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Assam Agricultural University, Jorhat
Assam Agricultural University is the first institution of its kind in the whole of North-Eastern Region of India. The main goal of this institution is to produce globally competitive human resources in farm sectorand to carry out research in both conventional and frontier areas for production optimization as well as to disseminate the generated technologies as public good for benefitting the food growers/produces and traders involved in the sector while emphasizing on sustainability, equity and overall food security at household level.
Genesis of AAU -
The embryo of the agricultural research in the state of Assam was formed as early as 1897 with the establishment of the Upper Shillong Experimental Farm (now in Meghalaya) just after about a decade of creation of the agricultural department in 1882. However, the seeds of agricultural research in today’s Assam were sown in the dawn of the twentieth century with the establishment of two Rice Experimental Stations, one at Karimganj in Barak valley in 1913 and the other at Titabor in Brahmaputra valley in 1923. Subsequent to these research stations, a number of research stations were established to conduct research on important crops, more specifically, jute, pulses, oilseeds etc. The Assam Agricultural University was established on April 1, 1969 under The Assam Agricultural University Act, 1968’ with the mandate of imparting farm education, conduct research in agriculture and allied sciences and to effectively disseminate technologies so generated. Before establishment of the University, there were altogether 17 research schemes/projects in the state under the Department of Agriculture. By July 1973, all the research projects and 10 experimental farms were transferred by the Government of Assam to the AAU which already inherited the College of Agriculture and its farm at Barbheta, Jorhat and College of Veterinary Sciences at Khanapara, Guwahati.
Subsequently, College of Community Science at Jorhat (1969), College of Fisheries at Raha (1988), Biswanath College of Agriculture at Biswanath Chariali (1988) and Lakhimpur College of Veterinary Science at Joyhing, North Lakhimpur (1988) were established. Presently, the University has three more colleges under its jurisdiction, viz., Sarat Chandra Singha College of Agriculture, Chapar, College of Horticulture, Nalbari & College of Sericulture, Titabar. Similarly, few more regional research stations at Shillongani, Diphu, Gossaigaon, Lakhimpur; and commodity research stations at Kahikuchi, Buralikson, Tinsukia, Kharua, Burnihat and Mandira were added to generate location and crop specific agricultural production packages.
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ThesisItem Open Access A study on the role of exopolysaccharide in conferring acid tolerance in Bacillus sp.(AAU, Jorhat, 2018-01) Deka, Priyadarshini; Barooah, MadhumitaSoil bacteria have evolved various mechanisms to adapt to stress environmental conditions such as temperature, salinity, drought and low pH condition of soil. Among the several environmental stress conditions, soil acidity an important factor influencing physicochemical and biological properties of soil along with microbial diversity and crop production is an emerging issue of immense concern due to its wide spread distribution across the globe. Although low soil pH restricts the number and diversity of bacteria, it is known that some soil bacteria are able to thrive in such conditions having evolved various mechanisms including production of biofilm to circumvent acid stress. Bacterial exopolysaccharide (EPS) are high-molecular-weight complex polymers composed of sugar moieties that form the main component of the biofilm which aid the bacteria to colonize substrata. In the present study, a total of 28 isolates were identified and characterized as acid tolerant EPS producing bacteria among which, B. amyloliquefaciens p16 produce the highest EPS (219.96 μg/ml). A culture medium containing sucrose (3.5%) as carbon source with pH 5.0 and incubated for 24 hrs was optimal for maximum production of EPS. The HPLC analysis of monomeric units of EPS produced by B. amyloliquefaciens p16 revealed the abundance of galactose at pH 7.0 which however, changed to arabinose when shifted to acidic condition (pH to 5.0 and 4.5). The isolate B. amyloliquefaciens p16, significantly improved soil physical