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M. Sc. Dissertations
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ThesisItem Open Access Micronutrient and molecular diversity analysis in mungbean [Vigna radiata (L.) wilczek] genotypes(CCSHAU, 2010) Aneja, Bharti; Yadav, Neelam R.Vigna radiata (L.) Wilczek, commonly called mungbean or greengram is the third most important pulse crop of India occupying nearly 3 million hectare. It is an excellent source of inexpensive and easily digestible proteins of low flatulence and is available in several edible forms. Iron and Zinc are important micronutrients for human health whose deficiency causes anemia and malfunctioning of immune system. Commercial cultivars contain low levels of iron and zinc and it is important to assess genetic variability in available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study SRAP polymorphism among 21 mungbean genotypes using 29 SRAP primers. A total of 121 amplified bands were produced which were polymorphic with an average of 4.65 bands per primer. The size of amplified bands ranged from 70-3000 bp. Unique bands were displayed by 8 mungbean genotypes using SRAP analysis. Six out of 29 SRAP primers were most useful in fingerprinting mungbean genotypes under study. The similarity coefficients between different genotypes ranged from 0.45-0.96 with an average similarity value of 0.71. At an arbitrary cut-off at 60 per cent similarity level on a dendrogram, the mungbean accessions were categorized into two major clusters. ML1108 and 2KM115 were found to genetically similar. SMH99-1A and ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. Recombinant inbred lines involving ML776 and Satya could be used for tagging gene for micronutrient content. The results indicated that SRAP markers were efficient for identification of Vigna radiata genotypes and for determination of the genetic relationships among them.ThesisItem Open Access Cloning and characterisation of sex-specific DNA marker in jojoba (Simmondsia chinensis L.)(CCSHAU, 2010) Surender; Kharb, PushpaJojoba (Simmondsia chinensis L. Schneider) is a slow growing, dioecious shrub of arid zones and belongs to family Simmondsiaceace. It is commercially valued for multipurpose uses of its oil present in seeds. Sex of the young jojoba plant cannot be judged until the first flower buds appear which may take up to 3-4 years. Bhardwaj (2008) identified a male specific ISSR marker in jojoba, amplifying a unique allele of 1100 bp in male genotypes only. This study was undertaken to clone and characterize this sex-specific marker and to convert this marker into more specific SCAR marker. PCR amplification of DNA, from ten male and ten females plants of jojoba, was carried out using primer IS52. The amplified products were separated by agarose gel (1.5%) electrophoresis. A fragment of size 1100bp size was found to be present only in male plants. This specific band was eluted from the agarose gel using Qiagen gel extraction kit and cloned in to pJET1.2/blunt Cloning Vector using CloneJETTM PCR cloning kit supplied by Fermentas USA. The ligation mixture was transformed into freshly prepared competent cells of E.coli JM107 strain. The transformed colonies were allowed to grow on LB (Luria Bertani) plates containing ampicillin (100μg/ml).Only recombinant colonies appeared on the LB plates. The plasmid DNA from one colony was isolated using modified alkaline lysis method and then checked on 0.7% agarose gel. The male specific cloned DNA fragment was got sequenced using automated DNA sequencer ABI 3130XL Genetic Analyzer (Applied Biosystem, USA) located at Department of Animal Biotechnology, COVS, CCS HAU, Hisar. The sequence generated was compared with the sequences available in NCBI database and the maximum homology search was done using BLASTn. The sequence did not have any homology with any known nucleotide sequences in the available databases. Three open reading frames (108, 40 and 36 amino acids) were revealed in the sequences. Out of these, two ORF (40 and 36 amino acids) did not show any homology in available databases. One ORF of 108 amino acids showed 32% similarity with monothiol glutaredoxin-5 and mitochondrial precursor of Aspergillus terrus. SCAR scF (Simmondsia chinensis forward) and SCAR scR (Simmondsia chinensis reverse) primers were designed based on the sequence of male specific fragment. This primer pair amplified a unique allele of 1000 bp in male genotypes only (Patent filed).ThesisItem Open Access Study on personality of adolescent boys from rural and urban disorganized families(CCSHAU, 2010) Rathour, Savita; Duhan, KrishnaThe present study was undertaken in Hisar district of Haryana state. Five senior