Central Agricultural University, College of Post Graduate Studies in Agricultural Sciences, Umiam
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ThesisItem Embargo Bulk Segregant Analysis for Aluminium Toxicity Tolerance in F2:3 Population of Rice(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-01) Sreeja, Akunuru; Tyagi, WrichaSoil acidity is a key limiting factor for crop productivity around the world. Other than the frigid zones, thirty percent of the world's land is acidic, accounting for about half of all potentially arable land, and rice is grown on about 12 percent of the world's cultivated acidic soils. Aluminium toxicity is a quantitative trait in rice and therefore, genes/markers need to be identified for molecular breeding. The current study was conducted to screen and identify superior and inferior performing bulks in a set of 197 F2:3 progeny derived from JR-31 × CAUS-107 cross treated with aluminium (Al) in one round and low phosphorous (Pi) followed by Al in the other round of hydroponics and then, to determine the markers associated with tolerance. The phenotypic traits shoot length, root length, and visual observations in the first round and shoot length, longest root length, average root length, shoot fresh weight, root fresh weight, root spread, root area, and hematoxylin scoring on a scale of 1 to 5. The parents were surveyed for polymorphism using 76 SSR and 56 candidate gene-based (CG) markers which revealed 31 monomorphic and 23 polymorphic markers. Of which, four genic SSR and five CG markers were used for the genotyping of the extreme bulks of 76 progeny identified by two rounds of screening. According to ANOVA (CRD), all the phenotypic traits showed a significant variation in the progenies under both rounds of screening at 1% level of significance with values ranging from 3.48 (root weight (RW)) to 33.2 (average root length (ARL). Correlation coefficient analysis indicated that the shoot length (SL) significantly correlated with root length (RL) (0.263) in the first round of Al toxicity tolerance screening at 1% level of significance. In the second round of screening for both low Pi and Al toxicity tolerance, correlation coefficient analysis between the phenotypic traits at a 1% level of significance revealed that the SL significantly correlated with LRL (0.606), average root length (ARL) (0.866), shoot fresh weight (SFW)(0.729), root fresh weight (RFW) (0.393), root spread (RS) (0.899) and root area (RA) (0.916). The LRL significantly correlated with ARL (0.716), SFW (0.55), RFW (0.395), RS (0.686), and RA (0.691). The ARL significantly correlated with SFW (0.649), RFW (0.282), RS (0.949), and RA (0.949). The SFW significantly correlated with RFW (0.385), RS (0.654), and RA (0.683). The RFW significantly correlated with RS (0.334) and RA (0.359). The RS significantly correlated with and RA (0.981). While hematoxylin stain score negatively correlated with the LRL (0.261) at a 1% level of significance. When phenotypic traits of both the rounds of screening of hydroponics were compared, SL (Al) significantly correlated with the SL (Al+Pi) (0.317) at 1% level of significance, and with RS (Al+Pi) (0.234) and RA (Al+Pi) (0.25) at 5% level of significance. While the RL (Al), significantly correlated with SL (Al+Pi) (0.255), LRL (Al+Pi) (0.489), ARL (Al+Pi) (0.338), RA (Al+Pi) (0.339), positively at 1% level of significance and with hematoxylin stain score (Al+Pi) (0.227) negatively at 5% level of significance. When the yield traits were compared with phenotypic traits of hydroponics, where only Al toxicity tolerance was screened, SL (Al) positively correlated with the FGPP (0.142), and negatively with TN60 (0.147). Root length (Al) positively correlated with the FGPP (0.179) and SF% (0.141) at 5% level of significance. The yield traits did not correlate with phenotypic traits of hydroponics, where both low Pi and Al toxicity tolerances were screened. A polymorphic survey of parents with 76 SSR and 56 Candidate gene-based markers revealed 31 monomorphic and 23 polymorphic markers. From those polymorphic markers, 4 genic SSR and 5 Candidate gene-based markers were used for the genotyping of the bulks from the phenotyping screenings. Allele bands were scored and the marker-trait association was performed using simple t-test and chi-square analyses. The t-test revealed that the marker, HvSSR01-34 associated with the longest root length (Al+Pi) (-Log10P = 1.5); marker HvSSR02-14 with TN60 (-Log10P = 1.28), SL (Al) (-Log10P =1.89), RL (Al) (-Log10P = 1.21), overall score (Al) (-Log10P= 1.6), ARL (Al+Pi) ((-Log10P = 1.33), SFW (Al+Pi) (-Log10P = 1.11), RS (Al+Pi) (1.65), and RA (Al+Pi) (-Log10P = 1.42). The marker RM12557 associated with RL (Al) (-Log10P = 1.9), overall score (Al) (-Log10P= 1.84), and RS (Al+Pi) (-Log10P = 1.14); marker AR051-2 associated with PN (1.05), the FGPP (-Log10P = 1.1), root length (Al) (-Log10P = 1.39), overall score (Al) (-Log10P= 1.21), overall score (Al+Pi) (-Log10P= 0.99) and RW (Al+Pi) (-Log10P = 1.32). The marker AU01-3 associated with SFW (Al+Pi) (- Log10P= 1.29) and RA (Al+Pi) (-Log10P = 1.01); FR033-3 with the LRL (Al+Pi) (-Log10P= 1.01); PR026-3 with RL (Al) (1.01) and hematoxylin stain score (Al+Pi) (-Log10P= 1.17) and PR062-3 with TN60 (-Log10P= 1.19), PN (-Log10P= 1.69), SL (Al) (-Log10P= 1.36), overall score (Al+Pi) (-Log10P= 0.99) and RW (Al+Pi) (-Log10P= 1.32). Chi-Square analysis based on 30 superior and 30 inferior progenies revealed that the CAUS-107 allele significantly associated with three markers at 5% level of significance. Markers HvSSR01-34; HvSSR02- 14 and RM12557 associated with SF% (5.554); SL (Al) (5.684) and RL (Al) (χ2 = 5.225) and overall score (Al) (χ2 = 4.139), respectively. While JR-31 allele for marker HvSSR02-14 associated significantly with the overall score (Al) (χ2 = 4.669). Chi-Square analysis based on 20 superior and 20 inferior progenies revealed that four markers were significantly associated with the phenotypic traits at 5% level of significance. AR051-2 associated with overall score (Al) (χ2 = 4.916) and with RL (Al) (χ2 = 5.121), respectively. While the marker PR062-3 showed allelic distortion with 71% of progeny having JR-31 type allele. To summarise the bulks based on phenotypic data were significantly different and genotyping on a set of 76 progeny revealed that 7 markers viz. HvSSR01-34; RM12557; AR051-2; AU01-3; FR033-3; PR026-3 and PR062-3 associated with various traits under hydroponics conditions. Three markers viz, HvSSR01-34; AR051-2, and PR062-3 associated with better performance under lowland acidic soil conditions. These markers and traits can be used for MAS after further evaluation.ThesisItem Open Access Bulk segregant analysis for blast resistance in F2 population derived from two contrasting rice genotypes of north eastern hill region(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2018) Sumpi, Hage; Rai, MayankRice