ThesisItem Open AccessOptimization of in vitro transformation protocol and RNAi based gene silencing for viral (Cucumber Mosaic Virus) resistance in Bhut jolokia (Capsicum chinense Jacq.)(2021) Deuri, Bharati; Bhorali, PriyadarshiniBhut jolokia (Capsicum chinense Jacq.), one of the most popular and hottest chillies in the world, is widely cultivated in Assam and other North Eastern regions in India. A member of the Solanaceae family, Bhut jolokia is characterized by very high pungency due to the presence of high amount of phenolic alkaloid ‘Capsaicinoids’. It is an ideal chilli variety for extraction of oleoresin and capsaicin, which have high market demand due to their industrial uses and medicinal properties. Bhut jolokia production is challenged by several biotic constraints, particularly viral diseases, which affect its quality and yield. Among the viruses, Cucumber Mosaic Virus (CMV) causes severe crop damage, leading to low productivity. Current control measures for CMV are mainly preventive through vector management strategies, which are not adequate in controlling the disease. An effective way to control the disease is the use of biotechnological tools such as RNA interference (RNAi) technology to engineer resistance against the virus. Plants expressing a copy of a viral gene in sense and/or antisense orientation have shown resistance upon infection with the virus via post-transcriptional gene silencing. In the present investigation, an in vitro regenerationtransformation strategy has been optimized for Bhut jolokia and, a hairpin RNA (hpRNA) based gene silencing construct has been developed using the Replicase gene from CMV. The study was initiated by callus induction from Bhut jolokia leaf segments in MS basal medium. Very good quality callus were induced in MS medium supplemented with 0.5 mg/l or 1 mg/l 2,4-D. Multiple shoot induction and regeneration from callus were obtained in MS medium supplemented with 8.5 mg/l KIN and 0.5 mg/l TDZ along with 5 mg/l AgNO3 with maximum shoot initiation frequency of 95% and regeneration frequency of 90%. Root regeneration was found to be optimum in half strength MS medium supplemented with 1.5 mg/l NAA within 4 weeks of culture with maximum rooting frequency of 70%. For standardization of an Agrobacterium-mediated genetic transformation system, the strain LBA4404 carrying pCAMBIA1301 binary vector construct with gusA as the reporter gene and hptII and nptII as selection marker genes was used. Transformation was carried out using 45 days old callus and also with intact Bhut jolokia seeds as explants. Hygromycin concentration of 9 mg/l was found to be optimum for efficient selection of putative transformants. From a total of 30 nos. of callus infected by Agrobacterium, 9 numbers of putative transformed shoots were regenerated in presence of selection agent. Finally, only 2 (6.66%) fully rooted plants survived out of which, only 1 plant finally survived during hardening in the green house. Moreover, out of 30 nos. of infected seeds, a total of 7 numbers of putative transformed seedlings were developed. Finally, only 1 (3.33%) seedling survived, which was transferred to the green house for hardening. Thus, both callus and seeds could be used as explants for transformation in Bhut jolokia, although the frequency of putative transformants obtained using callus explants was higher than that in seed transformation. The putative transformants were confirmed by GUS histochemical assay and PCR analysis. For developing the RNAi construct, a 323 bp Replicase gene sequence was cloned into pHANNIBAL vector both in sense and anti-sense orientations. The construct was then transferred to pBI121 binary vector, which was electroporated into Agrobacterium strain LBA4404 for plant transformation. Functional validation of the CMV Replicase hp-RNA construct was done through bioassay in model plant Nicotiana benthamiana by Agro-infiltration. Transgene expression in N. benthamiana was confirmed by RT-PCR analysis. The bioassay results indicated suppression of CMV infection in Agro-infiltrated N. benthamiana plants when mechanically inoculated with CMV sap. Further, DAS-ELISA established the functional efficiency of the hpRNA construct in providing considerable level of resistance against CMV infection. The in vitro regeneration-transformation strategy and the hpRNA based gene silencing construct, developed through this study would serve as a foundation towards future studies on engineering resistance against CMV in Bhut jolokia. ThesisItem UnknownMorphogenetic, Metabolic and Molecular Dynamics during Mycelial Interactions among Fungal species(AAU, Jorhat, 2021) Dullah, Samim; Boro, Robin ChandraIn nature, microorganisms interact/compete with one other for food and space and the type of interactions are unique to each interacting