Loading...
Theses
Browse
7 results
Search Results
ThesisItem Open Access COMPARATIVE EVALUATION OF ACARICIDAL PROPERTIES OF Datura stramonium and Datura metel(AAU, Jorhat, 2020) GOSWAMI, MANISHA PRIYAM; Nath, TankeswarTicks are group of arthropod vectors that not only cause disease but also transmit diverse pathogens that affect human and animal health. Indiscriminating use of synthetic acaricides have led to development of acaricide resistance in ticks. Several plant secondary metabolites are known to exhibit toxic affect on insects. Phyto-extracts are also environmentally friendly and have thus become attractive alternative in insect management strategies. Datura has long since been used in folklore medicine and is reported to have other pharmaceutical uses. My research involved the use of two different species of Datura, viz., D. stramonium and D. metel that grow abundantly in the North East India especially, in Assam. The two species of Datura were collected from different locations of Jorhat district and evaluated for their acaricidal properties. Chemo-profiling of both the plant species revealed the presence of some phyto-metabolites with known biocidal properties, more specifically acaricidal properties. Saponin, Flavonoids, Tannins, Phlobatannins and Steroids were analyzed in both the species and revealed that except for Saponin all others metabolites were present in both the species of Datura. Atropine and Scopolamine earlier reported for their acaricidal activity were assayed using HPTLC technique. Shoots of D. stramonium showed the presence of Atropine (0.04238%) and Scopolamine (0.21554%) whereas; the presence of Scopolamine (0.00754%) was detected only in the roots. Presence of Atropine (0.08083%) only was detected in shoots of D. metel. The variation in Atropine as well as Scopolamine in roots and shoots of the same plant species might be due to variance in tissue specific concerned gene(s) expression, and among the different species might be due to genetic variation or variation in eco-geographical conditions which are yet to be explored. Acaricidal properties of both the plant species were assayed in terms of LC50 as well as Inhibition in Oviposition (IO%) against a tick line of Rhipicephalus microplus collected from Assam. Between the two plant species, D. metel showed lower LC50 (6.37%) than D. stramonium (12.27%). In case of IO also, more IO value (76%) was observed in D. metel than D. stramonium (35.27%). The lower LC50 and higher IO values indicate that D. metel has more acaricidal potential than D. stramonium. Findings from the study also showed that shoots of D. stramonium contains both Atropine (0.04238%) and Scopolamine (0.21554%) whereas, D. metel-shoots contain only Atropine (0.08083%) and it is almost double of what D. stramonium contains. The present study indicates that Atropine may be directly associated with acaricidal activity with little or no role of Scopolamine. Although, the study did not involve the analysis of other compounds, their involvement in acaricidal activity cannot be denied. This research can be extended further to explore specific metabolites having acaricidal properties present in other plant systems including Datura for the better and greater interest of development of plant based-based acaricides/insecticides.ThesisItem Open Access Molecular characterization of the gut microbes of greater wax moth (Galleria mellonella)(AAU, Jorhat, 2020-01) Saikia, Silpi Shikha; Borah, Basanta KumarGreater wax moth (GWM), Galleria mellonella (Lepidoptera: Pyralidae) is a notorious honey-bee pest found throughout the world. The moth is very destructive to the honeycomb, lays eggs and their larva grows up eating beewax. Beewax is a natural polymer, mainly composed of saturated/ unsaturated, linear/ complex monoesters, hydrocarbons etc. The most frequent hydrocarbon bond in beewax is the CH2-CH2 which is found in polyethylene (PE). Meanwhile, biodegradation of PE by gut microbes of Indian meal moth, Plodia interpunctella and lesser wax moth (a close species of GWM) was reported. As wax-digestion is not a common character of animals, we hypothesized to encounter some microbes in GWM-gut, which could also be responsible for the PE degradation. Therefore, we aimed this investigation to isolate and identify