blast caused by fungus Magnaporthe grisea is one of the most devastating diseases worldwide. Use of resistant cultivar is the most effective way to control this disease. The identification of markers linked to blast resistance is very important as they can be used in marker assisted selection to develop new resistant cultivars. Bulk Segregant Analysis (BSA) is a method that works with selected individuals which hastens the process by reducing the number of required assays, providing rapid and simple alternative for identification of markers linked to a trait and gene mapping. The present study used this technique to select extreme phenotypes in a bi-parental population (F2) derived from two contrasting rice genotype LR5, also known as Lal Jangali (a local landrace of rice resistant to blast) and LR26 (Manipuri black rice traditionally known as Chakhao, susceptible to blast). Disease assessment for blast on progenies and parental genotype was carried out under natural disease occurrence. F2 progenies showing extreme phenotype were selected and subjected to genotyping with the polymorphic markers (including SSRs and SNPs and some previously reported blast markers) obtained from polymorphism survey on the parental genotype in order to identify markers linked to blast resistance. Association of the markers with phenotypic trait in the selected progenies was identified by statistical analysis. Chi square test for goodness of fit revealed that RM1337 (p-value=0.003, 0.002), RM7102 (p-value=0.002, 0.001), snpOS0310 (p-value=0.02, 0.007), snpOS0316 (p-value=0.001, 0) and snpOS0318 (p-value=0.002, 0.001) showed association with tolerance and susceptibility against leaf blast, whereas RM247 (p-value=0.025, 0.016) and snpOS0316 (p-value=0.0.05, 0.004) showed association with these two groups for neck blast. Except for snpOS0310 which is on chromosome 6, all other markers are present on chromosome 12. RM1337 and RM7102 co-localize with already reported Pi20t gene, suggesting its role in resistance to local pathotypes. Regression for blast resistance was also evaluated to estimate the relationship between the marker genotype and blast severity. Population structure analysis was also performed on the selected group of progenies to determine the ancestry, which revealed that the progenies showing resistance to both leaf and neck blast carried maximum percentage of ancestry from the resistant parent LR5 and those susceptible to blast carried more of LR26 ancestry. Larger population and more polymorphic markers can be used to further fine map the segment of chromosome 12 identified in this study.ThesisItem Open Access Characterization of a panel of contrasting rice genotypes for low phosphorus tolerance using morphological and molecular markers(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2018) Gympad, Ebenezar; Tyagi, WrichaRice (Oryza sativa) belongs to the family Poaceae has chromosome number of 2n=24. Rice is considered as a major food crop across major countries worldwide. There are many problems which affect rice productivity. Soil acidity, anabiotic factor, is one of the factors causing low yield in rice. 49 million ha of the total land area in India is affected by soil acidity. Major portion of acidity affected soil are concentrated in north-eastern part of India due to extreme levels of soil acidity. Phosphorus deficiency is a huge problem faced in NEHRs of India due to acidic soil. Phosphorus forms precipitation reactions with aluminium (Al) and iron (Fe) in acidic soils and the complex of P in Al-P and Fe-P minerals under acidic conditions tends to be stable. In this stable form, P is not available for uptake by plants and its availability is therefore, reduced. Therefore this affects rice functions like energy transfer, photosynthesis, transformation of sugars and starches, nutrient movement within the plants. The present study aimed at evaluating 60 diverse rice genotypes performance with respect to 15 different traits under lowland, acidic P deficient soil conditions using morpho-physiological parameters and to study the association between candidate gene based markers and phosphorus deficiency tolerance. A panel of 60 selected genotypes was selected based from the previous study on 110 genotypes and grown in the field. Data were collected on 58 plants only as the other 2 plants could not survive. On the basis of the 15 traits above, 20 top performing and 20 bad performing lines were selected for further evaluation. Genotypes like BAM 4115, LR 23, LR 21, BAM 8381, LR 19, LR 18-2 and LR 5 had the highest tiller no. at 30 days and BAM8315, BAM 6921, LR 5 and BAM 8381 showed the highest tiller no. at 60 days, LR 1 showed the highest grain yield. The genotypes LR 1 and BAM 4054 showed the largest PUE content. With respect to phosphorus uptake the genotypes BAM 742, BAM 2815, BAM 1098, BAM 8381, UR 29, BAM 811, BAM 56, BAM 712, BAM 1689, BAM 747, BAM 698, BAM 785, BAM 758 and BAM 759 were better performer.The correlation matrix showed that panicle length, leaf area and biological yield were significantly correlated grain yield. High correlation was also observed for tiller number at 60 days with traits like panicle number (0.736), biological yield (0.647), days to flowering (0.600) and grain yield (0.593) at 1% level of significant (0.325). P content was significantly correlated with TN30, TN60 and GY. PUE of flag lead was significantly correlated with DTF and BY. However, PUP of flag lead was negativelycorrelated with most of the traits including GY, TN and PUE. Significant correlation of our data with previous field data (2014) for 11 agronomic traits suggests that the genotypes and traits identified can be used for various breeding and crop improvement programmes for low P tolerance. Genotyping with candidate gene based makers showed polymorphism for markers like K46-2, PR 111-3 and HvSSR 06-06. Marker trait association revealed significant association of marker, PR111-3 with leaf area, grain yield and spikelet fertility. Marker K46-2 showed significant association with panicle length and test weigh.ThesisItem Open Access Characterization of advanced inbred lines for low phosphorus and iron toxicity tolerance(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2018) Rasul, Shaikh Akbar; Tyagi, WrichaRice (Oryza sativa) is the world’s most important food crop and it’s productivity is adversely affected by many biotic and abiotic factors. Nutrient deficiency and mineral toxicities are one of the major problems affecting rice productivity, these problems are mainly associated with soil pH especially acidity/sodicity. In India, major portion of soil affected by acidity are concentrated in north-eastern part of India. Phosphorus (P) deficiency and severe iron toxicity in acid soil leads to poor growth and yield reduction in rice. Therefore, it is necessary to develop the rice genotypes with tolerance to iron toxicity and phosphorus