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ThesisItem Open Access Dna Fingerprinting And Mapping Of Anthracnose Resistance Gene In Sorghum(Chaudhary Charan Singh Haryana Agricultural University; Hisar, 2005) Singh, Monika; Boora, K. S.ThesisItem Open Access Genetic and microsatellite marker analysis of azucena (japonica) x HBC19(Basmati) F5-F6 rice population(Chaudhary Charan Singh Haryana Agricultural University;Hisar, 2005) Pankaj Kumar; Chowdhury, V. K.ThesisItem Open Access Genetic improvement of Saccharomyces spp. for ethanol production from starch(CCSHAU, 2005) Bajaj, Anshu; Chaudhary, KamlaA study was undertaken to improve ethanol production from starch by yeast Saccharomyces spp. A total of 10 isolates of yeast were isolated. After secondary screening in submerged culture conditions six isolates were selected for further investigation. Based on Maximum (6.4U) amylase and ethanol (8.8g/l) production, the Saccharomyces spp. B6 was selected and mutagenized using UV rays and nitrous acid. The Saccharomyces spp. mutant B6U3 produced maximum amylase (8.9U) & ethanol 16.4g/l (in starch) and 26.8g/l (in dextrose) among various mutants and was selected for further studies. The optimization of culture conditions showed that Saccharomyces spp. B6U3 produced increased level of amylase as compared to parent strain. Ethanol production was found to increase with increase in the level of starch. At high concentration of starch the ethanol production was decreased. Starch at a concentration of 10% was found be the best for production of ethanol by Saccharomyces spp. B6U3. Peptone was found to be best nitrogen source for production of ethanol by Saccharomyces Spp. B6U3 producing a maximum 33.4 g/l of ethanol in 48h. In media containing ammonium sulphate, ammonium nitrate casein & urea less amount of ethanol was produced. Peptone was found to the best nitrogen source at 2% level and 34.4g/l of ethanol was produced in 48h of fermentation. Increase in the conc. of peptone led to decreased ethanol production. On increasing the inoculum size upto 20% the ethanol production also increased from 5.6g/l to 32.8g/l after 48h of fermentation. A little variation was found on using 20% of starch grown and dextrose grown inoculum. There was a slight increase in the ethanol production on using dextrose grown inoculum (34.8g/l) than starch grown (33.2g/l) after 48h of fermentation. Ethanol production was found to be maximum after 48h of fermentation. Out of different starch sources used Soluble starch, Sorghum, spoiled wheat, Potato and Cassava, soluble starch was found to be the best substrate for production of ethanol by Sacchromyces spp. B6U3.ThesisItem Open Access Cellulase synthesis in 2-deoxy glucose resistant mutants of trichoderma reesei p-2(CCSHAU, 2005) Tina Mittra; Chaudhary, KamlaA study was under taken to isolate 2-DG resistant mutants of Trichoderma reesei P-2 for cellulase synthesis. Spores of T. reesei were mutagenized with UV irradiation for a time period of 15 minutes. At this time maximum number of mutants were obtained.Antimetabolite resistant mutants were obtained on 0.05% 2-DG containing Reese medium plates. A total of 24 isolates which grew profusely, were purified and again patched on Reese medium contained 2-DG. Finally 11 mutants showed good growth on this medium. 2-DG resistant mutants of T. reesei mutants MU-22 and MU-24 showed maximum cellulase production and selected. T.reesei mutants MU-22 and MU-24 showed maximum FPase activity (1.54 IU/ml and 1.56 IU/ml) respectively, CMCase activity (6.4IU/ml and 5.5IU/ml) respectively in cellulose medium. However and -glucosidase activity was more in MU-22(0.28 IU/ml). Effect of different carbon sources on synthesis of cellulases were analysed in mutants MU-24 and MU-22. Cellulose was found to be the best carbon source whereas in cellobiose and cotton medium, cellulase activity was reduced. In phosphocellulose medium -glucosidase activity was high and sorbitol was not a good inducer for the synthesis of cellulases. The optimization studies for cellulase production by MU-24 showed that cellulose at 1% concentration produced high level of cellulases. Similarly