secondary schools of Hisar city and three higher/senior secondary schools of Kaimari, Mangali and Gangwa villages of Hisar I block were selected as per the demand of study. A sample of 45 rural and 45 urban respondents of 13-18 year age were taken, thus to make a total sample of 90 adolescents. Their parents also participated in study. Multi dimensional Assessment of Personality form for adults and teenagers, developed by Vohra, S. in 1993, 1996 was used to assess the personality of parents and adolescents. The finding revealed significant differences in boldness(Z=5.46*), general ability(Z=2.57*), guilt proneness(Z=2.52*), leadership(Z=5.90*), mental health(Z=1.81*), sensitivity(Z=2.50*),self sufficiency(Z=4.74*), social warmth(Z=2.46*) and tension level(Z=2.55*) of rural and urban respondents. Significant correlation existed between parental personality aspects and adolescent’s personality aspects. Adolescent’s adaptability, boldness, general ability, guilt proneness, innovation, leadership, maturity, self control, self sufficiency and tension had significant positive correlation with adaptability, boldness, general ability, guilt proneness, innovation, leadership, maturity, self control, self sufficiency and tension level of their parents. Whereas there was a significant negative correlation was observed between adolescent’s enthusiasm and parent’s tension, adolescent’s innovation and parent’s excitability, adolescent’s maturity and parent’s tension. Results further revealed significant association between age and enthusiasm level of the respondent. Economic status of family had significant effect on adolescent’s general ability, guilt proneness, leadership, mental health, self control and social warmth. Social variables viz. caste, family type, family size, parental sex and parental education were significantly associated with enthusiasm, general ability, guilt proneness, individualism, leadership, maturity, mental health, self control, self sufficiency and tension level of adolescents.ThesisItem Open Access Molecular analysis of thermotolerant and sensitive genotypes of wheat (Triticum aestivum L.em.Thell) using ISSR markers(CCSHAU, 2010) Richa; Dhillon, SantoshWheat is one of the most important staple food crops and is cultivated throughout the world. High temperature stress is one of the major constrains to wheat production world wide. This study was undertaken with the objective to assess polymorphism among 7 thermosensitive and 7 thermotolerant genotypes of wheat. DNA extracted from young leaves of 14 wheat genotypes was amplified by using 40 ISSR primers. Out of 40 primers, 26 ISSR primers showed amplification and were selected for allele scoring. ISSR data was analyzed to calculate various parameters such as the number of total bands, number of polymorphic bands, per cent polymorphism, bands per primer, polymorphic bands per primer, similarity matrices and dendrogram. ISSR generated a moderate level of average polymorphism i.e. 49.2%. An average of 7.19 bands per primer were generated. Overall size of PCR amplified products ranged between 220 bp and 3000 bp. Based on similarity matrix data, the value of similarity coefficient ranged from 0.73 to 0.87 with an average genetic similarity of 0.8. Dendrogram separated genotypes into two major clusters, one consisted of thermosensitive genotypes except S1(WH157) and other had all thermotolerant genotypes. Among all the 40 ISSR primers screened, primer ISSR-24 showed≈2200bp band which was present in all 7 thermosensitive genotypes but absent in all thermotolerant genotypes. Another differentiating band of ≈1750bp was observed with primer ISSR-29.This band was present in all thermotolerant genotypes and absent in all thermosensitive genotypes except S1. Primer ISSR-24 and ISSR-29 were found to be putative markers linked to thermotolernace in wheat crop and hence find application in wheat improvement program.ThesisItem Open Access Molecular characterization of poly hydroxy butyrate producing bacteria(CCSHAU, 2010) Shalini, Swati; Sikka, Virendra K.Polyhydroxybutyrates are the biopolymers and have a potential to be used as biodegradable plastics to replace synthetic plastics. The present investigation has been undertaken to isolate polyhydroxybutyrate producing bacteria and analyze the diversity among them related with sequence of PHB synthase gene. The soil and water sample for isolation of bacteria were collected from fourteen diverse sources. Out of hundred bacterial isolates forty were found PHB producing after initial fluorescence based screening on Nile blue A containing medium. By trying different combinations of carbon and nitrogen sources 2g glucose as carbon source and 0.65g potassium nitrate