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ThesisItem Open Access STRAIN IMPROVEMENT OF Calocybe indica THROUGH MUTAGENESIS AND ITS AGRONOMIC OPTIMIZATION(PAU Ludhiana, 2011) JATINDER KAUR; H. S. SodhiTremendous revolution in the mushroom technology in recent years with respect to types and strains of cultivated mushrooms has made Calocybe indica, a specialty mushroom, the third most popular and commercially grown mushroom in India. A lot of work has been done as far its cultivation technology is concerned but no systematic work has yet been undertaken for its genetic improvement. During present investigation nine strains of C. indica were cultivated on wheat straw using recommended technology. Strain Ci- 3 gave maximum biological efficiency of 81.28%. Basidiospores from all the nine strains were collected and subjected to germination trial on different media but no germination was observed. Improvement of high yielding strain, Ci-3 using mutagenic treatment on protoplasts was conducted. Four mutagenic treatments (one physical and three chemical) yielded 30 putative mutants. Growth studies of putative mutants indicated maximum growth rate of CMB-4 on wheat straw while CMN-11 gave maximum biomass on complete yeast extract medium. Mutant CMN-9 gave maximum endoglucanase (0.345 μg/min/ml) and xylanase (0.540 μg/min/ml) activity. Seven mutants were identified on the basis of growth and enzymes studies. A set of 14 primers were used for molecular characterization of mutants along with the parent using RAPD-PCR. Out of these only 6 primers resulted gave distinct amplification products. Six primers yielded 68 scorable bands ranging from 40bp to 280bp. Similarity coefficient of Ci-3 ranged from 0.706 for CMN-3 to 0.794 for CMU-2. Three mutants (CMU-2, CMU- 5 and CME-2) showed 4 bands each having similar relative mobility values (0.2, 0.35, 0.5, 0.65) during protein profiling using SDS-PAGE. Three bands were obtained for CMN-11, CMB-4 and Ci-3 and 2 bands for CMN-9. Only one band was obtained in case of CMN-3 with relative mobility of 0.2. Cultivation of the mutants was carried out on wheat straw following hot water treatment. Four mutants (CMN-3, CMN-9, CMN-11 and CMB-4) were found to give significantly higher yield than the parent while only three mutants (CMN-3, CMN-9 and CMN-11) gave better results on mixture of wheat straw and paddy straw and on steam sterilized wheat straw. During nutritional studies mutant CMN-9 was found to be nutritionally better with high protein, tocopherol, β- carotene and lycopene content. Postharvest treatment of mutants indicated cabinet drying as a better option in terms of colour and texture retention.ThesisItem Open Access STUDY ON ERGOSTEROL CONTENT AND PROTEIN PROFILE OF MEDICINAL MUSHROOM, Ganoderma lucidum(PAU Ludhiana, 2013) Anna Goyal; H. S., SODHIGanoderma lucidum, a specie belonging to the class basidiomycetes, family polyporaceae of the order aphyllophorales has been widely used as a source of potent nutracuetical products. Present study was planned to identify and characterize the role of proteins and ergosterol in the developmental process of Ganoderma lucidum. Four strains of Ganoderma lucidum (GL I - IV) showed a gradual increase in biomass to give 25.52g to 31.72g of biomass after three weeks of growth in mushroom complete medium broth with maximum in strain GL-III. Ganoderma lucidum strains were grown on wheat straw supplemented with 5% wheat bran with maximum biological efficiency for GL-I strain (31.27%) followed by GL-II (26.76%) and number of fruit bodies were 927 and 693 each weighing 33.7g and 38.6g, respectively. Ganoderma lucidum strain GL-I showed maximum ergosterol content (4601μg/g) whereas strain GL-III showed only 32μg/g. From spawn run, maximum ergosterol was obtained from GL-IV strain followed by strain GL-II while at pin head formation and fruit body formation ergosterol content was better for GL-II. Ergosterol content of fruit body of GL-1 was observed maximum (7009μg/g). Overall observation indicated that the ergosterol content increased with each stage of cultivation process i.e. from spawn run to pinhead and finally to fruit body formation. The intracellular and extracellular enzymatic studies have indicated enhanced activity during spawn run on solid substrate in comparison to that grown in the broth. The esterase and peroxidase activity significantly increased during the pinning of the cultures thus, indicating a positive role of these enzymes in fructification process. The FTIR analysis of proteins made during different stages of cultivation namely spawn run, pin head formation and fruiting indicated that the fruiting strains (GL-I and GL-II) have an ordered protein structure with hydrophobic amino acids. In case of GL-IV, unordered structure was obtained that could be related to the role of hydrophobin proteins in mushroom fructification process. Another observation on GL-IV indicated the presence of basic amino acids and aromatic amino acids with very low amount of acidic amino acids like aspartic acid and glutamic acid. The observation recorded during present study indicated a positive role of hydrophobic amino acids and hydrophobin proteins in mushroom fructification process. Ganoderma lucidum strain GL-II was also grown on selenium fortified mushroom minimal medium at different concentrations of 5ppm to 25ppm of sodium selenate. Scanning electron micrographs exhibited gradual decrease in hyphal diameter, spore number and spore diameter with increase in selenium concentration and the spore structures were altered. A significant decrease in spore diameter is observed in concentration of 20ppm and 25ppm (5.60 and 1.26 μm, respectively) as compared to control (10.04 μm). The SEM-EDS studies showed no selenium traces on the hyphal surface, however, on the contrary, SEM-EDS studies of crushate samples revealed selenium traces indicating selenium absorption as the cytosolic moieties as selenoproteins. Atomic absorption spectroscopy indicated