deficiency performing well in lowland acidic soil conditions.Towards this end, an attempt was made to evaluate two advanced breeding lines for iron toxicity and low P tolerance in lowland acidic field with the following objectives: 1) to characterize the set of advanced inbred lines with respect to yield contributing traits and 2) to genotype advance inbred lines by using DNA based markers. Out of 51(BC2F9) near isogenic lines (NIL) and recombinant inbred lines (RIL) (235 plants) (F3:4) grown in the lowland acidic field conditions, a set of top performing KM lines and ULRC-36 plants were selected based on the phenotypic data such as bronzing score at 45 and 60 days after transplanting (DAT), tiller number (TN) at 60 DAT, panicle number (PN), test weight (TW), spikelet fertility (SF). The selected lines showed variation for the traits under study. The highest test weight was observed in KM-194and ULRC-36-8 followed by ULRC-55 in NILs and RILs mapping population, respectively. In NILs population KM-194 performed well for P content, phosphorous uptake (PUP) and showed no bronzing. KM-608 performed better for phosphorous use efficiency (PUE).The highest tiller number at 60 DAT was recorded in KM-660. In case of ULRC-36, the highest P content, PUP and PUE was observed in ULRC-36-152, ULRC-36-242 and ULRC-105, respectively. Among the selected plants, no bronzing was found in ULRC-36-22, ULRC-36-157, ULRC-36-175 and ULRC-36-253. For tiller number at 60 DAT, plants ULRC-36-22, ULRC-36-107, ULRC-36-152, ULRC-36-157 and ULRC-36-278 showed performance better than the parents. Correlation study for the traits under study showed positive correlation at 1% and 5% level of significance for the key trait likes TN, PN, grain yield and other traits in the both populations. Six trait specific molecular markers for low P tolerance and Fe toxicity also showed variation for the selected genotypes and the desired alleles were present in some of the top performing lines.The selected KM lines can be tested for seed quality traits andthe ULRC-36 population further advanced before genetically fixed lines can be selected.ThesisItem Open Access Evaluation of curcumin content and genetic diversity using molecular marker in turmeric genotypes cultivated in north east India(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2019) Magar, Sayali; Chowdhury, V. K.The Genus Curcuma belonging to Zingiberaceae family consisting of about 133 accepted species worldwide. Turmeric (Curcuma longa L.) is a perennial rhizomatous commercially important spice crop which is native to Indian subcontinent and Southeast Asia. India contributes 82% of world production (532,353 MT in 2019) of turmeric. Golden spice turmeric possesses anticancer, anti-depressant, antioxidant and antifungal properties. Because of this, in the past few years there has been increasing demand for different purposes. As turmeric comes under category of highly recalcitrant species and high disease susceptibility, cost of production is higher, therefore it is necessary to standardize protocol for mass production and disease-free stocks through tissue culture technique and knowledge of genetic variability is essential for crop improvement programs and for conservation of plant genetic resources as well as there is need to identify the cultivars having high curcumin content that are adapted to different agroclimatic conditions of North East India. Considering these aspects, the present study was planned for analysis of curcumin content and genetic diversity using ISSR and RAPD markers as well as optimization of protocol for turmeric in vitro regeneration. For in vitro regeneration aseptic bud were cultured on 12 MS medium with varying concentrations and combinations of BAP and NAA to check response of explant for shoot initiation, length and number of shoots along with number of roots. Among 12 treatments BAP (3.5 mg/l) with NAA (1.5 mg/l) showed best results. Average number of shoots obtained were 2.90 per plantlet and average number of roots were 9 per plantlet. Per cent response (86%) and shoot length (Average 4.30 cm) observed were best in BAP (5 mg/l). A total of 42 ISSR and 10 RAPD primers were used out of which 23 ISSR and 5 RAPD primer produced distinct bands ranging from 400-1100 bp (ISSR) and 500-1400 bp (RAPD). The study revealed high to moderate genetic variability. A total 145 bands with 78% polymorphism and 20 bands with 76.7% polymorphism were obtained in ISSR and RAPD markers, respectively. The unweighted neighbour joining method was used to access clustering pattern. Darwin software based Dendrogram (obtained by ISSR and RAPD molecular data) categorized all 22 accession into three major clusters. The Dissimilarity coefficient value ranging from 0.218 to 0.848 was calculated from the allelic data using Darwin software. The PIC value ranges from 0.497 to 0.222 and MI value ranges from 2.88 to 0.30. Spectrophotometric method was used for study curcumin content and curcumin per cent was calculated by the standard formula. The curcumin content observed in 22 accessions varied from 0.29-8.9%. Curcumin content was highest in Lachin (8.9%) followed by Megha (8.3%) and lakadong (7.7%) in genotype collected from Jaintia Hills and RiBhoi district of MeghalayaThesisItem Open Access Genetic fidelity assessment of in vitro regenerated king chilli (Capsicum chinense Jacq.) using molecular markers(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2018) Nongrum, Careen; Choudhury, V. K.Chilli belongs to the family Solanaceae. It is a self-pollinated plant having diploid no. of chromosome (2n=24). King chilli is found to be a natural hybrid and occupies a taxonomic position between C. chinense and C. frutescens, clustering more closely with C. chinense group. Capsicum chinense Jacq. cv. is one of the hottest chillies. It is characterized by very high capsaicinoid content. They have various uses such as pungent flavor in food, natural plant colour and pharmaceutical ingredient. Lack of natural vegetative propagation in chilli limits the conservation of genetic purity and thus, micropropagation can be a remedy to this. Capsicum sp. has been found to be recalcitrant under in vitro conditions. The regeneration ability depends on the explant type, genotype and plant growth regulators. True-to-type clonal fidelity is however one of the most important prerequisites in the micropropagation of any plantspecies. High concentrations of different plant growth regulators may induce somaclonal variation at different developmental stages, especially in case of callus derived plantlets. The present study was carried out to develop efficient in vitro regeneration procedure using different explant materials and variable composition and combination of plant growth regulators and for assessing genetic fidelity using RAPD and ISSR markers. Out of 20 MS media supplemented with variable concentrations and combinations of