properties in terms of greater soil aggregation (80.59 mm diameter aggregates) and water holding capacity (53.90%) when inoculated into soil over the control (31 mm diameter aggregates and 18.21%, respectively). The differential expression of epsA and epsB, the first two genes of the eps operon showed a 7 and 9 fold increased expression in pH 5.0 compared to pH 7.0 respectively. Disruption of the epsB gene in B. amyloliquefaciens p16 using integration vector pMUTIN4 generated mutants that produced significantly lesser EPS (33.23 μg/ml) when compared to the WT (223.87 μg/ml). The generated mutant of B. amyloliquefaciens p16 lacking the wrinkled morphology had an extended lag phase of 24 hrs and was barely able to survive in acidic medium (pH 4.5) unlike that of the WT type. Soil inoculated with generated mutants formed smaller soil aggregates (42.41±1.70 mm) and had decreased water holding capacity (27.67±1.94%) compared to the WT (80.59± 0.22 mm and 53.90± 1.66%, respectively). This study indicates that EPS secreted by acid tolerant bacteria (B. amyloliquefaciens p16) imparts acid tolerance and also aids in improving the soil physical structure through increased soil aggregation and water holding capacity.ThesisItem Open Access MAPPING AND DISSECTION OF GENETIC EFFECTS INTO QTLs FOR GRAIN YIELD UNDER DROUGHT IN ELITE RICE VARIETY OF ASSAM(AAU, Jorhat, 2017-01) VERMA, RAHUL KUMAR; Modi, M. K.Two traditional drought tolerant cultivars were crossed with Ranjit in the present study in order to identify QTLs and improved lines for various yield traits under drought stress. The allelic distribution of 45 SSR markers associated with major grain yield QTLs under drought stress were studied in 115 ahu rice cultivars. The cluster analysis distinguished cultivars into 2 major clusters. The drought tolerant cultivars (ARC10372 and Banglami) were grouped with tolerant check (Nagina22) and high yielding cultivars were grouped with susceptible check (Ranjit). A total of 110 polymorphic SSR markers were identified across the parents, Banglami and Ranjit. These SSR markers were used for segregation analysis in 180 F2 plants. Among 110 polymorphic SSR markers, 88 fitted the expected Mendelian segregation, whereas 22 (20.0%) significantly deviated from it (P<0.01). Only 89 SSR markers could be assigned to 12 linkage groups covering a total of 1628.7 cM of the rice genome. The F2:4 population consisting of 2460 F4 plants were evaluated for various yield traits under two hydrological conditions viz., irrigated control (non stress) and drought stress created in rainout shelter. The drought stress was imposed from panicle initiation to panicle emergence period (reproductive stage). A total of seven QTLs, viz., qNOT2.1 (number of tillers), qEBT6.2 (effective booting tillers), qPNL1.1, qPNL6.1 (panicle length), qNGP12.1 (number of grains per panicle), qNCP6.1 (number of chaff per panicle) and qGYP7.1 (grain yield), were identified under drought stress whereas, four QTLs viz., qEBT6.1 (effective booting tillers), qNCP4.1 (number of chaff per panicle), qSFP6.1 (spikelet fertility) and qGYP9.1 (grain yield), were identified under irrigated conditions. The cultivar ARC10372 was crossed with Ranjit and 180 F2 plants were raised. Among these, only 36 F2 plants carrying tolerant parent allele for the SSR markers RM431 and RM11943 associated with the major grain yield QTL (qDTY1.1) were selfed to raise F2:3 population. Only 10 F3 plants were selected on the basis of yield and root traits. These were selfed to raise F4 population. The F2:4 population consisting of 217 F4 plants were evaluated for various yield traits under drought stress and irrigated conditions. Two F2:4 lines (B-42, B-106) from ‘Banglami x Ranjit’ and one F2:4 line (A-78) from ‘ARC10372 x Ranjit’ were selected on the basis of improved yield and grain quality traits under drought stress. The selected plants may be used for the development of drought tolerant rice variety.ThesisItem Open Access STUDY ON MOLECULAR MECHANISM OF BRUCHID BEETLE (Callosobruchus chinensis) RESISTANCE IN BLACK GRAM (Vigna mungo L.)