an increasing trend in the uptake by the hyphal biomass as the concentration of sodium selenate increased with maximum absorption at concentrations of 15 ppm and 25 ppm (9.9%). It was concluded that fortification till 15 ppm can be used as stress was not that prominent and culture could grow rapidly without significant alteration in structure and morphology to enhance its biomedicinal properties. Present study has indicated that during the mushroom development process, ergosterol content increases with a positive role of proteins like peroxidases and hydrophobins at each stage of morphogenesis.ThesisItem Open Access “BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ALKALINE PROTEASE OF BACILLUS CIRCULANS MTCC 7906”(PAU Ludhiana, 2011) Inderjeet Kaur; Gurvinder Singh, KocherThe present study was conducted to characterize alkaline protease of Bacillus circulans MTCC 7906. Cotton deoiled meal (2.1%) supported optimum enzyme production in 144 hours of incubation. Among the agricultural byproducts used to improve alkaline protease production, the combination of Soybean meal and Glucose supported maximum enzyme production in 96 hours of incubation which was comparable with the control Reese medium (Casein + Glucose) in terms of alkaline protease activity as well as incubation time required for enzyme production. The alkaline protease was purified (16.2 fold) to homogeneity from the culture supernatant by the combination of ammonium sulphate precipitation (30-60%) and DEAE-cellulose anion exchange chromatography. The biochemical characterization of partially purified alkaline protease displayed maximum activity at a pH of 9.0, temperature of 60°C, an enzyme concentration of 0.1 ml/3.0 ml and substrate concentration of 12 mg/ml. The Km and Vmax of partially purified alkaline protease were found to be 4.5 mg/ml and 5555 nmole tyrosine min-1 ml-1, respectively. The enzyme activity was activated by divalent ions whereas a metal chelator EDTA and ammonium hydroxide reduced the activity. The molecular weight of the enzyme was estimated to be 46 kDa on SDS-PAGE. For, molecular analysis, full length alkaline protease gene (BcAP, 1329 bp) was PCR amplified from B. circulans DNA and cloned into pTrcHisA vector. Sequencing and alignment of nucleotide and predicted amino acid sequences of alkaline protease region identified a number of substitutions (mutation) which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The multiple alignment of nucleotide and amino acid sequences of alkaline protease gene of B. circulans established major matches with three closely related subspecies of B. subtilis (B. subtilis subspecies subtilis strain 168, B. subtilis BSn5 and B. subtilis subspecies spizizenii strain W23). Molecular evolution of B. circulans strain was determined by phylogenetic analysis of alkaline protease gene and predicted amino acid sequences. This analysis also suggested that alkaline protease gene is novel gene, which has been accessioned in GenBank with accession number JN645176.1. Recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the protein with estimated molecular size of 46 kDa as observed earlier.ThesisItem Open Access Effect Of Bradyrhizobium Spp-, Sinorhizobium Fredit And Plant Growth Promoting Rhizobacteria On Soybean ( Glycine Max ( L.) Merrill ) Crop(Punjab Agricultural University ;Ludhiana, 2003) Ramesh Chand; Pandher, M. S.ThesisItem Open Access Studies On The Development Of Vesicular Arbuscular Micorrhiza (Glomus Fasciculatum Inoculum And Its Application To Micropropagated Plants(Punjab Agricultural University; Ludhiana, 2001) Guleri, Radhika; Gupta, R. P.ThesisItem Restricted Biochemical And Molecular Charracterization Of Pseudomonas Isolates For Degradation Of 4-Chlorobenzoic Acid(Punjab Agricultural University ;Ludhiana, 2004) Banta, Geetika; Kahlon, R. S.ThesisItem Open Access Elucidation of phorate metabolism by bacterial isolates from agricultural soils for bioremediation(PAU, 2013) Jariyal, Monu; Gupta, V. K.Fifteen phorate metabolizing bacterial species isolated from sugarcane field soils were identified using 16SrDNA sequence homology. Based upon relative phorate degradation, Brevibacterium frigoritolerans, Bacillus aerophilus and Pseudomonas fulva were found to cause more than 98 per cent phorate reduction. These bacterial species could grow over a wide range of pH (4.0-11.0) and temperature (25-37°C), but optimally at pH of 6.0-6.5 and 37°C, in shaking cultures. Whereas, B. frigoritolerans was salt sensitive, P. fulva & B. aerophilus grew optimally in 3.0 and 4.0 per cent NaCl, respectively. All the three bacterial species grew optimally in the presence of glucose and peptone (1.0 % each). Only B. aerophilus carried a plasmid of around 4 kbp, but curing of this plasmid did not affect the phorate degradation establishing that phorate degradation genes are borne on chromosomal DNA. Induction by phorate resulted in only nondifferentiating protein profiles in all the three bacterial species establishing that phorate degradation is a constitutive character. In soils amended with upto 300 mg kg -1 phorate, these bacterial species within 42 days actively metabolized phorate by between 89.81 and 95.62 per cent with maximum capacity shown by P. fulva. This phorate degradation was further improved to 98.31 per cent, by mixed cultures of all the three bacterial species which constituted most effective bioremediation consortia for significantly relieving soils from phorate residues. The investigations in this study has isolated three bacterial species and established their potential for active bioremediation of phorate both in liquid cultures and agricultural soils.ThesisItem Restricted Establishment Of Azospirillum In Ryegrass (Lolium Perenne) And Fodder Maize ( Zea Mays) Rhizosphere(Punjab Agricultural University ;Ludhiana, 2002) Chambial, Shweta; Gupta, R. P.ThesisItem Open Access Interaction Of Azorhizobia With Oryza Sativa(Punjab Agricultural University; Ludhiana, 2002) Kalia, Anu; Gupta, R. P.
