjay
    Bacterial L-asparaginase has been widely used as a therapeutic agent in the treatment of certain kinds of cancer, mainly in treatment of acute lymphoblastic leukemia. Moreover, L-asparaginase is used in food industry for the production of acrylamide free food, as model enzyme for development of new drug delivery system and L-asparagine biosensor for diagnosis of leukemia. The various side effects of this drug are mainly due to the presence of partial glutaminase activity of L-asparaginase. Hence, glutaminase-free L-asparaginase is highly desirable for its successful application. Among the tested microorganism, Pectobacterium carotovorum MTCC 1428 has the ability to produce novel glutaminase-free L-asparaginase. Hence, further studies were carried out using P. carotovorum MTCC 1428. Localization, production, purification and characterization of glutaminase-free L-asparaginase have been performed by P. carotovorum MTCC 1428. Production of glutaminase-free L-asparaginase was performed both in the shake flask and bioreactor (batch and fed-batch). Furthermore, Kinetic models were developed for dual substrate growth, substrates utilization and L-asparaginase production by P. carotovorum MTCC 1428 in batch bioreactor. The localization of L-asparaginase was carried out using cell fractionation techniques. The specific activity of L-asparaginase was found to be 85 and 77% in the cytoplasm of Pectobacterium carotovorum MTCC 1428 that was grown on medium containing L-asparagine, and a combination of both L-asparagine and glucose, respectively. Among the carbon sources tested, L-asparagine and a combination of both L-asparagine and glucose were found to be the most suitable carbon sources for maximum production of L-asparaginase. Yeast extract, L-asparagine and peptone showed significant effect on the production of L-asparaginase. L-asparagine was used as an essential carbon source for the maximum production of L-asparaginase by P. carotovorum MTCC 1428.........
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    Production, Structure analysis and biotechnological applications of dextran from a novel strain of pediococcus isolated from microbial diversity hot Assam
    (2010) Patel, Seema
    Dextrans are a class of homopolysaccharides composed of D-(1D6) glycosidic linkages in the main chains and D-(1D2), D-(1D3) and D-(1D4) branched glycosidic linkages. Dextrans may be linear or branched, with variable degree of branching. Dextran are used as food syrup stabilizers, matrix of chromatography columns, plasma substitute, anti-thrombogenic agent, biomaterials, paper and metal-plating processes, oil recovery. Porous dextran scaffolds have tissue engineering applications as drug delivery vehicles. Dextran derivatives as oligosaccharides have profound applications as neutraceuticals, prebiotics, immune stimulatory and anti cancer agents. Functionality of dextran depends on the strain of lactic acid bacteria synthesizing it. Dextran producing lactic acid bacteria have recently attracted much attention due to immense industrial potential of dextrans. The commonly known dextran producing lactic acid bacteria are Lactobacillus, Leuconostoc and Streptococcus. Biodiversity hotspots are promising niches for isolating commercially important strains of dextran producing lactic acid bacteria. North-East region, falling under the Indo-Burma biodiversity hotspot is expected to harbour such novel strains. With this objective, a high dextransucrase yielding strain of lactic acid bacterium was isolated from the soil collected from a place near Guwahati. 16s rRNA sequencing identified the isolate to be Pediococcus pentosaceus, belonging to lactic acid bacteria family. Certain strains of Pediococcus genus are industrially important as potent food preservatives for their pediocin production ability. But, the dextran production aspect of this genus has never been explored or reported before. This work, reports for the first time, the dextran...