species. Fungal-fungal interactions are complex, and different types of secondary metabolites are secreted during interaction. In this study, 14 fungal isolates were facilitated in 105 possible combinations to interact in potato dextrose agar (PDA). Ten interactions between different fungal isolates showed mutual replacement with each fungus; capturing territory from the other. Thirty-five interactions showed complete replacement as growth of one of the fungal partners was inhibited. In forty-six interactions, formation of barrage was observed leading to deadlock type of interaction wherein both fungi have restricted growth. The barrage formation during interaction was further studied with two fungal interactions viz., (i) T. coccinea vs. L. lactinea and (ii) T. coccinea vs. T. versicolor. Microscopic changes were observed in the hyphal growth during interaction like hyphal coiling, dense mycelial network, pore formation. Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. The metabolites produced during interaction of Trametes coccinea (F3) with Leiotrametes lactinea (F9) and Trametes coccinea (F3) with Trametes versicolor (F1) was analysed through Liquid Chromatography coupled with Mass Spectroscopy (LC-MS). Most of the metabolites secreted during interaction are associated with defensive response. The bipartite fungal interaction resulted in the production of a dark brown colour pigment – melanin as confirmed by the LC-MS, FTIR and NMR analysis. Moreover, the fungal-fungal interaction also led to increase in the production of laccase, a group of multicopper oxidases involved in detoxification of toxic compounds. Further increased activity of superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide anion to hydrogen peroxide was also recorded during fungal–fungal interaction. There was significant increase in the activities of hydrolytic enzymes including cellulase, xylanase and chitinase during in vitro fungal-fungal interaction, suggesting the importance of such interactions for commercial enzyme production. Quantitative real-time PCR revealed upregulation of lcc1 (encoding a laccase enzyme) and few other stress related genes of T. versicolor during its hyphal interaction with T. coccinea, suggesting a direct correlation between laccase production and melanin production. The study helped to gain a better understanding on the morpho-physiological, biochemical and gene expression profiles during in vitro fungal-fungal interaction. Such interactions induce the production and secretion of an array of metabolites and enzymes which can be prospected towards biotechnological applications. ThesisItem UnknownMAPPING OF QUANTITATIVE TRAIT LOCUS (QTL) FOR DROUGHT TOLERANCE IN RICE(AAU, Jorhat, 2021) CHANDRAKANT, KALDATE RAHUL; Modi, Mahendra KumarDrought is a major abiotic constraint for rice production worldwide. The reproductive stage drought stress (RSDS) leads to a huge loss in grain yield. The prospecting of new donor cultivars with identification and introgression of large effect drought tolerance related QTLs is essential to develop drought-tolerant rice varieties. The present study was conducted with the aim of mapping quantitative trait locus (QTLs) associated with drought tolerance in rice. With a total of 3417 GBS (Genotyping by sequencing) based polymorphic SNP (Single nucleotide polymorphism) markers distributed over the rice chromosomes a saturated linkage map spanning 1924.136 cM was constructed with an average marker density of 0.56 cM using an F3 mapping population developed by crossing traditional Ahu rice cultivar Koniahu (drought tolerant) with Disang (drought susceptible). Using inclusive composite interval mapping (ICIM) approach 172 genomic regions associated with grain yield and related traits were detected in 198 F3 and F4 segregating lines evaluated for two consecutive seasons under both RSDS and irrigated control conditions. Of which, 102 QTLs were identified under RSDS with LOD (Logarithm of odds) score value ranging between 2.53 to 7.51 and phenotypic variance explained (PVE) value of 1.97 to 12.42 per cent. While 70 QTLs with LOD score value ranging between 2.50 to 10.07 and PVE value of 2.35 to 11.46 per cent were detected under control condition. In total, nine QTLs were major QTLs having a PVE value 10. Among the QTLs identified under RSDS, 72 (70.59%) QTLs were novel whereas 30 (29.41%) QTLs were observed to be co-localized or overlapped with genomic regions previously mapped for the same trait/ QTLs. Five putative QTLs namely, qGY2.00, qGY5.05, qGY6.16, qGY9.19, qGY10.20 were found to be associated with grain yield under drought. Further, fourteen CAPS (Cleaved Amplified Polymorphic Sequences) were developed from selected eight novel QTLs linked SNP regions and validated in parental cultivars along with ten F5 generation