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M. Sc. Dissertations

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2005 - 2009262010 - 20111
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Subject: Biotechnology and Molecular Biology ×

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Now showing 1 - 9 of 27
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    ThesisItemOpen Access
    DNA fingerprinting of self-Compatible and self-Incompatible genotypes of sunflower (Helianthus Annuuns L.)
    (CCSHAU, 2005) Mehta, Deepa; Boora, K.S.
    Sunflower (Helianthus annuus L.) ranks second among oilseed crops in the world after soybean. Sunflower is protrandrous. Seed set in sunflower is a complex phenomenon and one of the mean to overcome this problem is by identifying self fertile and open pollinating lines. The present study was undertaken to identify molecular marker closely linked to self compatible and self incompatible gene(s) in sunflower using RAPD analysis. A total of 26 genotypes (13 self compatible + 13 self incompatible) of sunflowers were used. Protocols were optimized for DNA extraction and PCR amplification of self compatible and self incompatible genotypes of sunflower using RAPD markers. DNA was isolated using CTAB method with some modifications and among different genotypes, CMS 338 (C)A gave highest quantity (1034.5 μg/ml) of DNA whereas genotypes Acc-1445-6 gave lowest amount (317.5 μg/ml) DNA. Quality of DNA was tested by agorase gel electrophoresis and UV spectrophotometer. A single discrete band of high molecular weight showed that DNA was pure and free from contaminants. Optimum PCR amplification was observed on all DNA samples when reaction mixture contained genomic DNA (100 ng), MgCl2 (1.5 mM), Taq DNA polymerase (3 units), dNTPs (200 M), 10X Taq DNA polymerase buffer (1 μl), primer (10 μM) and annealing temperature of 400C. A total of fifty four random primers were screened and thirty eight primers produced polymorphism. These thirty eight primers were screened with self compatible and self incompatible bulks. Primer OPA-9 and OPE-2 produced a unique DNA band in self incompatible and self compatible bulks, respectively. Marker OPA-9 produced unique band in all the self incompatible genotypes except genotypes IHT-298 and HB-342. This marker is 7.6 cM away from the genes for self incompatible. Similarly, marker OPE-2 produced unique band in all the self compatible genotypes except IB-43, Acc-1351-3, HB-105. This marker is about 11.53 cM away from the genes for self compatibility. Marker OPA-9 produced a unique band in twelve self compatible genotypes. Marker OPE-2 produced unique band in eleven self incompatible genotypes. These markers may be proved useful in marker assisted selection for self compatible and self incompatible traits, respectively.
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    ThesisItemOpen Access
    In vitro studies on mass multiplication in stevia rebaudiana (Bertoni) Hemsl
    (CCSHAU, 2006) Narang, Jagriti; Yadav, R.C
    The present study was undertaken to develop an efficient micropropagation system for plant regeneration and transfer the regenerated plants to soil conditions in Stevia rebaudiance (Bertoni) Hemsl. Nodal and shoot tip explants were used for shoot regeneration on 39 different media supplemented with different growth regulators. Nodal explants gave best shoot regeneration response (58.70%) on MS medium supplemented with BAP (5.0 mg/L) and AgNO3 (2.0 mg/L) and MS medium fortified with BAP (5.0 mg/L)and AgNO3 (2.0 mg/L) gave highest percent of shoot initiation (71.37%) in shoot tip explants. The shoot were elongated on MS medium containing different concentrations of growth regulators using agar as gelling agent. Best shoot elongation was observed on MS medium containing BAP (4.0 mg/L), GA3 (1.0 mg/L) and AgNO3 (1.0 mg/L) out of the four media tried. Shoot tip explants are considered to be the best explant compared to nodal explants for shoot regeneration. Media supplemented with AGNO3 produce healthy shoots as compared to other media tried. Rooting of the regenerated shoots was observed (35.00%) on MS medium supplemented with IAA (2.0 mg/L) out of 9 different media tried. The regenerated plantlets were transferred to soil and vermiculite mixture (1:1 and 58.3 per cent) plants survived in the soil.
