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Master Degree Theses

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2011 - 20153
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    ThesisItemOpen Access
    Molecular Dissection of Phenylpropanoid Pathway During Ricinus communis - Fusarium oxysporum f.sp. ricini interaction
    (Biotechnology Department, N. M. college of Agriculture Navsari Agricultural University, 2011-10) Jadhav, Pritam Ramesh; Mahatma, M. K.
    Castor (Ricinus communis L.) is an important non-edible oilseed crop of India. The wilt caused by Fusarium oxysporum f.sp. ricini is the most serious disease of castor affecting its plant population and yield . Understanding resistance/ susceptibility in castor would facilitate the development of new control strategies and the identification of host factors required for resistance responses. Therefore, in the present investigation the plant pathogen interaction was studied in correlation with Phenylpropanoid pathway at molecular and biochemical level in the resistant and susceptible genotypes of castor at 0 h after infection (h.a.i), 24 , 48 and 0, 24, 48 and 72 h.a.i , respectively, in wilt pathogen infected and non infected genotypes. Phenol profiling using HPTLC showed the presence of three phenolic acids i.e. caffeic acid, ferulic acid , and salicylic acid in castor genotypes with and without infection at the interval of 0, 24, 48 and 72h. Higher caffeic acid and ferulic acid content in infected and non infected resistant genotypes at 0, 24 and 48 h.a.i was detected, whereas caffeic acid was not detected in susceptible genotypes at Oh. The phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), Cinnamate 4 hydroxylase (C4H) activities were studied in both resistant and susceptible varieties and higher activities of these enzymes were noted in resistant genotypes. The PAL activity was increased in all genotypes after 24h of infection but the increment of activity was more pronounced in resistant genotypes. Highest activities of enzymes PPO and C4H were recorded in resistant genotypes at 48 h.a.i. than the susceptible genotypes and the higher intensity of PPO-2 isoform was observed at this stage in infected resistant genotype that non infected, whereas it was absent in infected and non infected susceptible genotypes at this stage. PPO-2 isoform was present in infected and non infected resistant and susceptible genotype at 24h.a.i. Caumarate 3 hydroxylase (C3H) enzyme showed - higher constitutive (Oh) activity in wilt resistant genotypes of castor than the susceptible ones. Expression analysis was carried out using RT -PCR with PAL, C4H I and C4H2 gene specific primers. The expression of PAL gene and C4H-2 gene was appreciably higher in resistant genotypes at 48h interval than 24h interval compared to susceptible genotypes. The gene C4H-I was down regulated in susceptible genotypes after 24 and 48h infection while in resistant genotypes it was up regulated. Overall, resistant genotypes exhibited appreciably higher expression of PAL, C4H I and C4H2 genes, which are responsible for defense against wilt infection. These results suggest the resistance. cri tical role of phenols In castor disease
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    ThesisItemOpen Access
    In vitro regeneration and genetic transformation of finger millet (Eleucine coracana L.) genotype GN-4
    (Biotechnology Dept., N.M.College of Agriculture, Navsari Agricultural University, Navsari, 2015-11) Dabhi, Kirti A.; Jha, Sanjay
    Finger millet (Eleucine coracana L.) is an important cereal crop which constitutes about 81% of the minor millets produced in India. An efficient in vitro plant regeneration and Agrobacterium tumefaciens mediated genetic transformation protocol has been described for finger millet (Eleucine coracana L.) using callus as explants. Among different cytokinins used, kinetin (1.50 mg/l) in combination with BAP (0.50 mg/l) produced maximum number of shoots (11.00) and shoot length (9.8 cm). Maximum frequency of rooting and highest number of roots were produced on half strength MS basal with NAA (1.0 mg/l). Agrobacterium mediated genetic transformation, of finger millet genotype GN-4 was developed by using Agrobacterium Rs-AFP2 strain carrying marker gene hygromycine phosphotransferase II (hpt II) gene was used. The integration of the gene was confirmed by PCR. When the effect of the age of the callus and co-cultivation duration were evaluated, forty five days old co-cultivated callus for five days yielded 1.33% frequency of transformation. The morphological features of transgenic plants did not differ from those of non transgenic plants.
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    ThesisItemOpen Access
    ISOLATION AND CLONING OF PHYTASE GENE FROM DIVERSE SOURCES
    (PLANT MOLECULAR BIOLOGY & BIOTECHNOLOGY DEPT., N. M. COLLEGE OF AGRICULTURE, NAVSARI AGRICULTURAL UNIVERSITY, NAVSARI, 2014-12) Mogal, Chaitanya S.; Singh, Diwakar
    Phytase hydrolysis of (myo-inositolhexakisphosphate myo-inositol phosphohydrolase) hexakisphosphate (phytic acid) catalyzes to the inorganic monophosphate and lower myoinositol phosphates, and in some cases to free myo- inositol. Two bacterial strains, one fungal strain and one plant source were screened for phytate degradation viz. Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633), Aspergillus niger (ATCC 16888) and Glycine max (JS-335). Among these, B. subtilis (ATCC 6633) strain was found to produce maximum clearance zone of 2.7 cm on phytase screening medium containing sodium phytate as substrate, incubated at 370C for 48 hours. Phytase gene was isolated from B. subtilis (ATCC 6633), which was able to withstand temperature ranging from 400C to 500C, pH 4.0- 6.0 and showed maximum activity at 500C and 6.0 pH. However, phytase isolated from E.coli (ATCC 25922), A.niger (ATCC 16888) and Glycine max (JS- 335) were able to withstand temperature ranging from 37.50C to 450C only and at pH 3.5-5.0. A sequence of 904 bp characteristic of phy gene was obtained through PCR amplification. Sequencing of PCR amplified fragments and further analysis using NCBI-BLAST online homology search program revealed that phy gene of B.subtilis (ATCC 6633) and phy gene of Bacillus sp. YP1, complete genome and B. subtilis subsp. spizizenii strain YCJS phytase gene, showed expected (E) value 0.0. The phylogenetic analysis indicated that B. subtilis (ATCC 6633) had high similarities with the phytases phy gene of Bacillus sp. YP1, complete genome and B. subtilis subsp. spizizenii strain YCJS phytase gene, complete cds. It was also found that B. subtilis (ATCC 6633) had relative high similarities with Bacillus sp. BAB- 1 Phy gene, Bacillus subtilis HJ5 complete genome and Bacillus subtilis strain BC5 phytase gene, partial cds. Further 904 bp fragment was cloned in Ins T/A cloning vector and E. coli was successfully transformed and confirmed by colony PCR.