ammonium sulphate at a concentration of 0.14% was found to be essential for all the three enzyme activity. Since T. reesei is a mesophilic organism, at high temperature it did not grow. The optimum temperature for growth of T. reesei was found to be 30ºC. Saccharification of rice straw indicated the release of 50% reducing sugar which were more (35%) then the parents strain. The protein profile of culture filtrate of T. reesei mutants were determined by SDS PAGE. It was observed that the first set of proteins which appeared during cellulase induction were of 50KDa, 40KDa, 30KDa and 25KDa. Thus from this investigation it can be concluded that level of exoglucanase, endoglucanase and -glucosidase has been altered by mutation and such improvement in altering the level of individual component of cellulase complex is practically possible.ThesisItem Open Access Fingerprinting satawar (Asparagus racemosus) genotypes using RAPD markers(CCSHAU, 2005) Arora, Puneet; Dhillon, SantoshSatawar (Asparagus racemosus) is a medicinal plant growing in tropical climates and is useful in curing a wide array of ailments. This study was thus undertaken to prepare a DNA fingerprint database of selected varieties of Asparagus racemosus and to assess genetic diversity among them using randomly amplified polymorphic DNA (RAPD) markers. Twenty five RAPD primers were used to assess molecular polymorphism in fifteen Asparagus racemosus genotypes. A total of 211 amplified products were obtained out of which 50 were monomorphic and 161 were polymorphic. Average polymorphism across fifteen genotypes was found out to be 78.650%. For the genotypes tested, 5 to 17 bands were obtained, with an average of 10.55 bands per primer. The size of amplified fragments ranged from 230-2250 bp. Some primers also produced unique alleles in specific Asparagus genotypes which could be used to distinguish them. Analysis of this polymorphism profile, generated using suitable statistical programmes, grouped the fifteen genotypes into two major clusters at a similarity coefficient of 0.680. Varieties HAR-6 and Wild-3 were found out to be the most diverse and distant from other varieties. The second cluster again divided into two minor clusters, out-grouping the Nepali variety. The next large sub-cluster contained all the other genotypes. Varieties Wild-1 and Indian Yellow were genetically most similar. Genetic similarity matrices of the genotypes ranged from 0.630 to 1.00, indicating a moderate genetic variability among the genotypes. Wild-1 and Indian Yellow, showed a genetic similarity value of 1.00 while the genotypes HAR-1 and Wild-3 were found out to be genetically most diverse, at a value of 0.630. All other genotypes varied between these two extreme values. The results indicated that RAPD markers are efficient for identification of Asparagus racemosus genotypes and for determination of the genetic relationships among them. Fingerprint data obtained in this study can be further utilized in identification and development of improved Asparagus varieties.ThesisItem Open Access Fingerprinting satawar (asparagus racemosus) genotypes using RAPD markers(CCSHAU, 2005) Arora, Puneet; Dhillon, SantoshSatawar (Asparagus racemosus) is a medicinal plant growing in tropical climates and is useful in curing a wide array of ailments. This study was thus undertaken to prepare a DNA fingerprint database of selected varieties of Asparagus racemosus and to assess LITERATURE CITED -xivgenetic diversity among them using randomly amplified polymorphic DNA (RAPD) markers. Twenty five RAPD primers were used to assess molecular polymorphism in fifteen Asparagus racemosus genotypes. A total of 211 amplified products were obtained out of which 50 were monomorphic and 161 were polymorphic. Average polymorphism across fifteen genotypes was found out to be 78.650%. For the genotypes tested, 5 to 17 bands were obtained, with an average of 10.55 bands per primer. The size of amplified fragments ranged from 230-2250 bp. Some primers also produced unique alleles in specific Asparagus genotypes which could be used to distinguish them. Analysis of this polymorphism profile, generated using