  • No Thumbnail Available
    Item
    Characterization of candidate plus trees(CPTS) of pongamia pinnata(L), a versatile legume from North Guwahati, Assam
    (2010) Kesari, Vigya
    Plant source for fuel that replaces fossil fuels is a topical subject and has gained prominence as DDBiofuel cropsDD. Pongamia pinnata, commercially important tree species used to produce biofuels, is known for its multipurpose benefits and its role in agro-forestry. Pongamia development as a multi-potent legume crop in IIT Guwahati in collaboration with Sila Forest Range, North Guwahati, Assam was attempted in the present study. The overall objectives of the thesis were divided into 6 distinct chapters that includes systematic candidate plus tree (CPT) identification, growing nursery, mass multiplication of identified CPTs using vegetative propagation and tissue culture approach, diversity evaluation among elite genotypes using SDS-PAGE, RAPD, ISSR and AFLP, collecting seeds from identified CPTs, extracting oil, examining medicinal and biodiesel aspects, morpho-biomolecular characterization of seeds by exploitation of advanced biotechnological tools and exploring symbiosis between Rhizobium and Pongamia by identification of novel Rhizobium pongamiae from root nodules CPT. In first chapter, 10 CPTs belonging to populations 6 and 10 were identified based on morphological markers (vegetative and reproductive) using combined analysis over locations by CROPSTAT software. Identified CPTs were then multiplied using seed propagation technique in nursery bed. The best genotype identified was NGPP 46 with respect to pod-seed traits and total oil content. Hexane extraction yielded maximum oil content from seeds (33 %) compared to petroleum ether (30 %). When varying the seed to solvent ratio, no significant difference was noticed on the total oil yield for an individual tree, although the recovery of solvent and time taken for oil extraction reduced to significant level at higher ratio. Identified CPTs can be further included in programmes aimed at genetic improvement of the species. Second chapter has been divided into two sub-objectives. First one includes examining the amenability of vegetative propagation and effect of maturation in CPT, NGPP 46 through rooting of stem cuttings treated with varying concentrations and combinations of auxins. All auxin treatments promoted sprouting and at lower concentrations triggered/enhanced rooting of cuttings. The effectiveness was in the order of IBA>NAA>IAA when applied singly. IBA at 4.92 mM was found to be most effective as the rooting percentage and number of roots were significantly higher (p0.01) than in control. However higher concentrations of auxins above 7 mM in general inhibited the rooting of cuttings. The interaction among auxins was found to be effective in root induction and differentiation and the most stimulating effects were observed in three-component mixture (IAA 1.42 mM + IBA 4.92 mM + NAA 1.34 mM). For further study mature stem cuttings of ten CPTs were subjected to the best responding auxins application to compare their adventitious rooting ability. Significant differences were observed for sprouting (26 - 86.67%) and rooting (56.67 - 93.33%) behaviour. Rooting after nine weeks was more than 96% from NGPP 26, 27 and 46, in IAA 1.42 mM + IBA 4.92 mM + NAA 1.34 mM and was approximately 4 times that of control. Auxin treatment increased number of roots (2.32) and root length (22.23 cm) but has negligible effect in the number of shoots per rooted cuttings. Cuttings harv...
  • No Thumbnail Available
    Item
    STUDIES ON SOPHOROLIPIDS PRODUCTION AND PRETREATMENT OF HIGH FATS AND OILS CONTAINING DAIRY WASTEWATER USING CANDIDA BOMBICOLA
    (2010) Daverey, Achlesh
    Dairy industry is one of the major food industries in most of the countries of the world including India and this industry has grown rapidly due to the high demand of milk and milk products. Dairy industry generate large amount of wastewater which is very rich in biodegradable organics and nutrients. It also contains high level of fats and oils which are not easily biodegradable and often interfere with the normal biological treatment process. Therefore, pretreatment of dairy wastewater is essential to remove these fats and oils from the wastewater before subjecting it to final biological treatment. In comparison to the existing physical, chemical and biological methods for pretreaing the dairy wastewater biological pretreatment methods using microorganisms and/or their products are found to be the most effective and successful. Biosurfactant facilitated biodegradation of fats and oils, primarily by increasing its solubility has been identified to be a potential method to deal with high fats and oils in wastewaters. The present work focused on pretreatment of high fats and oils containing dairy wastewater by the sophorolipids (SLs, a glycolipids type of biosurfactant) producing yeast Candida bombicola. To reduce the SLs production cost two agro-industrial wastes namely cheese whey and sugarcane molasses were tested as a low cost hydrophilic carbon source in place of costly glucose for its production by the yeast in batch shake flasks as well as in a laboratory scale bioreactor.
