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M. Sc. Dissertations

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20097
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Subject: Biotechnology and Molecular Biology × End date: 2009 × Start date: 2009 ×
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Now showing 1 - 7 of 7
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    ThesisItemOpen Access
    Micropropagation studies in elite cultivars of gerbera (Gerbera jamesonii Bolus)
    (CCSHAU, 2009) Walia, Priyanka; Chowdhury, V.K.
    The present study was undertaken to develop an efficient protocol for micropropagation in Gerbera (Gerbera jamesonii Bolus) cultivars Doni and Danna Ellen. Capitulum, basal cutting and shoot-tip were used as explants in both the cultivars. Capitilum explants were surface sterilized with 0.4% bavistin for 30 min followed by treatment of 0.05% streptocyclin for 5 min and 0.1% (citric acid + ascorbic acid) treatment for 5 min which was followed by 0.1% -viii- HgCl2 treatment for 2.5 min which resulted in 80% sterilization of explants in both the cultivars. Shoot-tip explants were treated with 0.1% citric acid + 0.1% ascorbic acid for 10 min and then surface sterilized with 0.4% bavistin for 15 min, 0.05% streptocyclin for 5 min and then explants were washed with 70% ethanol for 30 sec followed by treatment with 0.1% HgCl2 for 2 min which resulted in 75% sterilization of explants in both the cultivars. Different sterilization protocols were tried for surface sterilization of basal cutting explants in both the cultivars. But none of the sterilization protocol was found to be effective to avoid contamination of basal cutting explants in both the cultivars. Maximum number of shoot buds (32) per capitulum explant was obtained on MS basal medium supplemented with 5.0 mg/l BAP and 0.1 mg/l IAA in cv. Doni, followed by cv. Danna Ellen with 28 shoot buds per explant on the same medium. Shoot bud induction was not observed in both the cultivars on the medium containing 1.0 mg/l BAP and 0.1 mg/l IAA. The shoot buds regenerated from capitulum explants in both the cultivars were transferred on various media combinations for shoot regeneration. Maximum number of shoots (9.0) was found to be regenerated in both the cultivars per shoot bud clump cultured on MS basal medium containing 1.0 mg/l TDZ and 0.1 mg/l IAA. Maximum number of shoots (on an average of 3.6 shoots) per shoot-tip explant was obtained in cv. Doni followed by cv. Danna Ellen (3.0 shoots/explant) on the MS basal medium supplemented with 5.0 mg/l BAP and 0.1 mg/l IAA. Shoot regeneration was not observed from basal cutting explants in both the cultivars as contamination was not controlled inspite of trying different sterilization protocols. In vitro flowering was observed in both the cultivars when capitulum was used as explant. Rooting (80%) was observed from aseptically regenerated shoots from shoot-tip explants on ½ MS basal medium containing 0.5 mg/l IAA in both the cultivars.
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    ThesisItemOpen Access
    In vitro establishment of nitrogen fixing azotobacter chroococcum with brassica juncea L. cv RH-30
    (CCSHAU, 2009) Trivedi, Abhishek; Kharb, Pushpa
    Brassica juncea is an amphidiploid with B. nigra (L.) Koch (2n = 16) and B. rapa L. (2n = 20) as parents, It is also known as, Indian mustard or leaf mustard, During 2006-2007 mustard occupied 6.7 million hectares with an annual production of 74 million tonnes in India. B. juncea cultivation is mainly confined to Uttar Pradesh, Rajasthan, Madhya Pradesh, Haryana, Punjab, and Bihar. B.juncea is mainly used in the production of mustard oil. Mustard oil is one of the major edible oils in India. Azotobacter chroococcum is a gram negative, free living nitrogen fixing bacterium and is used as biofertilizer. Several properties of Azotobacter are beneficial for its associated plants like its -IVability to produce various growth regulators and siderophores which results in an overall increase in growth of its associated plants. In the present study, attempts were made to study the establishment of A. chroococcum with calli of B.juncea. Calli of B.juncea were raised using cotyledonary explants. Maximum percent induction of calli from cotyledonary explants was observed in MS medium supplementd with 0.2 mg/l BAP and 0.2 mg/l NAA. To study the establishment of A. chroococcum with callus of B.juncea. Broth of A. chroococcum containing 106 and 107 cfu/ml were used to make 5, 10 and 15ul of A. chroococcum inoculum which were then injected in calli of B.juncea. Calli of B.juncea injected with 10 and 15 μl of bacterial inoculum did not survive because of overgrowth of bacterial cells. Therefore further studies were conducted with 5 μl of bacterial inoculum for injection into calli. The injected calli; after incubation of 4 and 8 days, were surface sterilized and crushed to make a suspension in sterilized water which was plated on Jensen’s nitrogen free medium. Bacterial count was found to increase after 4 and 8 days in calli injected with 5μl on bacterial inoculum, which confirms the associationship A. chroococcum with callus of B.juncea Scanning electron micrograph also confirms the establishment of A. chroococcum with callus of B.juncea.