suitable statistical programmes, grouped the fifteen genotypes into two major clusters at a similarity coefficient of 0.680. Varieties HAR-6 and Wild-3 were found out to be the most diverse and distant from other varieties. The second cluster again divided into two minor clusters, out-grouping the Nepali variety. The next large sub-cluster contained all the other genotypes. Varieties Wild-1 and Indian Yellow were genetically most similar. Genetic similarity matrices of the genotypes ranged from 0.630 to 1.00, indicating a moderate genetic variability among the genotypes. Wild-1 and Indian Yellow, showed a genetic similarity value of 1.00 while the genotypes HAR-1 and Wild-3 were found out to be genetically most diverse, at a value of 0.630. All other genotypes varied between these two extreme values. The results indicated that RAPD markers are efficient for identification of Asparagus racemosus genotypes and for determination of the genetic relationships among them. Fingerprint data obtained in this study can be further utilized in identification and development of improved Asparagus varieties.ThesisItem Open Access Screening of indica rice genotypes for shoot morphogenetic potential(CCSHAU, 2005) Kaswan, Vineet; Chowdhury, V.K.A number of commercially important rice (Oryza sativa L.) varieties belonging to indica, scented indica and japonica groups were compared for their callus growth and plant regeneration potential. Calli were initiated from mature seeds and immature embryos on Murashige and Skoog’s (1962) basal medium containing (2.5 mg/L) 2, 4-D, (560 mg/L) proline, (300 mg/L) casein hydrolysate, (30 g/L) maltose and (3 g/L) gelrite. Maximum per cent callus induction was obtained in varieties Taraori Basmati (97.4%) and TNG-67 (97.3%) and minimum in Dehradun Basmati (37.9%) in mature seed and in case of immature embryo variety, Pusa Basmati-1 (96.2%) showed maximum per cent callus induction. Three week old calli obtained from mature seed and immature embryo were subcultured on shoot regeneration medium MSK [MS basal medium containing kinetin (2.0 mg/l), NAA (0.5 mg/l) and (1%) agarose]. Remarkable variations due to genotype in plant regeneration were observed, maximum being in Japonica variety TNG-67 (90.6%) followed by scented Indica rice variety Pusa Basmati-1 (79.0%), Indica rice variety HKR-46 (71.7%) and minimum in Indica rice variety HKR-120 (9.3%) in mature seed derived calli and in case of immature embryo derived calli, maximum shoot regeneration frequency was observed in Pusa Basmati-1 (91.3%). In general, immature embryo derived calli showed higher shoot regeneration in comparison to mature seed derived calli. Well developed plantlets were hardened and transferred to the soil. Efforts were being made to further improve regeneration potential in four important Indica rice varieties, IR-72, IR-64, PR-106 and Govind using different growth regulators. Among the four rice varieties IR-72 consistently showed higher shoot regeneration (48.7% to 64.5%), maximum being in MSK medium. In IR-64, shoot regeneration frequency increased by using thidiazuron in addition to kinetin in the regeneration medium.ThesisItem Open Access Micro-propagation of air yam (Dioscorea bulbifera L.)(CCSHAU, 2005) Victoria; Kharb, PushpaThe present investigation was carried out to optimize conditions and develop an improved protocol for micropropagation in air yam (Dioscorea bulbifera L.). Nodal and shoot tip explants were used for shoot regeneration on different media supplemented with both synthetic phytohormones as well as cell free extract of phytohormones producing bacteria (Acetobacter diazotrophicus 767-50 and Azotobacter chroococcum-103). Response for shoot regeneration was observed within a week on all the media tested for nodal explants and from shoot tip explants shoot regeneration was observed only on medium supplemented with 15 mg/l adenine sulphate and 2 mg/l kinetin. Nodal explants gave best shoot regeneration (100%) on MS medium supplemented with adenine sulphate (15 mg/l) and kinetin (2 mg/l). On the same medium after subculturing of nodal explants, when shoot induction started maximum (15-20) multiple shoots