  • No Thumbnail Available
    Item
    Plant tissue culture and production of triterpenoids from medicinal plants azadirachta indicaa . Juss. and lantana cama L.
    (2010) Srivastava, Priyanka
    Medicinal plants are indispensible to human health. From times immemorial, the therapeutic benefits offered by these plants have been exploited by the common people and medical professionals alike. This is the reason why most of them have made their ways to scientific laboratories throughout the world as their large scale production and enhancements of their bioactive constituents have become a matter of fascination and interest to the scientists worldwide. Azadirachta indica A. Juss. (commonly known as Neem), an evergreen tropical tree of family Meliaceae, is one of the most fascinating trees of Asian subcontinent. It has many important medicinal, agrochemical and economic uses to its credit. Today, due to its remarkable biopesticidal properties shown by azadirachtin and other related triterpenoids, the tree has attained global importance. Though this property is not unique to neem, it holds a distinction as it provides a highly effective antifeedant, nontoxic and environment-friendly means of controlling or eliminating insect pests. Due to commercial potential and renewed worldwide research interest, the tree has now been introduced to other regions of the globe including Africa, South America and Australia. In spite of having valued properties, improvement of Neem by conventional methods is very limited owing to its highly heterozygous nature, long reproductive cycle and recalcitrant and poor seed yield. In this context, it is noteworthy that, studies utilizing gametophytic cells are in infancy, in this tree species. In vitro haploid production from gametophytic cells enables the establishment of completely homozygous lines in a shortened time frame compared to conventional methods and has many potential applications in plant improvement to establish inbred lines rapidly and to observe recessive traits. Therefore, the aim of the present study was to establish in vitro androgenic lines of Neem from anthers and check the contribution of these haploid cultures in the production of medicinally important azadirachtin, a tetranortriterpenoid, chiefly present in seeds. The presence of compound has been confirmed by chromatographic and spectroscopic techniques. Bioassays carried out further strengthen the aim of this investigation. Lantana camara L., commonly known as Red or Wild Sage, is an evergreen, strong-smelling woody shrub belonging to the family Verbenaceae. About 150 species are known to represent this genus. The plant is a native of tropical America but is now naturalized in many parts of India. All parts of the plant have been used traditionally for several ailments throughout the world. Several triterpenoids, napthaquinones, flavonoids, alkaloids and glycosides isolated from this plant are known to exert diverse biological activities including cytotoxic and anticancer properties. A number of potential uses of Lantana plant have been suggested but none has been exploited on the large scale. There is a wide scope of research on this plant. Owing to the prevalent heterozygosity in this genus and seasonal variation in secondary metabolite content, tissue culture offers a solution through which cell biomass can be raised and important compounds can be harvested from its biomass all the year round unaffected by the seasonal variation. The purpose of large scale production of pharmaceutically important metabolites c...