the gut-microbes from GWM following culture-dependant approach. We had characterized several bacterial and fungal species based on culture characteristics, Gram-staining and several biochemical tests such as carbohydrate utilization test, catalase test, citrate utilization test, MR-VP test, motility test etc. Using 16S-rDNA sequencing with conserved primer-sets from representative types, thirteen bacteria and one microalgae were obtained from the digestive tract of G. mellonella. These species includes Gram-positive Exiguobacterium aestuarii, Bacillus circulans, Microbacterium zaea, Microbacterium paraoxydans, Enterococcus faecalis and Gram-negative Agrobacterium sp., Sphingomonas pseudosanguinis, Sphingobium yanoikuyae, Acinetobacter radioresistens, as well as a microalgae (Picochlorum oklahomensis). Several of these species/ isolates have been reported to degrade polycyclic aromatic hydrocarbon, low density polyethylene, 2-methylphenanthrene etc. Interestingly, P. oklahomensis is a marine microalgae; it steals genes from bacteria and adapt themselves to abiotic stress. Further investigation will be required to find out more precise details about plastic degrading candidate microbes and their biotechnological applications.ThesisItem Open Access VALIDATION OF DROUGHT RESPONSIVE miRNA IN A DROUGHT TOLERANT RICE CULTIVAR(AAU, Jorhat, 2020-10) Debsarma, Oindrila; Sen, PriyabrataDrought like situation in non-irrigated agriculture system often limits rice production, necessitating introduction of drought tolerance trait into the cultivar of interest. In Assam, effect of dry spells on varieties cultivated on low lands was reported to be reduced up to 43.07%. miRNAs, a class of abundant small noncoding RNAs, have been identified as important regulators of gene expression in both plants and animals and are involved in many aspects of plant development, including the modulation of plant response to stress. Despite technical hurdles to miRNA functional analysis, there is a growing body of evidence that alteration of miRNA accumulation plays an important role in reprogramming plant responses to biotic and abiotic stresses. Assam having a diverse germplasm of rice, might have evolved an unique response to various stress. ARC-10372, a proven drought tolerant landrace may be a good source of drought related miRNAs and can provide insight into the role of miRNA in drought stress. In a previous effort in the Dept. to identify novel variety specific miRNA, small RNA sequencing had been employed to systematically investigate the tissue specific miRNAs responsible to drought stress, which are understudied in rice. The study revealed few novel miRNA in addition to known under drought stress. Differentially expressed miRNA as revealed by sequencing data are confirmed experimentally under drought situation using qRT-PCR. Out of 31 miRNAs, 7 known and 5 novel miRNAs were found to respond to drought stress. The identified target for these miRNAs revealed several conserved miRNAs targeting transcription factors like homeodomain-leucine zipper, MADS box family protein, zinc finger protein and Myb, well known for their importance in drought tolerance in plants. Drastic decrease in abundance of a few of the members of the novel miRNA ( nmiR4, nmiR25, nmiR32, nmiR84 & nmiR86) might suggests important role of these miRNAs in drought tolerance. Further work on revealing its confirmatory role shall yield more information in this regard.ThesisItem Open Access IDENTIFICATION AND VALIDATION OF DROUGHT-RESPONSIVE GENES IN THE UPLAND RICE CULTIVAR ‘BANGLAMI(AAU, Jorhat, 2020-10) Borah, Akankshita; Modi, M.K.Drought is one of the most important and highly unpredictable abiotic stresses causing drastic reductions in yield under rainfed rice environments, affecting 20% of the total rice-growing area in Asia (Pandey and Bhandari, 2008). Assam has diverse germplasm of rice which includes Banglami, a traditional drought-tolerant Ahu rice cultivar found locally in the state. Since 2013, crossing has been carried out between this drought-tolerant cultivar and Ranjit, a high yielding but drought-susceptible elite cultivar. Many drought-related QTLs were identified and reported from this cross-population. For the current study, twenty QTLs identified in the F4 population were selected