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    ThesisItemOpen Access
    Molecular characterization and genetic divergence analysis in date palm (Phoenix dactylifera L.) using RAPD markers
    (CCSHAU, 2007) Rekha Rani; Kharb, Puspha
    Date palm (Phoenix dactylifera L.) is a long living dioecious evergreen with 2n=36. It has the distinction of being one of the oldest fruit trees in the world. It is an important plantation crop due to nutritive quality of its fruit. This study was undertaken to assess polymorphism among different date palm genotypes (including male and female genotypes). Molecular polymorphism in 40 date palm genotypes (10 male and 30 female) was assessed by using 29 RAPD primers. A total of 223 amplicons were observed out of which 3 were monomorphic and 220 were polymorphic. Thenumber of amplified DNA bands varied between 2 and 17 with an average of 7.69±0.70 bands per primer. Average polymorphism across the 40 genotypes was 99.12±0.62. Three primers were identified which produced unique alleles capable of differentiating female l genotypes of Medjool from rest of the genotypes. Analysis of the polymorphism profile generated using NTSYS-PC software, grouped the 40 genotypes into 2 major clusters and thirteen sub clusters. Z2 and M6 were out grouped showing them to be genetically most diverse among all genotypes used in present study. Based on ‘Simqual’ sub programme, genetic similarity was calculated. It ranged from 0.28 to 0.93 indicating moderate genetic variability among the genotypes. The average similarity of male plants with all the thirty female plants was found to be 0.7. Thus RAPD markers detected a high level of polymorphism and the fingerprint data generated in this study can be used for the identification of Medjool variety of date palm.
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    ThesisItemOpen Access
    Molecular characterization and genetic divergence analysis in cowpea (Vigna unguiculata L. Walp.) using RAPD markers
    (CCSHAU, 2007) Suruchi; Dhillon, Santosh
    Cowpea (Vigna unguiculata) is a legume crop growing in almost all parts of world including tropics and subtropics. This study was thus undertaken for the molecular characterization of selected varieties of Vigna unguiculata and to assess genetic diversity among them using randomly amplified polymorphic DNA (RAPD) markers. Thirty five RAPD primers were used to assess molecular polymorphism in twenty four Vigna unguiculata genotypes. A total of 182 amplified products were obtained out of which 2 were monomorphic and 180 were polymorphic. Average polymorphism across twenty four genotypes was found out to be 99.35%. For the genotypes tested, 2 to 14 bands were obtained, with an average of 8.27 bands per primer. The size of amplified fragments ranged from150-2000 bp. Some primers also produced unique alleles in specific cowpea genotypes which could be used to distinguish them. Analysis of this polymorphism profile, generated using suitable statistical programmes, grouped the twenty four genotypes into two major clusters at a similarity coefficient of 0.50. The cluster-I was divided into 2 sub clusters involving 8 genotypes. Genotype CP-23 was out grouped and formed the 1st sub cluster of cluster I. The second cluster was again divided into two minor clusters. Subcluster-I involved two varieties CP-20 and FS68. The next large sub-cluster contained all the other genotypes. Genetic similarity matrices of the genotypes ranged from 0.3462 to 0.8681, indicating a high genetic variability among the genotypes. Genotypes CP-6 and CP-7 were genetically most similar with genetic similarity value of 0.8681 while the genotypes CP-3 and CP-11 were found out to be genetically most diverse, at a value of 0.3462. Similarity value for all other genotypes varied between these two extreme values. The results indicated that RAPD markers are efficient for identification of Vigna unguiculata 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 cowpea varieties.
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    ThesisItemOpen Access
    Studies on myb gene expression in Indian mustard (Brassica juncea L. Coss. & Czern) under salt stress
    (CCSHAU, 2007) Sunil Kumar; Yadav, R.C.