plant growth regulators, 12 media were seen to effective. The maximum number of shoots per explants was seen in MS18 (20 μM BAP + 10 μM IAA) with an average of 2.80 shoots per explant with an average response of 61.13%, the length of shoot was seen to be longest on MS20 (20 μM KIN and 10 μM IAA) having an average length of 2.19 cm and percentage response 78.87%. Analysis of variance showed significant difference for explants for no. of shoots per explant and highly significant for shoot length at 0.05, level of significance. For nodes and internodes, the shoots were not formed in MS media supplemented with both cytokinins and auxins. For fidelity assessment, 16 out of 28 selected RAPD primers generated a unique set of amplification products ranging in size between 200 and 2900 bp, total of 119 fragments within 200 bp and 2900 bp. The number of fragments in each primer ranged from 2 to12.10 out of 13 selected ISSR primers generated a unique set of amplification products ranging in size between 250 bp and 2000 bp, total of 58 fragments within 250 and 2000bp. The number of formed fragments in each primer ranged from 2 to 9. Shoot tips, nodes and internodes in vitro regenerated explants showed monomorphism except for leaf explant through indirect regeneration showed polymorphism corresponding to the mother plant. For standardizing a protocol for in vitro regeneration of King chilli, an effective procedure was found after trial with different explants and combinations and concentrations of growth regulators. The regenerated explants show true-to-type maintaining fidelity except for indirect regeneration. These explants can be used for micropropagation as they maintain the fidelity which would be beneficial as King chilli is an elite plant.ThesisItem Embargo Genetic fidelity assessment of In Vitro regenerated Sweet Flag (Acorus calamus) using molecular markers(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-01) Purushottam, Paraskar Shyam; Meitei, Ng. TombisanaSweet flag (Acorus calamus) is a perennial wild herb belonging to the genus Acorus, a member of family Acoraceae. It is widely known for its high medicinal properties, due to which it has been over-exploited from their wild habitat. As a consequence, the plant has now become endangered species. In order to conserve this species, tissue culture has been found as one of the best approach which can be capable to scale up the production at large scale in order to fulfil the demand of pharmaceutical/cosmetic industries. True to type clonal fidelity is however, important factor for conservation and micropropagation at large scale. Hence, present study was carried out to develop an efficient in vitro regeneration protocol for sweet flag by using different concentrations and combinations of plant growth regulators and to assess the genetic fidelity across micropropagated plants by comparing with the mother plant. For surface sterilization of explants, treatment of fungicide (0.2% Bavistin) for duration of 30 minute followed by immersion in 70% ethanol for 30 seconds and mercuric chloride (0.1%) treatment for 15 minutes was found effective and showed the least contamination after four weeks. MS solid media (0.8% agar) was found more promising than MS semisolid media (0.6% agar) in relation to explants establishment. Out of the 12 different MS media fortified with variable concentrations and combinations of plant growth regulators, 6 media were seen to be give a positive response on explant establishment. The maximum number of shoot per explant was observed in treatment T3 (MS+15 μM TDZ) with an average of 7.67 shoots per explants with an average explant response of 60%. The length of shoot was seen to be longest on treatment T11 (MS+10 μM BAP+5 μM NAA) having an average length of 6.88 cm with 80% explant response. The maximum number of roots (6.50) and root length (3.50 cm) was observed in MS medium containing 10 μM IBA. Analysis of variance showed significant variation among treatments for all the characters studied. For evaluating the genetic fidelity of the in vitro regenerated plants, 40 RAPD primers were used and compared with the mother plant. Of the total of 40 RAPD primers, 20 primers produced a total of 70 fragments ranging from 350bp to 2500bp. The amplified bands of all the samples of in vitro regenerated plants along with the mother plant were monomorphic. The results demonstrated the genetic stability of the in vitro regenerated plants using rhizomatous buds as the explant. Thus, the protocol standardized in the present study can be recommended for future large scale production of true to type plants and subsequent conservation of A. calamus.ThesisItem Open Access Genetic variability analysis of Faba bean (Vicia faba L.) genotypes of North East hill region of India using morphological characteristics and SSR Markers(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2020-10) Balaji S.; Meitei, Ng. TombisanaFaba bean (Vicia faba L.) (2n=2x=12) belonging to Fabaceae family is the sixth most important leguminous crop in India. The world production of faba beans is 4.3 Million tons from total cultivated area of 2.55 Million hectares. It is popularly knowns as Kala Matar and Bakla (Hindi) and Hawai- mubi (Manipur). In India, the potential of this crop is not fully utilized. It is grown in small area, in states of Bihar, Madhya Pradesh, Eastern Uttar Pradesh, Haryana, Manipur and Nagaland. In North Eastern Hill region of India, it is mainly cultivated as winter season crop and it gives better yield when grown as intercrop with maize. In India, diverse genetic variability of this crop is naturally presented. Therefore, the present study was undertaken with 21 genotypes including two check varieties (Pusa Udit and Pusa Sumeet) collected from different districts of Manipur, Nagaland and Meghalaya using different morphological traits and 30 SSR markers. Highly significant variation was observed among the genotypes for all traits except Biological yield. The highest GCV and PCV percentage was recorded for the traits of 100 seed weight, seed yield/plant, Harvest Index. Shannon Diversity Index (SDI) showed maximum diversity for the trait of biological yield (2.846) and minimum for harvest index (0.773). From dendrogram, cluster A (5) & cluster B (16) number of genotypes formed in morphological studies. Genetic distance showed maximum of 9 and minimum of 2. Based on molecular work, the genetic variability was assessed with 30 SSR primer pairs, while 15 primer pairs showed polymorphic bands from 2 to 3 alleles per locus. The results showed highest PIC, major allele frequency, observed heterozygosity, gene diversity for the 15 markers as 0.558, 0.905, 0.762 and 0.571 respectively. Genetic distance among the genotypes ranged from 4 to 34. From dendrogram, cluster A (6) & cluster B (15) number of genotypes formed in molecular studies. From analysis of molecular variance, source of variation