for mapping on the parental genome. The results revealed that 17 of the 20 QTLs were present on the parent genomes where 11 of the 20 QTLs (qEBT3.1, qEBT6.2, qNOT6.1, qNOT2.1, qPL1.1, qPL1.2, qPL9.1, qPL9.2, qPL1.1, qNOG9.1, qNOG12.1) were found common to both the cultivars. From these, two of the QTLs (qGY1.1, qGY7.1) were found to be exclusively contributed by Banglami while four of the QTLs (qRLWC9.1, qRLWC9.2, qPH7.1, qDTF12.2) were found to be contributed by Ranjit to the cross-population. Genes associated with the QTLs were identified from whole-genome sequence data of both the cultivars. In total, 2454 genes were located in the 13 QTLs found in Banglami while 1585 genes were found in Ranjit in the 15 QTLs. The differential expression of the candidate genes in water-stress and irrigated conditions were calculated with the help of available RNA-seq data. Differential gene expression was calculated for each of the candidate genes under drought and control. Fifty DEGs in Banglami and seventy-two DEGs in Ranjit having a Log2 Fold change greater than ± 2.5 up to ± infinity were considered significant. Few of the differentially expressed genes having a significant Log2Fold change as revealed by the sequencing data, were confirmed experimentally under drought situation using qRT-PCR. Stress-responsive candidate genes involved in nitrate uptake, DNA repair, ubiquitination, enzymatic activities, cell cycle activities, etc. which can play a significant role in conferring drought tolerance were identified in the drought-related QTLs. The expression pattern of the candidate genes in qRT-PCR was found similar to transcriptome studies, signifying important role of the candidate genes in drought tolerance mechanism in rice.ThesisItem Open Access GENOMIC STUDIES FOR AROMA IN JOHA RICE OF ASSAM(AAU, Jorhat, 2020-09) DAS, KINGSUK; Baruah, A. R.The badh2 locus for aroma in Basmati and Jasmine rice has been extensively studied and the present study was intended to target the functional marker for aroma, badh2 to determine the fidelity of the marker and validate the same to group the joha rice of Assam in particular. A total of 90 small-grained rice cultivars including 60 joha rice was included in the study. All the fieldwork and laboratory work were carried out at the ICR farm and in the Department of Agricultural Biotechnology, AAU, respectively. The study revealed that the majority of the joha cultivars (44) and 22 non-joha types including four chakhow, three jeera rice were detected with the aroma allele for badh2. The cultivars could be grouped into three based on the results of marker profile using the primer pairs for badh2 (Group I: cultivars possessing aromatic badh2 allele, Group II: cultivars with non-aromatic badh2 allele and Group III: Heterozygous for badh2). The cultivars were genotyped further using 23 markers linked to aroma (other than badh2), and those loci (mostly minor) were mapped previously flanking the regions of badh2. The markers used in the study detected 43.47% polymorphism, and only those polymorphic marker loci were analysed for allelic composition among the three groups of cultivars. The number of marker alleles ranged from 2-4 with a mean of 3.11. Except for 10L03, RM223, RM282, the rest of the markers showed higher homozygosity, indicating that high resolution of allelic difference among the groups of cultivars would be possible. The cultivars of Group I, i.e., with aromatic badh2 are distinctly unique from the other two groups, suggesting the allelic composition within the Group I was intact (share the same allele most of the time) and could be different from Groups II and III at many times. The Group I was detected a significantly higher number of expected alleles as compared to the other two groups except for RM 282, indicating the determinant of aroma might be governed by badh2 along with some other minor loci in the case of Group I. The present study could throw some light that in joha rice cultivars, the involvement of badh2 along with few other minor loci is mainly responsible for the aroma.ThesisItem Open Access GENETIC DIVERSITY ANALYSIS OF MILD INSECT RESISTANT WILD & CULTIVATED CHICKPEA (Cicer arietinum L.) GENOTYPES(AAU, Jorhat, 2020-10) Mahanta, Ankur; Sarmah, Bidyut KumarChickpea (Cicer arietinum