were observed in 76.66 per cent of the shoots while in the remaining shoots, 3-4 shoots per explant were observed Maximum (97.77%) rooting of shoots was observed on MS medium supplemented with 0.5 mg/l IAA. Healthy and strong rooting was observed on all the media tested within 15 days of culturing. Survival rate of regenerated plantlets was observed to be more than 90 per cent on transplantation to soil. The earliest shoot regeneration response was observed on 7th day on all the media supplemented with cell free extract of A. diazotrophicus, A. chroococcum and combination of both. Maximum 82.22 per cent and 88.88 per cent shoot regeneration response was observed on MS media supplemented with 15 mg/l adenine sulphate + 1.5 ml/l cell free extract of A. diazotrophicus and 15 mg/l. Adenine sulphate + 1.5 ml/l cell free extract of A. chroococcum, respectively from nodal explants. Shoot regeneration response was observed maximum (88.88%) on MS medium supplemented with 15 mg/l adenine sulphate + 0.5 ml/l cell free extract of A. diazotrophicus + 0.5 ml/l cell free extract of A. chroococcum. Root formation alongwith shoots was also observed on all the media supplemented with cell free extracts of both the bacterial strains. The number of shoots per explant regenerated on MS medium supplemented with cell free extracts of A. diazotophicus as well as A. chroococcum was found to be 2-3, whereas 3-4 shoots per explants were observed on the media supplemented with combination of cell free extracts of A. diazotrophicus and A. chroococcum.ThesisItem Open Access Studies on plant regeneration and Agrobacterium-mediated genetic transformation in cotton(CCSHAU, 2005) Harjeet Kaur; Yadav, Neelam R.Experiments were conducted to optimize conditions for callus induction, somatic embryogensis and shoot tip proliferation in four varieties i.e. H-1117, H-777, H-1098 and HS-6 of cotton (G.hirsutumn L.). Efforts were made to explore the possibility of Agribacterium mediated genetic transformation in cotton. Seeds were cultured on ST (Steward's) medium for germination. Callus were initiated in cultured hypocotyl explants from 10 to 12 days old seedling by culturing on seven MS based callus induction media containing glucose as carbon source and auxins (NAA, 2,4-D) along with cytokinin (Kinetin, Zeatin). Callus induction percentage varied between 43.57 to 97.26 per cent using hypocotyl explants depending upon genotype used. Out of four cultivars studied, H-1117 gave maximum frequency of callus induction (97.26%) on MS1 medium containing NAA (2.0 mg/l) and 2,4-D (0.1 mg/l). Two facterial CRD revealed that variance due to genotype, medium and their interaction was significant at 1 per cent level. The induced callus generated from hypocotyl explants was cultured on eleven media for somatic embryogenesis. H-777 gave maximum (50.26%) embryogenesis among the four cultivars used for study. Various additive like Putrescine, Spermidine, TDZ, Glutamine etc. were used along with MS basal medium for somatic embryogenesis. Medium containing TDZ (2.0 mg/l) and 2,4-D (0.1 mg/l) showed maximum embryogenesis in all the four cultivars. For shoot tip proliferation, excised shoot tips of above four genotypes were cultured on various media containing different concentration of auxin (NAA), cytokinin (BAP and kinetin) and gibbrellin (GA3). Per cent shoot proliferation on different media varied between 37.81 to 78.76 per cent. Maximum being on medium containing BAP (2.5 mg/l) and kinetin (2.5 mg/l) in genotype H-777 Agrobacterium tumefaciens strain EHA 105 harbouring plasmid pCAMBIA 1301 (Gus, KanR) was used to optimize agrobacterium mediated transformation in four selected genotypes. Hypocotyl and cotyledon explants were co-cultivated with the strain EHA-105 containing plasmid pCAMBIA 1301. Per cent gus expression ranged between 6.82 to 25.55 percent depending on tissue type and genotype. Cocultivation time was 72 hours and acetosyringone concentration used was 100 M. Variety H-1117 gave maximum gus expression and was found to be the best genotype for agrobacterium mediated transformaton. Hypocotyls were found to show maximum percent of transient gus expression.