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    ThesisItemOpen Access
    Expression of myb gene in brassica tournefortii L.under drought stress
    (CCSHAU, 2009) Pardeep Kumar; Yadav, Neelam R.
    Brassica tournefortii, a highly drought tolerant Brassica species was used to study myb gene expression under drought stress. Seeds of Brassica tournefortii were grown on MS medium. Then 14 days seedlings were uprooted and given drought stress by subjecting them to air drying, PEG (-2 to -8 bar) and mannitol (100 mM to 400 mM) treatments. Total RNA isolation from stressed seedlings was carried out using Trizol reagent yielding 17.52-36.8 Fg/ml of total RNA. One- step RT-PCR was carried out using total RNA as template with BjMyb-1 primer designed from conserved domain of AtMyb2 and its homologous. BjActin primers were used in RT-PCR which served as control, as actin gene is constitutively expressed in all tissues. Exposure to drought stress for 15 minutes and 30 minutes (air drying) gave no amplification showing air drying upto 30 minutes does not induce any myb expression. An amplified product of 250 bp was obtained on exposure to drought stress for 60 minutes with air drying, PEG (-2 to -8 bar) and mannitol (100 mM to 400 mM) treatments. The transcript level was found similar in all treatments irrespective of drought treatments. cDNA was eluted out from the gel and purified cDNA was transformed using pDrive cloning vector (Quigen) in XL-blue strain of E.coli using blue- white selection. Transformed clones were characterized by plasmid DNA isolation, PCR amplification of plasmid DNA with gene specific primers. Plasmid DNA from transformed clones showed higher molecular weight than untransformed plasmid DNA on agarose gel electrophoresis confirmed the insertion of DNA fragment into the plasmid. PCR amplification of plasmid DNA also confirmed the successful cloning.
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    ThesisItemOpen Access
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    ThesisItemOpen Access
    Development of efficient protocols for plant regeneration and agrobacterium-mediated transformation in okra (Abelmoschus esculentus (L.) Moench
    (CCSHAU, 2009) Arora, Apoorva; Yadav, R.C.
    Present study was undertaken to evaluate the most suitable concentration of growth regulators for plant regeneration of okra cultivars Varsha Uphar and Hisar Unnat and to attempt to optimize conditions for Agrobacterium-mediated transformation. After optimization efforts were made to introduce cry 1A(c) gene of Bt in one of the okra cv. Varsha Uphar. Experiments were conducted to develop an efficient and reproducible plant regeneration system in two cultivars. Explants (hypocotyls, cotyledon and cotyledonary node) were excised from aseptically grown 5-15 days old okra (Abelmoschus esculentus) seedlings. No shoot regeneration was obtained from cotyledon and hypocotyl explants on any of the media combinations. Highest percentage (100%) of callus production was obtained on MS medium supplemented with Zeatin (0.5mg/l or 1.0mg/l) in both the cultivars from both the explants . Percent plant regeneration was achieved from cotyledonary node explants cultured on MS medium supplemented with NAA (2mg/l) and Kinetin (0.1 mg/l) in both the cultivars. The highest percent (98.89%) regeneration was in cv. Varsha Uphar. Agrobacterium mediated transformation conditions were optimized using EHA 105 strain carrying pTOK233 plasmid which had GUS as a reporter gene for both the cultivars of okra, Varha Uphar and Hisar Unnat. Different factors were worked out to optimize Agrobacterium mediated transformation viz. Inoculation time (5, 10 mins), co-cultivation period (48, 72 hours), bacterial concentration (OD600 2.0, 0.5) and pre-culture of explants (freshly cut, 3days). Best conditions were found to be 10 mins of inoculation time, 72 hours of co-cultivation, bacterial concentration with OD600 2.0 and freshly cut explants. Efforts were made to introduce Bt gene in one of the okra cultivars, Varsha Uphar.