    The investigation was carried out to study the myb expression under different levels of salinity in Brassica juncea cv. CS52 in the Department of Biotechnology & Molecular Biology, CCS Haryana Agricultural, Hisar during the year 2006 and 2007. The myb gene expression was checked by RT-PCR approach. Primers BjMYBR-1 and BjMYBR-2 were designed from the conserved DNA binding domain of Atmyb2 by analyzing this gene sequence with other myb gene sequence available in database using ClustalW programme. The expression of Bjmyb1 became detectable within 15 minutes of salinity exposure, which increased and was maximum in 30 minutes of salinity treatment. Thereafter it declined and stabilized in 2 hr of salinity treatment. The expression was not affected by the different concentrations (100 mM, 200 mM, 300 mM and 400 mM) of NaCl used. Sequencing of amplified cDNA fragment revealed a 176 bp sequence. ClustalW analysis of Bjmyb1 with other eight myb genes from different organisms available in database showed its homology with Atmyb2 and Chmyb2.
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    ThesisItemOpen Access
    Genetic divergence analysis in date palm (Phoenix dactylifera L.) using ISSR markers
    (CCSHAU, 2007) Deepak; Dhillon, Santosh
    Date palm (Phoenix dactylifera L.) is a dioecious, monocotyledonous woody perennial belonging to the family Palmae (Arecaceae) and grown in the arid regions of the world for its nutritious fruit. It has the distinction of being one of the oldest fruit tree of the world. This study was thus undertaken to assess polymorphism among ten male and thirty female genotypes of eight varieties of date palm viz. Zaglool, Egypt No. 2, Hillawi, Zaidi, Sayer, Khadrawy, Shamran and Medjool using 40 ISSR primers. A total of 160 amplified products were observed of which 8 were monomorphic and 152 were polymorphic. Average polymorphism across the 30 genotypes was 89.46±6.8 %. The number of amplified DNA bands varied from 2 to 13 with an average of 7.27±0.77 bands per primer. One ISSR primer (BH-08) amplified a unique allele of 630 bp which was present in all male plants but was absent in all female plants included in the study, suggesting it to be a useful marker for early detection of sex in date palm. Primer BH-02 amplified an allele of 700 bp size which was unique to Medzool genotypes and could distinguish them from all other varieties. Analysis of polymorphism data using NTSYS-PC software, grouped the 40 genotypes into 2 major clusters. Almost all the genotypes of a particular variety were present together in their corresponding groups. However, one female genotype Z2 was outgrouped from all the other genotypes showing it to be the most diverse. One male genotype (M8) showed 85% similarity to SH2 indicating it to be a male of Shamran or a close relative of it. Genetic similarity based on ‘Simqual’ sub programme among various genotypes ranged from 0.41 to 0.89% indicating moderate genetic variability among genotypes. The average similarity between males and the thirty females was found to be 0.69. Thus ISSR markers detected a high level of polymorphism and the fingerprint data generated in this study can be used for further cloning and sequencing of sex-linked markers in date palm.
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    ThesisItemOpen Access
    Effect of rhizobium and rhizosphere extract on in-vitro regenerated plantlets of chickpea (Cicer arietinum L.)
    (CCSHAU, 2007) Manjeet Kumar; Sikka, V. K.
    Experiments were conducted to improve transplantation efficiency of in-vitro regenerated plantlets of chickpea. Three chickpea cultivars C 235, HC 5 and HC 1 were cultured for shoot regeneration and subsequently for root regeneration. A total of five Rhizobium strains were screened for IAA production out of which Strain M 113 was found to secrete maximum IAA (2.4 μg/ml) whereas strain M 25 showed a maximum IAA level of 2.2 (μg/ml) in culture. Rhizobium supplemented culture medium helped in better survival of transplanted explants in comparison to regenerated plantlets on normal control medium. Rhizobium consortium treatment and adding Rhizobium culture over roots did not help in rooting and survival of the regenerants. Instead of it adding culture extract in the rooting medium with MS salts helped a lot in improving survival. Among MRC 113, MRC 25 and MRC 104 varietal response shown was maximum in cultivar C235 where it was 90% and it was minimum in cultivar HC 1 where it was 66%. In MRC 113, MRC 25 and MRC 104 no growth hormones were added but only Rhizobium culture was added showing that culture is having plant growth promotion abilities, which may be due to phytohormone IAA production in culture. Cultivar C 235 gave better response in comparison to other cultivars on MRC 113 proving that Rhizobium strain 113 is best plant growth promoting among these strains. In medium MSE no rooting hormones were added but still it showed good rooting quality showing that rhizosphere extract may also promote root development and hence rooting quality. Rhizobia proved promising in improving survival of regenerated chickpea. This line of treatment needs to be further refined to enable seed set in the regenerated plants. Other plant growth promoting microbes in combination with Rhizobium inoculation are further expected to improve the response.