was higher among the individuals within population (72%). Significant and positive correlation was exhibited between the genetic distance of morphology traits and molecular markers. These results indicated that faba bean genotypes used in the study have a narrow genetic basis.ThesisItem Open Access In vitro regeneration and genetic diversity analysis in soybean using SSR markers(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2019) Theunuo, Sakuonuo; Khanna, V. K.Soybean (Glycine max) is classified in the family, Fabaceae. It is the most important legume in the world, contributing to 25 % of edible oil. Its production is however limited as it is highly susceptible to diseases and pests. Also, soybean has a narrow genetic base. An in vitro regeneration protocol was standardized using auxin (IBA) and cytokinin (BAP, KIN) in two genotypes of soybean, CSB 10084 and RVS 2011-3. Ten treatments each supplemented with various concentrations and combinations of PGRs and two explants, single coty-node (SCN) and double coty-node (DCN) were taken. MS containing 2 mg/l BAP gave 100% response in the explants of both the genotypes. MS containing 1 mg/l KIN took the least amount of time i.e. an average of 1.83 days to establish, in double coty-node of RVS 2011-3 and 2.50 days in single and double coty-node of CSB 10084. The longest shoot length of 4.27 cm on an average and 3.77 cm was recorded in double coty-node of CSB 10084 and RVS 2011- 3, respectively, in MS containing 2 mg/l BAP. Single coty-node of CSB 10084 gave the highest number of shoots with a mean of 2.67 in MS augmented with 1.75 mg/l BAP and 1 mg/l KIN. Multiple shooting was also observed in double coty-node of CSB 10084 in MS containing 2 mg/l BAP. After the regeneration of shoots, the plantlets were transferred to MS medium containing different rooting concentrations and MS containing 0.75 mg/l IBA gave the longest roots and MS containing 0.5 mg/l IBA gave the maximum root number. Molecular diversity analysis can broaden the genetic base and provide an insight about genetic variability which can go a long way in improving the crop. Therefore, diversity assessment was carried out in 30 soybean accessions with the help of 20 SSR markers. All the markers showed polymorphism producing a total of 79 alleles. Each marker generated 2 to 5 alleles. The PIC values ranged from 0.324 to 0.649. The PIC value was highest in Satt571, Satt230 and Satt129 with a value of 0.649, 0.645 and 0.638, respectively and thus were more informative than the rest of the markers. Dissimilarity values ranged from 0.05 to 0.95 which reveal that greater variability exists between MACS 13 and PS 1024 and NRC 137 and ADT 1. Based on the dissimilarity values, a dendrogram was constructed by using neighbor joining UPGMA. It revealed three major clusters with 13 genotypes each in the first two major clusters and in the third major cluster, 4 genotypes.ThesisItem Embargo In Vitro regeneration and genetic fidelity assessment of Red Ginger Lily (Hedychium rubrum)(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-02) Kishor B.; Kishor B.; Meitei, Ng. Tombisana; Meitei, Ng. TombisanaHedychium rubrum is a rare species native to India's North Eastern area. It's a small plant with a reddish stem, green lanceolate glabrous leaves, and vivid red orbicular flowers that can grow up to 4 feet tall. It has no aroma and is a potential decorative plant because its flower has a tall spike with crimson bracts, which is appealing on its own. The plant is listed as an endangered vascular plant species in India on the red list. Because of its potential as an ornamental herb, and because it is collected from the wild, further methods of multiplication are needed to ensure its survival. True-to-type clonal fidelity is however one of the most important prerequisites in the micropropagation of any plant species. Hence, the present study was carried out to develop an efficient in vitro regeneration procedure using rhizome buds and variable composition and combination of plant growth regulators and assessing genetic fidelity using RAPD markers. For explant sterilization 0.2% Bavistin (30 min) treatment followed by 70% ethanol treatment and 0.1% mercuric chloride (15 min) treatment was found most effective. Also, MS solid media gave a better response as compared to semi-solid media, which resulted in poor explant establishment. Out of the 12 different MS media supplemented with variable concentrations and combinations of plant growth regulators, 6 different media were found to be giving good response. The maximum number of shoots per explants was seen in T12 (15 μM BAP + 5 μM NAA) with an average of 4.25 shoots per explant and, the length of shoot was seen to be longest in the same medium with an average length of 7.56 cm. IAA and IBA was used for rooting and recorded maximum root length (7.75 cm) and number of roots (11) in10 μM IAA. Simultaneous shooting and rooting were observed in treatment T10 (5 μM BAP + 5 μM NAA) and T11 (10 μM BAP + 5 μM NAA) with average of 2.25 and 3.3 number of shoots respectively. ANOVA was performed for all the characters under study at 0.01, level of significance and showed significant difference among the treatments. For fidelity assessment, 44 RAPD markers were used of which 19 RAPD primers generated unique set of amplification products with a total of 83 fragments ranging in size between 250 bp and 3100 bp. The number of fragments in each primer ranged from 1 to 8. Similar banding patterns were observed in all the in vitro regenerated plants when compared with their mother plant thus concluding that the genetic fidelity is maintained among the in vitro regenerated plants. Thus, from the study, an efficient in vitro regeneration protocol for Hedychium rubrum has been developed which also produced true to type plants. The protocol developed in the study can be recommended for future micropropagation work.ThesisItem Open Access In vitro regeneration and genetic variation analysis using molecular markers in selected banana cultivars of Meghalaya(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2018) Marak, Flamia Chimachi; Choudhury, V. K.Banana (Musa spp.) is one of the important fruit and vegetable crop of India. Limitation of low multiplication, damage and diseases prone problems with conventional propagation can be overcome by plant tissue culture method which has potential for rapid multiplication of disease free plants from a single plant and independent production of planting material all year round. The genetic variation study can help in utilizing the genetic potential of genotypes in crop improvement programmes such as for stress resistance or disease resistance. Such studies will enhance the knowledge about the genotypes and help in germplasm conservation of important Musa spp. Therefore, the present study aimed at developing a protocol for in vitro regeneration of two banana genotypes (Songdu