Theses

Browse

Search Results

Now showing 1 - 9 of 11

Open Access
Optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin
(AAU, Jorhat, 2017-07) Das, Panchashree; Sen, Priyabrata
Citrus species are the most widely grown fruit crops within the whole world. India is the fourth largest producer of orange in the world. North-Eastern India is considered as one of the centres of origin of many citrus species. Among them Khasi Mandarin is the most widely grown citrus species. According to Ministry of Agriculture and Irrigation, Govt. Of India, the yield of Khasi Mandarin is declining day by day drastically due to biotic and abiotic stress. Conventional breeding for overcoming this problem is limiting due to non-availability of resistant sources. Recent advances in genetic engineering have made it possible to incorporate desirable genes from alien sources to elite genotype mainly through Agrobacterium-mediated genetic transformation. Citrus cultivars vary in their response to in vitro organogenesis and genetic transformation. This results in need for a cultivarspecific optimization of an in vitro regeneration and transformation protocol. Most of the plant regeneration processes in citrus, through tissue culture, involve use of Cotyledon, epicotyl segment, shoot-tip, internode, root meristem as explants. A study was conducted to develop a regeneration and Agrobactetrium mediated transformation protocol for Khasi Mandarin using zygotic seedling as explants obtained from six-week-old in vitro grown seedlings. Modified MS media containing 1 mg/l BAP, 0.5mg/l NAA and 0.4mg/l Kinetin shows the best result for multiple shoot induction with an efficiency of 68%. The number of multiple shoots developed was on an average 5. The modified MS medium containing containing 0.25 mg/l BAP,0.5 mg/l NAA, 0.5 mg/l IBA shows best result for rooting with an efficiency of 82% with an average root length 2 cm. Zygotic explants with injured shoot tip were used as explant for transformation with Agrobacterium strain AGL1, harbouring plasmid pCAMBIA1301 containing hpt as selectable marker gene and gus as a reporter gene. Modified MS media containing 100mM Acetosyringone was fund to be most effective medium for co-cultivation. Regeneration and selection media containing 1 mg/l BAP, 0.5mg/l NAA, 0.4mg/l Kinetin and 30mg/l hygromycin and 250mg/l timentin shows the best result. In vitro regenerated shoots that survived upto 3rd selection cycle were considered as putative transformants. Some of the putative transformed shoots showed positive result for gus in PCR analysis. The present investigation is a preliminary study on optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin. More and more concentrated effort is needed to establish a most efficient regeneration and transformation protocol considering various factors affecting genetic transformation and regeneration efficiency.
Open Access
Validation of selected SSR markers of QTLs associated with anaerobic germination trait in deepwater rice (Bao) of Assam
(AAU, Jorhat, 2017-01) Rajpoot, Sachin; Das, P.K.
India is one of the hot spots of vast genetic resources as well as secondary centre of origin of rice in the world. Exploiting genetic diversity could aid in providing useful information in the selection of material for breeding. Flood in rice field is due to heavy rainfall after seeding and early seedling emergence results in partial to complete crop failure. Primarily reason is high sensitivity of rice to the anaerobic conditions during germination (Yamauchi et al. 1993).Thus, tolerance to flood at early seedling growth improves crop establishment.The present study was conducted to evaluate anaerobic germination (AG) tolerance in Bao (deep water) rice germplasms of Assam compared to positive controls (Khao Hlan On, Ma Zhan Red) and negative control (IR-64). Under present study, genetic diversity was evaluated amongst 31 Bao rice germplasms of Assam using 38 SSR markers linked to Anaerobic Germination(AG). Assessment of allelic distribution showed three major groups in which, Rangdha Kekua Bao clustered with other four cultivars under cluster I while MaZhanRed and Khao Hlan On were grouped together with 12 of 38 Bao cultivars under major cluster II. Another half of Bao cultivars grouped together under cluster III separately indicating their distinctiveness. Regression test, validated only two markers viz.RM 553_170 linked to qAG 9, RM 5378_160 linked to qAG 2 that are significantly linked to the AG trait. These markers may be employed to evaluate the molecular diversity that can be used in aspects of plant genetics concerning germination studies under hypoxia. Present study also indicated that more markers are needed for lucid understanding of AG associated QTL and Marker assisted selection.
Open Access
SCREENING OF HYDROGENASE ACCESSORY GENES hypA AND hypB IN FEW ACID TOLERANT BACILLUS SP
(AAU, Jorhat, 2017-07) POOJA, S; Barooah, Madhumita
The present investigation reports on the screening of hydrogenase accessory genes viz., hypA and hypB in previously isolated acid tolerant Bacillus sp. followed by differential expression study (pH 7.0 and pH 4.5) of the both genes in three selected strains and subsequent cloning of hypA and hypB gene of Bacillus megaterium into pET28b+ vector. A total of 50 previously isolated acid tolerant Bacillus strains were considered for screening of hypA and hypB genes. Among the 50 isolates, 10 isolates tested positive for hypA & hypB genes. Three isolates viz., B. megaterium (GG1), B. cereus (GG2) and Lysinibacillus fusiformis (GG11) were considered for further study due to their ubiquitousness and higher acid tolerance. Bacterial RNA was isolated followed by synthesis of cDNA. A q-RT-PCR was performed in the above mentioned isolates to analyse the expression level of the said genes in acidic condition. The expression of hypA and hypB genes were significantly (P ≤ 0.05) up-regulated at pH 4.5 than in pH 7.0 in all the 3 isolates viz., GG1, GG2, GG11. An 18 fold increase in the expression of hypA and a 6.1 fold increase in expression of hypB were observed in the isolate GG1 at pH 4.5 compared to pH 7.0. The expression of hypA and hypB genes in GG2 was found to be 4.6 and 3.8 fold higher respectively at pH 4.5 than pH 7.0. In GG11, hypA showed 5.3 fold and hypB showed 2.8 fold increased expression at pH 4.5 compared to pH 7.0. Maximum expression of both the genes was observed in Bacillus megaterium. These genes were further cloned into pET28b+ vector. The transformation frequency obtained for the hypA and hypB was 1.5 x 106 and 3.2 x 104 CFU/μg respectively. Successfully transformed colonies were further validated through colony PCR as well as PCR on isolated plasmid DNA. The cloned hypA gene was sequenced and BLAST analysis was performed. BLAST analysis revealed that the genes belonged to the respective genus. The results of the present study revealed that hypA and hypB genes may have role in conferring acid tolerance to soil bacteria.
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.
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 Kumar
Bruchid 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.
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.
Open Access
Macrofungal Diversity of North East India and Development of Nanoparticle Based Detection of Mushroom Toxin
(AAU, Jorhat, 2017-11) Parveen, Assma; Barooah, Madhumita
Mushrooms 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.
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 Kumar
The 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.
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 Kumar
Rice 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.

Theses

Browse

Thesis Type

Type

Author

Date

Subject

Search Results