(AAU, Jorhat, 2017-01) Kakati, Indrani; Sarmah, Bidyut KumarBruchid beetles, (Callosobruchus spp.,) are a devastating stored grain pest of black gram. The interaction between bruchid and black gram genotype has not yet been demonstrated. In the present investigation, an attempt has been made to understand the molecular basis of resistance in a mild tolerant genotype (IC8219) of black gram through two molecular techniques, Suppression Subtractive Hybridization (SSH) library preparation and RNA Sequencing (RNASeq). This is the first report on elucidation of the molecular basis of defense during black gram-bruchid interaction. The changes related to generation of ROS and expression of defense related genes in a mild tolerant (IC8219 genotype) of black gram was studied after releasing bruchids. After 7 days of releasing bruchids, RNA from developing seeds of the pods oviposited by bruchids were collected. The generation of ROS was detected by using 3, 3’ diaminobenzidine (DAB) staining on the pods oviposited by bruchids. For suppression subtractive hybridization (SSH) the pods oviposited by bruchids were used as tester population, while RNA from seeds of control plants were used as driver population. A forward subtractive cDNA library was prepared from tester and driver population and subtracted cDNAs were cloned and transformed in JM109 competent cells. In all, 277 clones in an EST library were sequenced and analyzed. High quality ESTs (244) were submitted to the NCBI database (Acc. No. JZ917400-JZ917463). Based on CAP3 assembly, 134 genes were computationally annotated. The majority of the ESTs belonged to ‘Biological Process’ of gene ontology category. A total of 18 defense related genes were identified and were subjected to quantitative PCR analysis (qPCR). Of these 12 genes showed up-regulation in developing seeds. Few major defense genes like defensin, pathogenesis related protein (PR), lipoxygenase (LOX) showed high expression in the oviposited plants when compared with the non-oviposited plants. In order to obtain a greater representation of defense genes, de novo transcriptome assembly of RNA extracted from the pods oviposited by bruchids was also performed. RNASeq analysis revealed a total of 12,081 differentially expressed genes (DEGs) out of which 258 were up-regulated and 310 were down-regulated during black gram- bruchid interaction. In all, 20 defense related genes were identified of which 9 were represented both in the SSH library and RNASeq data. This is the first report on defense related gene expression study in developing seeds of black gram during upon egg laying by bruchid beetles.ThesisItem Open Access OPTIMISATION OF ETHNIC FERMENTED RICE BEER (XAJ) PRODUCTION OF ASSAM, INDIA(AAU, Jorhat, 2017-07) Keot, Jyotshna; Barooah, M.“Xaj”, the popular rice based alcoholic beverage is produced by the Ahom community of Assam using fermentation starter, Xaj pitha. The methodology of Xaj brewing is almost similar among the Ahom community residing in different localities of Assam, however, the concoction of the fermentation starters differ in terms of varieties and number of herbs or plant parts resulting in variation of the quality and acceptability of the final product. Fermentation starters are mixed cultures of fungi, yeasts and bacteria that are maintained on substrates like rice powder, supplemented with various herbs. Both fungi and yeasts present in the starter cultures are involved in rice based alcoholic beverage fermentation. Fungi are primarily involved in the initial liquefaction and saccharification process that breaks down the rice starch to fermentable sugars that is subsequently converted into alcohol by the yeasts. Based on traditional starter manufacturing method, defined fermentation starter culture was developed with the compatible microbial cultures consisting of fungus Amylomyces rouxii (NCBI accession number KP790015) Wickerhomomyces anomalus (NCBI accession number KX904346 ) and yeasts Saccharomyces cerevisiae (NCBI accession number KX904349) isolated from collected Xaj pitha samples. Selected plant extracts of Leucas aspera Spreng. Lygodium flexosum Vent. Polygonum strigosum L., Centella asiatica Urban and Alstonia scholaris (L.) R. Br. was added as representative herb for standard production of rice based alcoholic beverage based on their use value and frequency of use. Performance of defined starter culture in alcohol production and other biochemical properties of alcoholic beverage produced by defied starter culture were compared with that of Xaj produced using traditional starter culture, Xaj pitha. The rice based alcoholic beverage brewed using defined starter culture contained 10.23% (v/v) alcohol with 23.99% protein, 61.79% antioxidant activity, 0.48% acidity and a pH value of 3.79 after one month of fermentation