L.) is one of the most important pulse crop grown all over the world and India is the major producer. Despite being the largest producer, India still imports chickpea from other countries because of its low productivity. In Assam, the chickpea growers suffer as the cultivars available in the state are susceptible to various insect pests which appear to be the most important reasons for low productivity. In order to enhance the genetic potential of cultivated chickpea one has to assess the extent and the pattern of real diversity available in the existing cultivated and wild accessions (Croser and Ahmad et al. 2003). In the present study, we have characterized 18 chickpea genotypes belonging to C. arietinum, C. reticulatum & C. judaicum, based on morphological traits and molecular data of 48 SSR markers to analyze their genetic diversity and phylogenetic relationship. Morphological diversity analysis showed a significant variation for seed yield/plant, growth habit and flower colour. Considerable diversity was recorded from the SSR data analysis with a polymorphic information content ranging from 0.12 to 0.47 with a mean of 0.31. Most of the diversity was confined to the C. judaicum genotypes with respect to its average Jaccard’s similarity coefficient (0.246) and average Euclidean distance (9.098). The dendrogram generated for both morphological & SSR data reflects similarities between the cultivated and C. reticulatum genotypes while genotypes of C.judaicum were diverse and distinctly unique from the other genotypes. A better understanding of the diversity and relationships within and among the genotypes will contribute to identification and utilization in breeding program to widen the genetic base of this cultivated species, for the development of elite lines with superior yield and improved adaptation to diverse environments.ThesisItem Open Access DEVELOPMENT OF CAO-1 MUTANT IN RICE USING CRISPR/Cpf1 TECHNOLOGY(AAU, Jorhat, 2020-10) Nayak, Suprava Priyadarsini; Singh, SalvinderCRISPR (Clustered Regularly Interspaced Short Pallindromic Repeats) is originally a bacterial immune system against virus based on RNA guided bacterial defence mechanisms designed to recognize and eliminate foreign DNA of invading bacteriophage and plasmid. Although the most successful genome editing tool used at present is CRISPR/Cas9 (Jaganathan et al., 2018), the newly developed CRISPR/cpf1 (CRISPR from Prevotella and Francisella 1) is a single RNA guided endonuclease of a class-II CRISPR-Cas system that has several advantages over CRISPR/Cas9 (Zetsche et al., 2015). Cpf1 needs single guiding RNA (sgRNA) while Cas9 requires two guide RNAs (crRNA and tracrRNA). While Cas9 make a blunt end cut of the DNA molecule after recognizing a PAM sequence NGG (Moon et al., 2018), the Cpf1 endonuclease makes a staggered cut of the DNA after recognizing a PAM sequence viz. NTTT, offering researchers more option when selecting an editing site. The current study employed CRISPR/Cpf1 construct pSS09 harbouring sgRNA of CAO1 gene of rice that was developed at Prof. Okita’s Lab, Institute of Biological Chemistry, Washington State University, Pullman, USA. The main objective of the present study was to validate the efficacy of CRISPR/Cpf1 system in rice. Chlorophyllide-a-oxygenase (CAO1) is an enzyme that converts chlorophyll a to chlorophyll b. Knockout of the gene using CRISPR technology is expected to produce “pale yellow” leaf phenotype. We introduced CRISPR/Cpf1 binary vector harbouring sgRNA of CAO1 gene into Japonica rice cv. Kitaake through Agrobacterium genetic transformation. Embryogenic calli were induced from mature seed with efficiency of 96.3% and regeneration efficiency of embryogenic calli was observed as 63.3%. 43 putative transformed lines were generated in the current study. Out of 43 transformed lines, 20 lines were selected based on varied level of pale green colour of the leaves and subjected to PCR analysis using gene specific primers for the targeted sequence of CAO1 gene. Transformed lines number 8, 9 and 13 showed mutations in the CAO1 gene with 9 bp, 12 bp and 25 bp deletions, respectively. We observed fairly high efficiency of mutation rate (2.22%) with CRISPR/Cpf1 system and the mutations occurred at very near the targeted sgRNA sequence of CAO1 gene.