  • No Thumbnail Available
    Item
    Development of Transgenic Cowpea Overexpressing Btcry 1 Ac and Btcry 1 Ab
    (2011) Bakshi, Souvika
    Cowpea (Vigna unguiculata L. Walp) is an important grain and fodder legume widely cultivated in Africa, India, Middle East and South America. Insect pest infestation, prevalently perennial damage of cowpea pods by pod borers, Maruca vitrata and Heliothis armigera is most severe. Breeders lack resistance background for incorporation to cultivated varieties to control the diseases. It has been demonstrated that the introduction and expresssion of crystalline toxin genes (cry) derived from Bacillus thuringiensis (Bt) through transgenic approaches are effective mechanisms for protecting crops against insect infestations. In cowpea, absence of an efficient system for shoot multiplication and plant regeneration amenable to Agrobacterium-mediated transformation, mechanisms to increase T-DNA transfer to regenerating cells and strategy for efficient selection of transformed shoots have been identified as major bottlenecks for implementing transgenic approaches for genetic improvement. This investigation was carried out to find out the role of seedling preconditioning with cytokinin on shoot proliferation efficiency of cotyledonary node explants. A significantly higher shoot proliferation (7.1 shoots per explants), and mean shoot length (2.6 cm) were obtained with cotyledonary node explants derived from seedlings preconditioned in 10 DM TDZ for 4 days. The preconditioned explants were employed for efficient regeneration of transgenic cowpea plants. Btcry1Ac and cry1Ab overexpression constructs were prepared and mobilized to Agrobacterium tumefaciens vir helper strain EHA105. Method for efficient recovery of transgenic cowpea plants using an improved kanamycin selection regime was established that showed 46.1% increase in efficiency as compared to the existing transformation methods. Agrobacterium-cocultivated cotyledonary node explants were selected on medium containing 150 mg/l kanamycin for 20 days and surviving explants were shifted to kanamycin-free media supplemented with reduced dosage of BAP (2.5 DM) that resulted in profuse proliferation of kanamycinresistant shoots with 63.6% increase in shoot length within 15 days of culture. Transgenic cowpea plants with introduced cry1Ac were recovered that showed presence, integration, expression and inheritance of transgene. The role of sonication and vacuum infiltration on increase in T-DNA transfer efficiency to cotyledonary node explants were investigated. A combination of 20 s sonication followed by 5 min vacuum infiltration treatments were found to significantly...
  • No Thumbnail Available
    Item
    Establishment of in vitro cultures of Azadirachta Indica A. Juss. and Spilanthes acmella Murr. and their potential for the production of secondary Metablites
    (2011) Singh, Mithilesh
    Medicinal plants have always had an important place in the therapeutic arsenal of mankind. In the last few years as the demand for medicinal plants is increasing; their exploitation by the local population as well as pharmaceutical companies is also increasing continuously. It is, therefore, imperative to develop efficient methods for its large scale propagation. Besides micropropagation, in vitro production of useful bioactive compounds, their identification as well as quantification is also highly warranted. In this context, plant cell cultures have been proved handy for the production of high value secondary metabolites owing to the consistency in quality and quantity of the desired product. Plant cells are biosynthetically totipotent which means that each cell in culture retains the complete genetic information to produce the range of chemicals found in the parent plant. Azadirachta indica A. Juss. is a remarkable multipurpose, evergreen tree of the mahogany family, Meliaceae. The history of commercial use of neem tree is shrouded in the mystery and tradition of Vedic period of India. In the last two decades, neem has become the focus of attention due to its medicinal, agrochemical and economic uses. These properties can be attributed to several secondary metabolites present in the genus most of which, chemically, belong to the class of terpenoids. Among the entire range of diverse chemicals, one which remains the most sought after by scientists is azadirachtin, a highly oxidized tetranortriterpenoid, present in the neem seed kernels. This molecule has emerged as the safest, environment friendly and biodegradable biopesticide, unlike the chemical ones that pose problems of bioaccumulation in food chains upon long term usage. Current supply of azadirachtin from neem tree will not meet the increasing demand if the extractions from seeds remain the only source, hence, there is a need for the development of a commercially viable alternative for its enhanced and continuous production. Chemically, azadirachtin is a complex molecule and due to this complexity it is difficult to accomplish its total chemical synthesis in the laboratory. Keeping in consideration all these factors, plant cell culture can be seen as a potential alternative production system. With the cell culture methods, production can be more controlled and the product quality and quantity can be ensured, independent of geographical and climatic barriers. In cell culture, the culture conditions and process variable can be more easily optimized. Cell culture can also offer better selectivity and yield of the desired bioactive compound. In the present study, we made an effort towards systematic selection and screening of elite in vitro cell lines for constant and improved production of azadirachtin. Spilanthes acmella Murr. is an indigenous herb belonging to the family Asteraceae. The genus contains several secondary metabolites which are of therapeutic value and have been widely used in traditional medicine throughout the world, since time immemorial. In spite of being a plant of potential medicinal interest there are only a few scientific reports on the properties of this species. All the biochemical studies carried out, so far, have been done using natural population. As we are aware that environmental fluxes...