whereas, the traditional starter culture was able to produce 12.3% alcohol, 22.27% protein, 60.23% antioxidant activity, 0.789% acidity and pH value of 3.33 after one month of fermentation. Fermentation was stabilised in the laboratory prepared rice based alcoholic beverage through the addition of sulphur dioxide (20ppm/L) and turbidity of standard alcoholic beverage was removed by filtration techniques that could be stored for 3 months without any major changes in the physical and chemical properties or taste. It also scored higher in Hedonic sensory attributes such as acidity, colour and overall acceptability. This study indicates the potential of defined starter to produce high quality standard Xaj pani which will pave the way to produce the product in commercial scale the near future.ThesisItem Open Access Macrofungal Diversity of North East India and Development of Nanoparticle Based Detection of Mushroom Toxin(AAU, Jorhat, 2017-11) Parveen, Assma; Barooah, MadhumitaMushrooms have been an important part of the diet for the people worldwide due to its nutritious and delectable taste since ancient times. The ethnic communities of the Northeast India have extensive traditional knowledge of the edible mushrooms which they forage from the wilds. Unfortunately, increasing population pressure and consumer demand for exotic mushrooms have led to indiscriminate collection and sale of unidentified varieties leading to lethal cases on several occasion. Development of diagnostic kits to detect toxins present in wild mushrooms in situ for prevention and detection of mushroom poisoning is therefore very important and can aid in rapid detection of the toxins in affected patients for early treatment. A systematic collection of mushrooms and their detailed study of diversity and distribution of wild mushrooms in the state of Assam based on phenotypic and molecular characteristics led to a collection of 44 samples out of which, 17 mushrooms species were found predominantly during summer season, 11 species during autumn, nine species during monsoon, three species in winter and eight species during spring season. Soil was preferred habitat followed by tree/hardwood tree. Molecular characterization based on rDNA-ITS sequences revealed 16 macrofungal families. Out of the 44 samples, 23 samples were reported to be edible and for the other 21 non-edible strains, five strains had medicinal properties, six strains were earlier reported poisonous, two had industrial application while the properties of the rest are yet to be ascertained. Phallacidin, a bicyclic heptapeptide of the phallotoxin family is highly hepatotoxic and found in many of the poisonous mushrooms. This study generated antibodies against the Phallacidin (PCN) toxin present in poisonous mushrooms using Phallacidin-BSA conjugate in New Zealand white rabbit. The antibodies showed high sensitivity and detection limit of 11.89 ng/mL for phallacidin and 8.6 ng/mL for α-Amanitin. The detection limits with reduced assay time for these two toxins were further improved to 10.87 ng/mL (Phallacidin) and 11.09 ng/mL (α-Amanitin) through generation of HRP-PCN conjugate. A lateral-flow-based dipstick immunoassay format using antibody-gold conjugate for rapid field screening of Phallacidin with a detection limit of 25 ng/mL was further developed. The present study reports development of three methods viz. ELISA, HRP-PCN and lateral flow immunoassay for detection of Phallacidin & α-Amanitin. This is the first report of development of immunoassay to detect Phallacidin toxin. The immunoassays developed through this study can be convenient quantitative tool for screening of toxin in wild mushrooms.ThesisItem Open Access Study and characterization of the DNA virus diseases in few important crops of Assam(AAU, Jorhat, 2018-01) Baldodiya, Gajendra Mohan; DebNath, PalashPlant viruses cause many devastating diseases and are responsible for the huge loss in crop production and quality. The Infected plants may show a range of symptoms like leaf yellowing, leaf distortion, growth abnormalities and yield reduction. Although the majority of the plant viruses are RNA viruses, in the recent years, DNA viruses have emerged as a serious problem for vegetables and fiber crops, more specifically in tropical and subtropical regions of the world. In India, Begomoviruses are known for major yield loss in a variety of crops. So far, very few reports on DNA virus diseases are available