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    ThesisItemOpen Access
    Agrobacterium mediated transformation of indica rice variety IR-72 using Amaranthus seed albumin gene (AmA1) driven by a seed specific promoter
    (CCSHAU, 2007) Chowdhary, Ritika; Jain, R.K.
    Experiments were conducted to transfer Amaranthus seed albumin gene (AmA1) driven by GBSS promoter in indica rice variety IR-72 and Pusa Basmati by Agrobacterium-mediated transformation. During the present investigation, 14-18 days old calli induced from mature seed scutella and immature embryos on modified MS medium were used. The calli were II co-cultivated for 10 minutes in liquid co-cultivation medium with Agrobacterium strain EHA105 (pSB8G) and then transferred onto the solidified co-cultivation medium for 2-3 days. Calli were screened on selection medium containing cefotaxime (250 mg/l) and G-418 (50 mg/l) for 2-3 cycles of selection of 15 days each. A total of 160/1790 (mature seed) and 37/213 (immature embryo) calli of IR-72 and 124/831 of Pusa Basmati 1 showed sustained proliferation on the selection medium. A total of 22 IR72 and three Pusa Basmati 1 plants were regenerated from the selected calli by using the MS based regeneration media supplemented with antibiotics. Fifteen of these plants (IR72, 12 plants; Pusa Basmati 1, three plants) were successfully transferred to the transgenic greenhouse. Five of the seven-rice plants showed the amplification of 1.1 kb fragment indicating the presence of AmA1 gene. One of these PCR positive plants has already flowered and set seeds.
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    ThesisItemOpen Access
    Cloning of cellulase gene(s) of trichoderma reesei in sacchromyces cerevisiae
    (CCSHAU, 2007) Rochika; Chaudhary, Kamla
    A total of seven Trichoderma reesei cultures were purified by reculturing on potato dextrose agar medium. Cellulase production by T. reesei cultures varied from 0.73 IU/ml to a maximum of 3.11 IU/ml in cellulose medium. Among all the strains evaluated, T.reesei culture 5A showed the maximum cellulase activity (3.11 IU). Cellulase activity of T. reesei 5A was monitored at different interval of time (12-96 hrs.) and maximum activity was observed after 96 hrs. of growth. Genomic DNA of T. reesei 5A was isolated and checked for its quality by agarose gel VI electrophoresis and by UV spectrophotometer. The quantity of DNA was found to range between 195 μg/ml to 2607.5 μg/ml. Different parameters i. e. growth period of 36 hrs., CTAB concentration 1.5% and incubation period of 90 min., were found to be optimum during DNA isolation. Partial digestion of genomic DNA was done with Sau 3A and the digested DNA was ligated to Bam H1 treated Yeast-E. coli shuttle vector YEpFLAG-1. The construct was used for transformation of E. coli. Recombinant clones of E. coli were screened on carboxy methyl cellulose and ampicillin containing Reese medium. The recombinant plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo were isolated from E. coli recombinant clones and checked for amplification of cellulase gene by PCR using the standard conditions. PCR amplification was observed for exoglucanase and endoglucanase gene. Protoplast of yeast Saccharomyces cerevisiae were transformed with the recombinant plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo isolated from E.coli. The recombinant clones of yeast were screened on YEP medium containing 1% phosphocellulose and ampicillin. Expression of cellulase gene(s) in S. cerevisiae transformants was checked by monitoring the cellulase activity and ethanol production. CMCase activity was observed in all the three yeast transformants as 0.80 IU/ml, 0.50 IU/ml and 0.70 IU/ml respectively. No FPase activity could be detected in any of the yeast transformants. Different concentrations of phosphocellulose (2% and 5%) were used for ethanol production by the yeast transformants. Yeast transformant-6 produced maximum 1.5% alcohol when inoculated in 5% phosphocellulose containing YEP medium. Yeast transformant-2 and transformant-4 also produced ethanol in rice straw medium.