RILs. Putative gene identification within eight QTL regions detected a total of 3341 genes in which 1516 (45.63%) genes were annotated to at least one gene ontology (GO) term. The putative QTLs and candidate genes identified in the present study need to be further validated, which will be helpful for the improvement of drought tolerance in rice. ThesisItem Open AccessAgrobacterium mediated genetic transformation of Citrus reticulata cv. Khasi mandarin(2020-02) Bhandari, Sangeeta; Singh, SalvinderCitrus is number one fruit of the world on accounts of its high nutritional value. India is the fourth largest producer of Citrus in the world. The north-eastern region of India is a rich treasure of various Citrus species. Khasi mandarin is the most economically important one and plays a vital role in the socio-economic development of the people in this region. Khasi mandarins are declining at a very high rate due to its vulnerability to different pathogen and insect/ pest. Conventional breeding for overcoming these problems are limited in Citrus and are directly associated with the reproductive biology of Citrus. Recent advances in genetic engineering have made it possible to incorporate desirable genes from elite genotype mainly through Agrobacterium-mediated genetic transformation. Citrus species showed varied response to in vitro regeneration and genetic transformation. Cultivar specific optimization of in vitro regeneration and transformation protocol is very important. In the present investigation, in vitro regeneration and Agrobacterium mediated genetic transformation protocol for Khasi Mandarin was optimized using different explants like epicotyl, hypocotyl, nodal and inter nodal segment obtained from six-week-old in vitro grown zygotic seedling. Explants were transformed wih Agrobacterium strain LBA4404, harbouring plasmid pBI121-AtSUC-GUS containing nptII as a selectable marker and gus as a reporter gene. Hypocotyl was found to be the best explants for khasi mandarin transformation and regeneration. MS medium supplemented with BAP (2mg/L), NAA (0.5 mg/L), 2, 4-D (1mg/L), MES (0.5g/L), sucrose (30g/L) and acetosyringone (100μM) was found to be best medium for co-cultivation. Modified MS medium containing BAP (4mg/L), MES (0.5g/L), sucrose (30g/L), phytagel (4g/L), kanamycin (50mg/L) and timentin (150mg/L) showed highest regeneration efficiency (18%). Modified MS medium containing BAP (4mg/L), GA3 (0.5mg/L), MES (0.5g/L), sucrose (30g/L), phytagel (4g/L), kanamycin (50mg/L) and timentin (150mg/L) showed highest multiple shoot induction (6%). In vitro regenerated shoots that survived up to 3rd selection cycle were subjected to GUS assay for confirmation of GUS expression in the phloem tissues. Present investigation is a preliminary study for optimization of an in vitro regeneration and genetic transformation protocol in Khasi Mandarin. ThesisItem Open AccessGENETIC STUDIES FOR IMPROVING YIELD UNDER DROUGHT STRESS ENVIRONMENTS IN RICE OF ASSAM(2019-07) PARRAY, ROUF AHMAD; Baruah, Akhil RanjanDrought is a major limiting factor for rice under rainfed ecosystem in Assam. In this context, thirteen rice cultivars with varied level of drought tolerance were chosen from a set of 272 different rice genotypes based on a field experiment conducted during 2014-15 season under drought. The thirty days old seedlings of 13 cultivars were tested for extensive morpho-physiological, biochemical parameters, relative transcript accumulation and global gene expression using next generation sequencing (NGS) method, and data were recorded at fifth, tenth and fifteenth day of withholding water (DWW) in order to obtain detail trait based gene architecture and to improve high yielding variety of Assam using transcript dynamics. Among the physiological traits studied, stomatal conductance decreased as the dehydration stress increased but the effect was minimum in Apo, Dumai and Tepi Dumai compared to others. Photosynthetic rate decreased with increasing water deficit, but the effect was less pronounced in Apo, Dumai and Tepi Dumai. The rate of transpiration decreased upto 5DWW but gradual increase was observed in later stage. Moreover, the fall in transpiration rate was less in Apo. Water use efficiency (WUE) of rice plants was enhanced significantly under moisture stress at all the three periods of stress (5DWW, 10DWW, 15DWW) in Apo, Tepi Dumai and Dumai. Reduction in RWC was experienced across all genotypes but the decrease was less prominent in Apo, Dumai and Tepi Dumai. Drought stress condition led to increased proline content across genotypes as compared to irrigated condition. Apo, Tepi Dumai, Dumai and Kali Murali showed rapid increase compared to others. Increase in root length was observed across all cultivars with Apo being the longest followed by Dumai and Ranjit. Then, five drought