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    ThesisItemOpen Access
    Analysis of diversity for micronutrient content and DNA fingerprinting in Greengram [Vigna radiata (L.) Wilczek]
    (2009) Jyoti; Yadav, Neelam
    Vigna radiata (L.) Wilczek, commonly called mungbean or greengram is the third most important pulse crop of India occupying nearly 3 million hectare. Mungbean provides inexpensive, low flatulence and easily digestible protein available in several edible forms that complements staple rice diet in Asia. India is the primary greengram producer and contributes about 75% of world production. 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 -IIvariability in available germplasm for improving micronutrient content in commercial cultivars. The present study was undertaken to study RAPD polymorphism among 16 mungbean genotypes using 33 decamer primers in PCR reaction. A total of 148 amplified bands were produced out of which 142 were polymorphic and 6 were monomorphic. For the genotypes studied, upto bands were produced with an average of 6.16 bands per primer. The size of amplified bands ranged from 150-3000 bp. Some unique bands were also produced, which could be used to distinguish these genotypes. The similarity coefficients between different genotypes ranged from 0.40-0.89 with an average similarity value of 0.65. At an arbitrary cut-off at 60 per cent similarity level on a dendrogram, the mungbean accessions were categorized into two major clusters. Satya and Asha were found to genetically similar. ML776 showed high iron and zinc content while Satya was poor in iron as well as zinc content. OPB7 primer showed 900 bp amplified product in ML776 that was found absent in Satya. These two genotypes can be used in developing population for linkage mapping. The results indicated that RAPD markers were efficient for identification of Vigna radiata genotypes and for determination of the genetic relationships among them.
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    ThesisItemOpen Access
    In vitro regeneration studies in garlic (Allium sativum L.) cvs. HG-17 and G-282
    (CCSHAU, 2009) Sakthivel, Pavitra; Kharb, Pushpa
    Garlic is used worldwide as a spice and a condiment. It has very high nutritive value and is a valuable foreign exchange earner for India. It is well known throughout history for its medicinal properties. It is sexually sterile, hence it is propagated vegetatively. During present investigation, in vitro regeneration in garlic cv. G-282 and HG-17 was studied. Basal part of a vertically cut clove was used as an explant. 2iP was used at 1, 1.5, 3 and 3.5 mg/l while TDZ was used along with 2iP at 0.1, 0.2 and 0.3 mg/l keeping concentration of 2iP constant at 0.1 mg/l. Highest percentage of multiple shoot formation in both G-282 (90.3%) and HG-17 (80.5%) was observed with 3 mg/l 2iP. The average number of shoots produced was highest with MS + 3 mg/l 2iP for both G-282 (8.9 shoots/ explant) and HG-17 (7.5 shoots/explant). IAA (1.5 mg/l) induced maximum percentage of roots for both G-282 (75.0%) and HG-17 (77.4%). Average number of roots was highest in MS + 1.5 mg/l IAA for G-282 (9.9 roots/shoot) and HG-17 (9.5 roots/shoot). 0.5 mg/l NAA + 0.5 mg/l IBA was found to be the ideal concentration for induction of callus using young leaf explants. MS basal medium containing 10% sucrose induced maximum bulblet formation for both G-282 (87.5%) and HG-17 (75.0%). 41.7% of the regenerated plants of the cultivar G-282 and 50.0% of the regenerated plants of the cultivar HG-17 survived under pot conditions.