and Malbhog) and to assess the genetic variability among 10 banana genotypes (Malbhog, Songdu, Samol, Saheb, Watre, Rekbok, Champa, Champa gisim, Monaratchi and Atigola) collected from different region of East Garo Hills district of Meghalaya which will definitely help insubsequent crop improvement programmes. Thirteen various concentrations and combinations of plant growth regulators and one control (only MS media) were used, out of which only 9 media composition gave response. MS11 (50 μM BAP + 30 μMIAA) produced the longest shoot length with an average of 9.5 cm in both the cultivars which Songdu accounts with an average of 9.73 cm and Malbhog with an average of 9.26 cm. ANOVA performed for shoot length for different concentration and combinations of PGR showed significant difference at 0.05, level of significance. More number of leaves was observed in MS8 (40 μM BAP + 15 μM NAA) and MS10 (20 μMBAP + 5 μM IAA) with an average of 3.16 number of leaves in both the varieties. The highest number of roots in Songdu and Malbhog was observed in RM5 (10 μM IBA) than in RM4 (10 μM BAP+ 5 μM NAA) with an average of 3.5 and 2.5 number of roots respectively. Higher root length was also observed in RM5 than in RM4. The genetic diversity analysis was done using RAPD and SSR markers. The generated genetic distance ranged from 4.429 to 6.701. The Euclidean distance showed that the highest genetic distance of 6.701 was found between Champa and Atigola. While the lowest genetic distance of 4.429 was found between Champa and Malbhog. The dendrogram generated based on both the markers (SSR and RAPD) indentified two major clusters which were again subdivided and grouped according to how closely related the genotypes are. The studied in vitro regeneration can be helpful regarding the choice of concentration and combination of plant growth regulators and MS11 and MS5 (20 μMBAP + 5 μM NAA) can be recommended for shoot elongation after subculture in micro propagation studies. The genetic diversity studies conducted will help in further crop improvement programmes in identification and germplasm preservation.ThesisItem Open Access Isolation and characterization of tissue specific promoter from Pigeon pea(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2017-12) Verma, Satish Kumar M.; Tyagi, WrichaPigeon pea belongs to legume family which has a specific physiology including organs like nodule and pod. The understanding of genes/transcripts involved in development of various organs of pigeon pea is limited. The present study was conducted to identify tissue specific transcripts from pigeon pea by ortholog-based approach and to isolate and characterize 5'UTR region of tissue specific transcript(s) from pigeon pea. An in silico search was conducted using phytozyme database and plant transcription factor database (PTFDB) separately for four tissues viz, seed, flower, leaf and pod. A total numberof 39 putative transcription factors (TFs) belonging to 18 different TFs families were identified for pod. The highest numbers of transcripts were observed for GATA and C2H2 (Cys (2) His (2)) zinc finger family members, and similarly, 54 TFs belonging to16 different TFs families were identified for flower tissue. Higher transcript numbers were observed for MYB (Myelobastosis), MIKC (MADS Intervening K-box Cterminal) family. Likewise, a total number of 92 TFs belonging to 28 different TFs families were identified for leaf tissues. In this case the highest number of TFs belonged to C2H2 and NAC (No Apical Meristem CUC domain) containing families. Similarly, a total number of 23 TFs with 12 different TFs families were identified expressing in seed were like MYB, NAC. Based on in silico analysis 10 transcription factors for each of the four tissues were identified. Out of the 10 transcription families (36280, 13238, 20528, 25279, 13634, 00220, 11572, 11926, 13354, 19191) identified for seed specific expression, five (00220, 11572, 11926, 13354, 19191) were targeted for RT-PCR using five different tissues (seed, flower, leaf, pod and stem). Simultaneously, members of auxin and TCP transcription family were also targeted for validation. Internal gene for studying tissue specificity in pigeon pea was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This gene was selected based on RT-PCR of ß-actin (ACT), (GAPDH), ubiquitin family (UBI), elongation factor 1-A (EF1A) and ß-tubulin (TUB) on five different tissues (seed, flower, leaf, pod and stem). Our RT-PCR data led to conformation of 2 transcripts, 00220 and 11572 as seed specific pigeon pea transcripts. While 00220 is a member of MADS (MCM1/AGAMOUS/ DEFIEIENS/SRF) transcription family,11572 is a member of TALE (Three Amino acid Loop Extension) transcription family.Thermal asymmetric interlaced PCR (TAIL PCR) approach led to identification of 229 bp 5' UTR (Untranslated Region) of 00220 transcript. Upon sequencing and analysis using Plant CARE database presence of cis-acting elements like ARE,CAAT-box, G-box, GT1-motif, Skn-1 motif, and TATA-box was detected, three unnamed motifs were also identified at -10 and -66 position. The transcripts identified and putative promoter isolated in this study after further characterization might generate useful information regarding development in pigeon pea.ThesisItem Embargo Molecular characterization of advanced breeding lines of rice using candidate gene based markers(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2022-01) Bora, Jayanta; TyagiRice (Oryza Sativa) is one of the most important cereals which constitute the staple food of two-third of the world population. The North Eastern Hill region is the hotspot for the blast pathogen Magnaporthe grisea along with low phosphorus availability which result in low yield of rice. With the development of genomics and large number of QTL/genes reported for various traits along with markers validation it is become easier to develop haplotype for various genotypes among rice having all the favourable genes for higher yield against all biotic and abiotic stresses. The present study was carried out to determine the allelic status of the candidate genes in the advanced breeding lines of rice. A total of fifty-four ULRC (Upland Rice Cultivars) advanced breeding lines were selected and genotyping was done for candidate genes reported for high yield (SPIKE and Gn1a), low phosphorus (P) tolerance (PsTOL1, OsMLO8, OsWD40-2 and Os02g08018) and blast resistance (Pi 54 and Pita) which were then amplified using reported polymorphic/functional markers.The comparison of molecular data with yield data revealed that the presence of (type-5 allele) for the SPIKE gene shows a significant advantage in terms of yield while the presence of other genes does not shows a significant difference in yield. The frequency of desirable allele NAL1 for the SPIKE gene was the lowest (10.26%) among all the other genes in the ULRC advanced breeding lines. Haplotype