from the northeastern region, including Assam. In the present study, three important single-stranded DNA viruses, banana bunchy top virus (BBTV), squash leaf curl China virus (SLCCNV) and tobacco curly shoot virus (TbCSV) were identified from Assam infecting banana, pumpkin, and chilli crops respectively. The crop cultivating areas were surveyed and 89 plant samples showing symptoms of geminivirus and BBTV infections were collected from 16 different crops. The broad-spectrum antibody combination, tomato yellow leaf curl virus (TYLCV) in DAS-ELISA (double antibody sandwich ELISA) assay revealed the all possible Begomovirus infections. The molecular detection method by using universal and gene-specific primers confirmed the presence of viral genome in positively screen samples through DAS-ELISA. The full-length genome amplification of detected viruses was achieved by designing walking primers from both ends of the partial genome and rolling circle amplification (RCA) followed by cloning process. The in silico analysis (genomic feature analysis, multiple sequence alignment and phylogenetic grouping) revealed the molecular properties of identified viruses. The BBTV-As-JOR was found closely related with BBTV-Lucknow isolate, although it stands out for few genomic features. Each genomic segment of the BBTV-As-JOR isolate was comprised of an ORF in sense orientation, encoding specific protein like other BBTV isolates. The common region major (CR-M) of DNA-R shows 26nt deletion like BBTV Lucknow but deletion of 36nt in DNA-U3 was found different. The BBTV-As-JOR clustered with PIO (Pacific Indian Ocean) isolates in the phylogenetic analysis based on the R-segment. Similarly, the clustering of other BBTV segments was also found with previously reported BBTV isolates of PIO group. The genome of SLCCNV-As-JOR-G8 pumpkin shared high nucleotide sequences and proteins similarity with SLCCNV-pumpkin-Varanasi DNA-A. The phylogenetic clustering also conferred these findings. The sequence analysis confirmed the presence of seven ORFs viz, AC1, AC2, AC3, AC4, AC5, AV1/CP and AV2 in SLCCNV-As-JOR-G8 pumpkin. The ORFs showed the typical begomoviral genome organization; however, they displayed similarity to different begomoviruses. The TbCSV-As was identified as a close relative of TbCSV)-[SC118] sharing the similarity of two major proteins, AL2 and AL3. The occurrence of TbCSV in chilli (Capsicum frutescens) host plant and causing upward curling of leaves is the first report from India in our knowledge. This study presents the first report of identification and characterization of BBTV, SLCCNV, and TbCSV from Assam which may open up a new insight into the understanding of DNA viruses infecting important crops of the state.ThesisItem Open Access IDENTIFICATION, ISOLATION AND CHARACTERIZATION OF FLOWER AND POD WALL SPECIFIC PROMOTER FROM CHICKPEA FOR TISSUE SPECIFIC EXPRESSION OF TRANSGENE(AAU, Jorhat, 2017-07) Vasantrao, Jagadale Mahesh; Sarmah, Bidyut KumarThe transgene expression is, in part, a function of the promoter to which the coding region is fused. Constitutive over-expression of transgene occasionally interferes with normal growth and developmental processes in plants. Tissue-specific promoter can regulate transgene expression in a particular organ and developmental stage. In the present investigation, an attempt was made to isolate and characterize a flower and pod wall specific promoter from chickpea. The aim was to use the promoter to drive Bacillus thuringiensis (Bt) Cry gene in these organs of chickpea for enhanced resistance to a key pest, Helicoverpa armigera. For isolation of flower and pod wall specific promoter, a forward Suppression Subtractive hybridization (SSH) library was prepared using flower and pod wall (tester) and leaves (driver). Subtracted cDNAs were amplified, cloned and transformed into E. coli competent cells. In all, 226 clones of SSH library were sequenced and analyzed. After removing adaptors, vector sequences (<100bp) and low quality sequences, 179 high quality ESTs sequences were deposited in the NCBI GenBank database under the Accession numbers JZ923200-JZ923378. Based on CAP3 assembly of 179 ESTS, 126 genes comprised of 97 singletons and 29 contigs were computationally annotated. The mapping of 88.26% ESTs (158 out of 179 ESTs) was done based on Gene Ontology (GO) annotation, which distributed 751 GO terms into three categories: cellular location, molecular function and biological process. Within the biological process category, the 158 ESTs were classified into seven primary functional categories, including metabolic process (113; 28%), cellular process (103; 26%), single organism process (77; 19%), biological regulation (38; 9%), regulation of biological process (33; 8%), response to stimulus (22; 6%) and cellular component organization or biogenesis (17; 4%). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was also performed to understand functions and utilities of these ESTs in the biological system and a total of 45 ESTs involved in 49 different biological pathways were identified. Moreover, 67 ESTs were also identified encoding four different classes of enzymes such as oxidoreductases (29), transferase (20), hydrolases (16) and isomerese (2). To identify the genes exclusively expressed in the flower and pod wall, the 179 EST sequences of the pod wall were searched using BLASTN of the Chickpea Transcriptome Database (CTDB) database to obtain CTBD ID. The CTDB IDs for pod wall ESTs were used to obtain the gene expression profile in the flower. The candidate genes were selected based on 9 their high levels of expression in the flowers and pod wall. A total of eight (8) flower and pod wall specific genes were identified and subjected to quantitative PCR (qPCR) analysis. Of these, 3 genes (FHG: Floral homeotic AGAMOUS-like isoform X2, MADS1: MADS box transcription factor and CEP: chickpea expressed protein) showed significantly high levels of up-regulation in the flower and pod wall when compared with leaves. These are the differentially expressed genes in the chickpea pod wall that have been identified for the first time. In order to obtain a regulatory sequence of selected flower and pod wall specific genes, 1000 bp region upstream of the start codon of FHG and MADS1 gene was obtained by Genome Walking and subjected to in-silico analysis using PlantCARE promoter prediction database. Sequence analysis of these promoter regions revealed certain tissue specific cis-acting elements that may regulate transcript accumulation in the flower and pod wall. Finally, the isolated promoters were cloned in a binary vector, pBI121, harboring the GUS as a reporter gene in order to study the efficiency of the promoters. Thus, for the first time the transcript dynamics of the chickpea pod wall were revealed and the transcript profile demonstrated various differentially expressed genes in the pod wall, which may be participating in metabolic build up of not only the pod wall but also seeds. The transcript library was useful to identify two novel promoter of genes that exclusively expressed in the flower and pod wall. These information of pod wall transcripts and isolated promoter may be valuable for chickpea improvement.ThesisItem Open Access SCREENING OF INDIGENOUS RICE (ORYZA SATIVA L.) GERMPLASMS OF ASSAM FOR TOLERANCE TO ANAEROBIC CONDITION DURING GERMINATION(AAU, Jorhat, 2017-07) Kumar, Dhananjay; Sarmah, Bidyut KumarRice is the staple food for the people of India and the major source of livelihood to the farmers. One of the most serious problems that adversely affect rice production in Assam is the recurrence of devastating floods. Even though more than 60% of summer rice is planted by the month of June, flooding after transplanting could completely devastate the crop. Furthermore, in the case of direct seeded rice, occurrence of flood delays sowing of seeds. Rice seeds can germinate under hypoxic conditions, but may fail to extend their coleoptiles and develop roots and leaves. Thus, there is an urgent need to provide farmers with rice varieties that besides being highly adoptive to local environment, also have the additional trait of tolerance to hypoxic condition. Breeding for hypoxic tolerance for germination was attempted but with limited success, because highly tolerant donors were unavailable and knowledge of the physiological and molecular basis of tolerance was inadequate. The precise and stringent control of physiological responses of deep water germplasms to flooding indicates that plant must possess sensible oxygen sensing mechanisms. Despite the wealth of molecular and phenotypic data on plant responses to water logging, very less information about how declining oxygen levels are sensed, and how the complex and extensive expression changes are controlled. So, in this study deepwater germplasms from Assam were screened against the proven tolerant lines KHO, MZ Red and Khaiyan (obtained from IRRI).Phenotypic characterization of 160 germplasm from Assam, based on a set of physiological parameters identified Rangadhar Kekua Bao (RKB) with highest frequency of germination (78%) in anaerobic condition. Physiological basis of tolerance involved uninterrupted supply of energy through catabolism of starch offset in sugar homeostasis by increasing the sugar sink towards coleoptiles elongation. Biochemical analyses revealed, enzymes involved in starch catabolism, alcohol fermentation and ATP and PPi dependent substrate level energy generation were significantly higher under hypoxia in tolerant germplasm. Transcript studies conducted on tolerant rice germplasms using GADPH as stable endogenous gene revealed that genes involved in sugar signaling such as TPP7, CIPK15 and SnRK1A that regulate Ramy3D transcription involving the transcription factor, MYBS1 under hypoxia were significantly upregulated in the RKB compared to proven tolerant line, KHO and susceptible line IR-64. 8 Group-VII members of Ethylene response factor family (ERFs) in rice namely ERF71 and ERF63 that are substrates for N-end rule of proteolysis also showed significant up regulation indicating towards an ethylene or low oxygen based hypoxia sensor that is yet to be identified in rice. Thus it could be inferred that hypoxia during germination of RKB is regulated by O2 sensing mechanisms involving ERFs that in turn activates sugar signalling pathways involving TPP7 a master regulator of sugar homeostatic, thus providing uninterrupted supply of energy, increasing the sugar sink towards coleoptile elongation possibly through action of EXPA7 and EXPA12.ThesisItem Open Access Analysis of the putative promoter of Indian Cassava Mosaic Virus, a Geminivirus(AAU, Jorhat, 2017-07) Baruah, Geetanjali; Sen, PriyabrataGeminiviruses are single-stranded DNA viruses, considered as the largest group of plant pathogenic viruses having nine genera. Geminiviruses are considered as a rich source of promoter elements as the intergenic region (IR) of their genomes harbor a bi-directional promoter driving expression in the viral-sense and complementary-sense directions. Indian cassava mosaic virus (ICMV; genus: Begomoviridae) is a bipartite (having two circular genomes, DNA-A and DNA-B) geminivirus; and in this study, we tried to define and delineate its bi-directional promoter of the DNA-A. This promoter drives the expression of Coat Protein (CP) in the viral-sense and Replication associated protein (Rep) genes in complementary-sense direction. Four sequential deletion-constructs for each of these promoters were made, after a prior in silico analysis using plantCARE to ensure that no key regulatory motif such as TATA box get deleted, driving expression of Gus gene in pBI121. In transient expression assay in Agrobacterium, and tobacco, the deleted versions (del-1) showed higher expression than the full-length promoters of both CP and Rep. Transgenic Nicotiana tabacum plants were raised using the full-length CP, full-length Rep and their del-1 constructs and same observations were made. Besides, their phloem-specific activity of the CP promoter constructs was also observed. Subsequently, Arabidopsis transgenic plants were raised for all the constructs and a similar expression pattern was observed. However, visually higher Gus expression in Arabidopsis flowers was observed. In silico analyses showed that the transcription factor (TF), CDC5 (a known transcription enhancer), was over-represented in CP del-1 construct showing highest expression. Besides, another transcription factor, the MADS Box 13, was over-represented in the CP promoter constructs; this TF plays role in development of gametophytes and embryo. Copy number, as determined by quantitative PCR, was found to be 2, 1, 4 and 2 for CP, CP del-1, Rep and Rep del-1, respectively. The expression was also quantified, that showed a similar pattern. Based on the observations, putative positive and negative regulatory elements of the promoters were also identified. Two transcripts were mapped in the viral-sense direction; the longer starting at position 138, and the shorter at position 170; while the longer could express both the AV1 and AV2 ORFs, the shorter transcript could express only the AV1 ORF. It is the first report of a comparison of deletion constructs of viral-sense and complementary-sense cassava mosaic virus promoters and their phloem-limited expression.