responsive pathway genes (OsDREB2, OsNAC1, bZIP16, OsbZIP 23, OsbZIP72) were chosen to check the differential expression patern in the cultivars at the same data point as mentioned above. Expression profiling of OsDREB2 showed significant increase in gene expression with increase in drought stress in the case of Apo and Dumai. Significant expression of the OsNAC1 was found in Apo, Dumai at different time points of dehydration stress whereas expression of ARC 10372 was prominent in 15DWW. Apo showed significant difference in expression of bZIP16 under all the three stages of water stress whereas Dumai and Ranjit showed enhanced expression compared to other cultivars. Expression profile of OsbZIP23 showed significant accumulation of transcripts in Apo in all stages followed by Dumai. Significant expression of OsbZIP72 was observed in Apo at 10DWW and 15DWW followed by Ranjit and Dumai. Based on the results of morpho-physiological, biochemical and expression analysis, three cultivars, viz., Ranjit, Apo and Dumai were chosen to study the detailed transcriptome at only 10DWW. Transcriptome profile revealed highest mapped genes in Dumai followed by Ranjit and Apo, however, only 14.5% genes were in common. Ranjit was found to be more responsive to abiotic stimulus including water stress. Gene ontology (GO) suggested no significant change of pathway genes upto 10 DWW among the three cultivars. The transcriptome data were validated using five differentially expressed genes in these three cultivars along with a F4 mapping population. It revealed similar trend, suggesting the present transcriptome data set was in good fit. However, detail transcriptome study in vital plant parts at different stages under drought stress will throw more light about the interaction of pathway genes to adress the problem better. ThesisItem Open AccessMolecular cloning and in silico characterization of linalool synthase gene from Cymbopogon winterianus(AAU, Jorhat, 2018-07) Saha, Oliva; Sen, PriyabrataCitronella (Cymbopogon winterianus), an aromatic and medicinal grass from Poaceae family, is grown for the commercial and industrial purpose. It has the large repository of isoprenoid compounds known for its anti-tumoral, anti-bacterial, anti-fungal, anti-viral, anticancerous, detoxifying and natural insect repellent properties. Out of the different components of citronella oil, linalool (3,7-Dimethylocta-1,6-dien-3-ol) is one of the significant aromatic ingredient. Formation of linalool is catalysed by linalool synthase (LIS) which is encoded by linalool synthase gene. This gene has been widely studied in many medicinal plants along with sorghum, rice, maize, setaria, etc.However, till date, no studies on molecular cloning, characterization and tissue-specific expression profiling of LIS gene from C. winterianus (CwLIS) has been reported.Therefore, the present study aims at cloning and characterization of full-length cDNA of LIS gene from C. winterianus. The full-length sequence of CwLIS was obtained by primer walking using several pairs of degenerate primers and the sequence fragments were aligned to acquire full length sequence. Cloning of the desired gene (1758bp) was performed using TOPO TA vector (3.9kb), and the transformation was done in E. coli DH5α competent cells. The sequence analysis revealed 1758 bps cDNA, which has a Coding Sequence (CDS) of 1422 bps that encodes a protein of 473 amino acids. The domain analysis revealed that CwLIS is a single domain protein under terpene synthase superfamily. The multiple sequence alignment (MSA) based on PSI-BLAST search against RefSeq with 62% sequence identity revealed the presence of two aspartate-rich regions, which are supposed to coordinate 3 Mg2+ ions. The phylogenetic analysis revealed that the CwLIS is closely related to Sorghum bicolor linalool synthase. To understand the molecular mechanism behind Mg2+ and linalool binding, first, the 3D model of CwLIS was generated and validated with their stereo chemical parameters. Further, to find out the expression profile of CwLIS in different tissues, Reverse Transcriptase-PCR was performed and the results revealed a higher expression in leaf sheath followed by leaf, root and flower and further conformed by qRTPCR analysis. The result from the present study provide basic information for further research about linalool synthase and comprehensive sequence resource for study, such as gene expression, genomics and functional genomics in Cymbopogon winterianus. ThesisItem Open AccessAssessment of Genetic Diversity For Ideal Plant Architecture in Rice Cultivars of Assam With Special Importance to Aromatic Group(AAU, Jorhat, 2018-08) Debnath, Neelakshi; Baruah, A.R.The development of super rice to break the yield plateau is one of the goals for rice breeding in recent years. Ideal plant architecture (IPA), which is under polygenic control, has been proposed as means to enhance rice yield potential. A study directed towards assessing the presence of desired alleles for IPA related genes/quantitative trait loci (QTL) in rice cultivars grown in diverse agroecological situations, specifically the aromatic rice of Assam (joha rice) that usually bears weaker plant type should be of prime importance. The present study was conducted to detect allelic variation for IPA in traditional, high yielding and joha rice cultivars of Assam and to compare nucleotide sequence variation for aroma gene in cultivars possessing IPA. A total of 80 cultivars comprising sali, ahu, boro and joha rice of Assam were assessed for markers linked to five IPA related QTLs (Sol1, PAY1, IPA1, Gn1 and LAZY1). Although a total of 15 markers (M3, M6, M8, M10 for Sol1; RM339, RM223, SP5, SP7 for PAY1; RM149, RM1345, M4, M5 for IPA1; PD56, M265 for LAZY1; 16BPDEL for Gn1) were employed to check for polymorphism, only five markers (GN1, M10, M265, RM 1345, SP5) showed reproducible and polymorphic results. The Gn1 and Sol1 were most abundant in cultivars as evident from the observation that 46 numbers of cultivars harboured desired alleles for Gn1 and Sol1 followed by PAY1( 31 cultivars), LAZY1 (12 cultivars) and IPA1(12 cultivars). Among HYVs, the varieties such as Ranjit, Mahsuri and Bahadur were found to possess four numbers of desired alleles, however, IPA1 allele was other than the expected. Among Joha cultivars, Bengoli joha, Goalporia joha, Kartica joha were with the maximum of four numbers of positive alleles. As Ranjit missed the expected size of the IPA1 allele, differential expression for the gene in Ranjit and one cultivar possessing the desired IPA1 allele (Chakaw Poireton) was conducted. It revealed that although Ranjit did not possess the desired IPA1, however, the expression pattern revealed that 25-30 fold more accumulation of transcripts for the gene in Ranjit than Chakaw Poireton, suggesting the functional differences in the coding regions. The allele specific analysis for aroma gene, badh2 revealed that the markers could efficiently group 53.66% of the cultivars corresponding to the existing phenotype, indicating the power of the marker to be utilized in a large germplasm screening for aroma. The sequencing for badh2.hapG9 detected a non-synonymous substitution (AG) which was not being reported elsewhere; however, novelty of which needed to be confirmed and validated. The study will enhance our knowledge to formulate breeding strategies for combing IPA with aroma. ThesisItem Open AccessOptimization of an in vitro regeneration and transformation protocol in Khasi Mandarin(AAU, Jorhat, 2017-07) Das, Panchashree; Sen, PriyabrataCitrus 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. ThesisItem Open AccessHOST RANGE, INCIDENCE AND GENETIC VARIABILITY OF watermelon mosaic virus IN CENTRAL SPAIN(AAU, Jorhat, 2018-01) Paswan, Ricky Raj; Acharjee, SumitaWatermelon mosaic virus (WMV, Potyvirus) is an economically important pathogen common in cucurbits of temperate and Mediterranean regions worldwide. The presence of WMV in cucurbits in the Mediterranean basin has been known for decades. More recently, an emergent strain that causes more severe symptoms compared to classic strains has been recognized in France. The cultivation of melon is threatened by the spread of emergent strains of WMV. Diagnostic methods for detecting the host range, incidence and evolution of these emergent types are critical for developing control strategies to optimize agricultural production. In this study, next generation sequencing and RT-PCR approaches are combined to investigate the epidemiology of WMV in an agro-ecosystem of Central Spain. Four vegetation types, or habitats, including cultivated and adjacent land-use types were surveyed in the summer and autumn of 2015. Forty-three plant species were screened for WMV, 15 of which were WMV-positive across two habitats other than crops. The results indicated an increase in the extent of the WMVs known host range. The incidence of WMV ranged from 64% in Cucumis melo to 5% in a weed species, Datura Stramonium. Genetic analyses of the coat protein gene of 30 isolates from melon and 3 other ‘weed’ species sampled in crops showed population variation in nucleotide diversity, but pairwise fixation indices indicated negligible distinctions between them. Phylogenetic inferences showed both negligible and large branch length differences between isolates from different host species. When sequences of a number of different strains were added to the isolates from the melon crops, one clad clustered with an emergent group previously identified from elsewhere in Europe and Asia. This study reports the first instance of an emerging (EM) strain in Central Spain.