analysis was carried out for fifty-four ULRC lines in which three lines have all the desirable alleles fixed for high yield, low P and blast resistance. A minimum of three desirable alleles were fixed for all the ULRC lines, all total nine haplotypes were found among the fifty-four ULRC lines. Ten lines have all the desirable alleles fixed for yield, twenty-three lines have all the desirable alleles fixed for low P tolerance and thirty-one ULRC lines have both the desirable alleles fixed for blast resistance. The highest Euclidean genetic distance was 2.44 and least was 1 as calculated between the fifty-four ULRC advanced breeding lines using Roger’s estimation. Cluster analysis for fifty-four ULRC advanced breeding lines using Ward’s method revealed two major clusters represented by lines 36-184 and 36-285 which were further sub-divided into small group of clusters. From the overall findings the marker SPIKE-indel3 detecting the type-5 allele can be facilitate in MAS selection for rice improvement programme.ThesisItem Open Access Molecular characterization of mineral stress responsive rice genes and identification of their putative orthologs(2021-08) Boobana P.; Tyagi, WrichaPlants majorly experience low phosphorous (P) and high iron (Fe) levels in acidic soils (pH≤5), which leads to reduction in crop productivity. These stresses lead to various changes at physiological and molecular level and induce various stress responsive genes. The aim of this study was to analyze the expression of already reported mineral stress (low P) responsive rice genes such as PHR3, PHO1.2 across diverse and contrasting genotypes such as LR11, LR23, LR26, KMR3, BAM350 and BAM811. Expression of genes like AAO2, PRI reported in high Fe stress response were also analysed in these genotypes using qPCR. The stress conditions such as low Pi (Pi = 0.015 mM/L) and high Fe (Fe2+ = 2.56 mg/L) was given through hydroponic sand culture method along with control (Pi = 0.35 mM; Fe2+ = 0.02 mM) and differential gene expression (analysed by qPCR) after 24 hrs, 48 hrs and 7 days of treatment was evaluated in shoots. The physical parameters such as shoot length, root length, fresh shoot and root weight, dry shoot and root weight were also measured. The selected low P tolerant genotypes performed well under both low P and high Fe conditions. In response to low P conditions, all the tolerant genotypes had lower malondialdehyde (MDA) levels while susceptible genotypes had higher levels. In response to high Fe conditions, tolerant LR23 showed very high levels of MDA. Relative downregulation in expression of PHR3 in response to 24 hrs of low Pi treatment was observed for all the genotypes except BAM811 and LR11. However, in response to 48 hrs of low P stress, the low P tolerant genotypes LR23 and LR11 maintain the expression of PHR3. LR26 show a slight decrease and KMR3 shows a significant increase. The two low P susceptible genotypes BAM350 and BAM811 show a drastic reduction in PHR3 expression levels. PHO1.2 (responsible for transport of P from roots to shoots) expression in response to 24 hr low P treatment in low P tolerant genotypes (LR23 and LR26) was significantly upregulated whereas in LR11 the expression levels were significantly higher and maintained under both abundant and low P conditions. The expression levels were significantly downregulated after 48 hrs of low Pi treatment. KMR3 showed reverse trend for PHO1.2 in response to 24 and 48 hrs of low Pi stress. The expression levels of PHO1.2 in low P conditions were significantly low as compared to control in susceptible genotype BAM811. Genotype BAM350 showed high levels of PHO1.2 only in response to 48 hrs of low P treatment. Our data suggests that the expression of key genes involved in low P tolerance response is either delayed or downregulated. OsPHR3 and PHO1.2 expression has not been checked under high Fe conditions, till date. Our data suggests that PHR3 and PHO1.2 are highly induced in shoots after 48 and 24 hrs of high Fe stress, respectively. The expression levels of PRI (responsible for high accumulation of Fe in plants) in response to 24 hrs of high Fe treatment was significantly upregulated in all the genotypes with expression levels many folds more in LR26 and LR11. After 48 hrs of high Fe treatment, levels of PRI were downregulated in low P tolerant genotypes LR26, LR11 and KMR3. However, LR23 showed high levels of PRI expression. Susceptible genotype BAM350 the levels of PRI were many fold higher in high Fe conditions. Our experiments suggest differential expression of PRI in low P conditions as well. Under high Fe treatment for 7 days, oxidative stress as measured by levels of AAO2 was observed in BAM811. Genotypes LR11 and KMR3 also showed higher levels of AAO2 under Fe stress. The expression levels of AAO2 were higher in LR23, LR11 and KMR3 after 7 days of low P treatment. In low P tolerant genotype LR26, AAO2 downregulation was observed. Low P susceptible genotype BAM350 also showed significant downregulation whereas no change in AAO2 levels was observed for BAM811. In order to understand the role of rice genes involved in mineral stress in other species, orthologs of selected genes were identified in Brassica and linseed using bioinformatics tools. Orthologs of PHR3, AAO2, PRI in both Brassica and linseed and ortholog of PHO1.2 only in Brassica could be identified. The functional domains such as MYB, Myc, multicopper oxidase, SPX-EXS were all conserved across rice, Brassica and Linum and proteins from these three species could be aligned using ClustalW. The phylogenetic tree generated showed 3 distinct groups where Brassica species (B. napus, B. oleracea, B. rapa) formed one closely related group, and Linum and rice proteins were placed as two separate groups. An attempt to amplify the putative orthologs was made but further standardisation of the primers designed is required. This implies opportunities for functional characterisation of these mineral stress genes in Brassica and linseed for future use in molecular breeding.ThesisItem Open Access Molecular characterization of selected RILs derived from two low phosphorus tolerant rice genotypes(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2018) Sharma, Takhenchangbam Oshin; Tyagi, WrichaRice (Oryza sativa L., 2n=2x=24) is one of the most important cereal crop of the world. Phosphorous (P) being one the most important nutrients for growth and development, efficient phosphorus uptake capacity (PUP) and internal phosphorus use efficiency (PUE) is highly desirable. Production of rice in the North Eastern region of India is highly affected due to deficiency of P in soils resulting in low productivity. Therefore, it is necessary to develop rice genotypes which are tolerant to P deficiency. PSTOL1 is the only known gene successfully deployed in the field for better PUP in poor soils. Previously, data generated in our lab had identified