was found as most suitable for intracellular PHB accumulation. Six best PHB accumulating strains were selected and characterized for optimization of PHB production media. The selected strains were producing 59-29% PHB of dry weight of cell pellet. Different combinations of Agri-byproducts were tried as carbon source for cost effective PHB production and 3g molasses was found as best carbon source for PHB production (60% of cell dry weight).PCR amplification was performed by PHB synthase gene specific primers with genomic DNA of selected strains. Diverse pattern of amplification was observed which indicate towards diversity of phbC gene sequence. The gene specific band was eluted and cloned in TA sequencing vector.ThesisItem Open Access Development and molecular marker analysis of F1 and/or backcross plants from crosses between aerobic and high yielding rice cultivars(CCSHAU, 2010) Jogender Singh; Jain, R.K.The present investigation has been undertaken to: (i) carry out field evaluation of various Basmati (Pusa1121, HBC19 and Pusa Basmati 1460), indica (PAU201, HKR47) and aerobic (MAS-ARB25, MASARB868, MAS25, MAS26 and MAS109,) rice varieties for various agronomic traits under submerged field conditions, (ii) develop F1 and/or backcross plants from crosses between aerobic and high yielding rice cultivars, and (ii) assess the molecular status of F1 and parental rice genotypes for some markers linked with the root traits, abiotic stress tolerance and BAD2 aroma gene. Aerobic rice varieties had small panicle length and low yield per plant compared to Basmati and high yielding indica rice varieties when grown under submerged cultivations in the field. A DNA fingerprinting database of ten parental genotypes has been prepared using 29 SSR markers and a BAD2 aroma gene specific marker. High level of polymorphism was observed among the aerobic, Basmati and indica rice varieties. A total of 117 alleles were detected at 30 loci. Number of alleles per locus ranged from 2 to 6 with an average of 3.9 alleles per locus. The overall size of PCR products amplified range from 70-585 bp. The molecular size difference between the smallest and largest allele at a locus varied from 2 to 328. The polymorphism information content (PIC) values, which are indicative of genetic diversity, ranged from 0.180 to 0.820 with an overall average of 0.616 per locus. The NTSYS-PC UPGMA tree cluster as well as PCA analysis clearly grouped the 10 rice genotypes into two major groups one with three Basmati rice cultivars and other with aerobic as well as indica rice genotypes. Polymorphism for seven microsatellite markers (RM205, RM206, RM242, RM440, RM447, RM562 and RM7075) and BAD2 aroma gene specific (Bradbury et al. 2005) markers with a base difference of >30 bp in the amplified products, could be clearly visualized on 2.5% w/v agarose gels. The two F1 plants HBC19 x MAS26 and Pusa1121 x MAS25 showed lower levels of seed setting (8.0 and 20.6%, respectively) that too only in end of October and starting of November. No backcrossed seed was obtained. A total of 15 crosses were made between aerobic and high yielding indica /Basmati rice varieties; crossed seed were recovered from 11 crosses. Seven plants obtained from the HKR47 x MAS26 and MASARB25 x Pusa Basmati crosses were confirmed as F1 hybrids with the help of SSR RM242 and BAD2 aroma gene specific markers.ThesisItem Open Access To develop an efficient micropropagation protocol for Stevia rebaudiana(CCSHAU, 2010) Naveen Kumar; Chowdhury, V.K.The present investigation has been undertaken to: (i) study the effect of various concentrations of different growth regulators on in vitro shoot multiplication and growth in Stevia rebaudiana and (ii) to develop the micropropagation protocol for efficient and quick multiplication of Stevia rebaudiana. The shoot-tip and nodal segments were used as explants. Different sterilization protocols were tried for surface sterilization of the explants in Stevia rebaudiana. The nodal and shoottip explants were surface sterilized with 0.2% bavistin for 20 min and 70% alcohol treatment for 50 sec followed by 0.1% HgCl2 for 3 min which resulted in 100% sterilization of the explants. The nodal and shoot-tip explants were best regenerated on MS medium+BAP 2.0mg/l. The per cent explants regenerating shoots on nodal and shoot –tip explants were 90% and 50% respectively. The average no. of shoots was 2.00 and 3.2 from nodal and shoot-tip explants respectively. The primary proliferated cultures were transferred on to various MS media supplemented with different concentration and combination of growth regulators for shoot multiplication. The best shoot multiplication medium was MS medium supplemented with BAP (0.3mg/l) + Kinetin (0.3 mg/l), on which an average 50.20 number of shoots developed from nodal and shoot-tip explants after 40 days on culture medium. The other growth regulators viz. zeatin, TDZ and GA3 were also tried to check the effect on in vitro shoot multiplication at different interval of time but the response was not satisfactory. The multiple shoots were surgically separated and transferred on to various rooting media. The frequency of root regeneration was 100% on half MS medium containing IBA (0.5 mg/l) while the rooting frequency was 50.00% on half MS medium containing IAA (2.0 mg/l) and 42.86% on ½ MS+IAA (0.1 mg/l). Regenerated plantlets were shifted to pots containing sterile soil and sand mixture (3:1) for propagation. The survival percentage of plants after hardening and transfer to potted soil was 80%.ThesisItem Open Access SSR marker analysis of thermotolerant and sensitive genotypes of wheat (Triticum aestivum L. em. Thell)(CCSHAU, 2010) Pardeep Kumar; Dhillon, SantoshHexaploid bread wheat (Triticum aestivum L. em. Thell) is an important cereal food crop for majority of world’s population. SSR markers show high level of polymorphism even in species with narrow genetic base, such as wheat. High-temperature stress is one of the major constrains to wheat production worldwide. This study was undertaken with the objective to assess polymorphism among seven thermosensitive and seven thermotolerant genotypes of wheat. DNA extracted from young leaves of 14 wheat genotypes was amplified by using 45 SSR primers. Out of these primers, 37 showed amplification and were selected for further investigation. For SSR assays, data was analyzed to calculate various parameters such as the number of total bands, number of polymorphic bands, per cent polymorphism, bands per primer, polymorphic bands per primer, similarity matrices and dendrogram construction. The polymorphism percentage ranger from 33.3% to100%, giving an average percentage of polymorphism of 77.8%. The SSR primers yielded average 2.43 bands per primer. Overall size of PCR amplified products ranged between 95bp and 1120bp. Based on SSR similarity matrix data, the value of similarity coefficient ranged from 0.60 to 0.90 with an average genetic similarity of 0.76. At a similarity coefficient 0.751, two group were formed which separated all thermotolerant and thermosensitive genotypes in the dendrogram. Two and three dimensional PCA (Principle Component Analysis) showed similar clustering as evident from cluster tree analysis. Primer Xgwm 46 showed a ≈200bp band which was present in all the thermotolerant genotypes and absent in all thermosensitive genotypes. While WMC 170 produced three unique alleles of 215bp (WH 730 &WH 1021), 217bp (GW 173 & NIAW 34) and 220bp (RAJ 3765 & WH 1076) which were present in all the thermotolerant genotypes (except WH1022) but absent in all the thermosenstive genotypes. Xgwm46 and WMC 170 primers may probably be thermotolerance specific and may have potential for use in marker assisted selection programs for wheat production improvement.ThesisItem Open Access Development of interspecific hybrids between Indian mustard (Brassica juncea Coss. & czern) and white mustard (Brassica alba Moench) through embryo rescue(CCSHAU, 2010) Asha Rani; Yadav, R.C.Wide hybridization is an important method of incorporation of desirable character. The present study was undertaken to develop an interspecific hybrids between B. juncea cv. RH30, cv. RH8812 and B. alba to incorporate the Alternaria blight resistant character from B. alba to B. juncea. Ovules were excised from siliqua and cultured on seven different media supplemented with different growth regulators. In cv.RH30 X B. alba,the hybrid siliquae were 1.2 to 3 cm in length whereas in case of parent plants of cv. RH30, siliquae length was between 4.1 to 4.5 cm. In cv. RH8812 X B. alba, the hybrid siliquae ranged from 2.8 to 3.4 cm in length while in parent plants of cv. RH8812, the siliquae length was between 4.9 to 5.7 cm. In cross of RH30 X B. alba and RH8812 X B. alba, 48% and 39% pod setting was observed respectively. In case of cv. RH30 X B. alba, highest percent response for ovule germination (79.46%) was observed on MS medium supplemented with kinetin (2.5mg/l) and CH (0.5g/l). In case of Brassica juncea cv. RH8812 X B. alba, highest response (45.63%) of ovule germination was on MS + BAP (2.5 mg/l) + CH (0.5mg/l). Six rooting media were tried for root formation in regenerated shoots. Maximum rooting response was 100% on MS medium supplemented with IAA (0.5mg/l) in both the hybrids. The regenerated hybrid plants were transferred to a mixture of sand: soil in 1:1 ratio in pots and 80 percent hybrid plants survived in the soil.