two rice genotypes performing well under acidic soils having low P; LR23 (Sahbhagi Dhan; PsTOL1+) and LR26 (Chakhao Poreiton; PsTOL-). A recombinant inbred line (RIL) generated from LR23 X LR26 was phenotyped and genotyped with the following objectives: a) to characterize a set of top performing RILs with respect to morpho-physiological characters and b) to determine the parental allelic contribution in selected RILs with respect to SSR markers. A set of 1600 individuals (F5) were grown in the lowland acidic field and 17 different phenotypic traits were taken. On the basis of tiller number at 30 days and 60 days after transplanting, spikelet fertility %, 100 seed weight, total number of filled grains, a set oftop 20 performing lines were selected. These lines showed variation for the 17 phenotypic traits with lines like 172 (3) had higher panicle length and leaf area; line 34 (3) had higher tiller no. at 30 days, dry weight and total grain. Similarly, 81 (4) performed well for PUE and panicle number. Correlation analysis revealed that filled grain, 100 seed weight, dry weight and total grain were significantly correlated with grain yield (P value=0.57). Parental polymorphism survey performed using 157 SSR markers and 34 SNPs revealed 25 and 17 polymorphic markers, respectively. The STRUCTURE analysis to identify percentage ancestry (cut off >80%) in the 20 lines revealed that lines 33 (3), 34 (3), 65 (3), 122 (3), 81 (4), 59 (4) and 222 (3) are LR26 type whereas 114 (2), 188 (4) and 193 (1) are LR23 type. Chi-square test for goodness of fit revealed that HvssR02-14, RM527, snpOS0303, snpOS0304, snpOS0305, snpOS0306, BADH2 and Chalk5 showed significant values, suggesting preponderance of LR23 allele in markers 02-14 and chalk5 and LR26 allele other six markers. A sand based screening using Yoshida solution on these 20 lines along with the parents revealed significant differences in control and treatment conditions for all the six traits. Correlation for the sand culture data revealed significant association of total dry weight with total fresh weight and of root dry weight with total dry weight under normal phosphorus condition. Root dry weight under low P conditions was significantly correlated with total fresh weight. Thus, the selected top performing lines were distinct for both phenotypic and genotypic traits and therefore, can be used for further selection of best lines under lowland acidic soil conditions.ThesisItem Open Access Molecular mapping for low light intensity tolerance using the contrasting rice genotypes of Eastern India(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2017-05) Dutta, Suvendhu Sekhar; Rai, MayankRice (Oryza sativa L.) is the most important cereal crop of the Eastern and North-Eastern India. Low light induced problems include high tiller mortality at vegetative stage, reduction in spikelet number, spikelet sterility and reduced dry matter production. A set of contrasting genotypes in the NEHR mini core set during kharif-2014 and a set of 110 rice genotypes from different parts of eastern India were screened for low light intensity tolerance during kharif-2015. Total grain yield, spikelet fertility, biological yield, number of effective panicles, specific leaf weight and chlorophyll content were found to be the key traits response to low light intensity (30 % less than normal). A panel of forty six genotypes made up of top twenty three tolerant and susceptible genotypes each, based on field screening in kharif-2015 along with previously reported checks (Swarnaprabha- tolerant and IR 8-suceptible) was used for mapping study. By using the online rice databases, a set of ninety eight genes previously reported for light response were identified, which was then narrowed down to twenty. Molecular markers (forty five HvSSRs and newly designed twenty eight gene based primers) were used for molecular mapping for low light intensity tolerance. Standardization of these seventy three primers in a set of eight contrasting genotypes led to identification of fifteen HvSSR and two gene based polymorphic (CAU-CG-ILA1-3 and CAU-CG-RK3) primers. These polymorphic primers were run on the mapping panel. Marker-trait association studies based on t test and regression analysis revealed seven HvSSR and one gene based markers associated with the key traits viz., HvSSR01-66, HvSSR02-54 and CAU-CG-ILA1-3 with grain yield, HvSSR02-52, HvSSR06-56, HvSSR06-69 and HvSSR09-45 with spikelet fertility and with biological yield HvSSR02-44, HvSSR02-52, HvSSR06-69, HvSSR09-45. Expression analysis for the genes used for mapping (LGD1, PNH1, ILA1, CAB2R and LP2) was performed on a panel of eight contrasting rice genotypes grown under normal light and low light intensity (75 % of ambient) at two time points (one hour and two days). Relative expression of the five selected genes in the leaf next to flag leaf showed that there was significant down regulation of transcripts after one hour of low light treatment insusceptible genotypes for genes LGD1 and PNH1, whereas the transcript levels were maintained in tolerant genotypes. Transcription was significantly down regulated and up regulated, respectively for ILA1 and LP2 genes specifically in tolerant genotypes under one hour low light treatment. However, no significant differences in gene expression levels for the five genes were observed after two days of stress treatment for tolerant and susceptible genotypes. The genotypes and markers associated with traits identified in this study, after further evaluation, can be used to develop more productive rice varieties for low light affected regions.ThesisItem Embargo Molecular signature on phloem sap of the enhanced host susceptible phase to Liphaphis erysimi in Brassica species.(College of Post Graduste Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2022-12) Devi, Ahanthem Malini; Mondal, Hossain AliThe importance of oil and by-products from oilseeds crop in the lives of humans and cattle cannot be underestimate as oilseed crops are lucrative crop with great potential for enhancing human diets, preventing hunger and food insecurity. However, oil seed crops like rapeseed-mustard are extremely susceptible to aphid attack mainly mustard aphid (Liphaphis erysimi) and it serves at the key limiting factor in achieving a surplus productivity with reported losses in yield up to 90%. Mustard aphid is exclusively phloem feeders and they utilized their highly specialized mouthparts to suck and ingest simple sugars, proteins, and amino acids enriched phloem sap from plant sieve elements. The precise access of the host plant's Sieve Element (SE) cell by the aphid stylet is a crucial factor for clonal proliferation. The objective of the present study was to identify optimized factors enhancing host susceptibility toward aphid clonal proliferation and uncover molecular signature of the aphid herbivore phloem sap in comparison to control. Among the 5, 10 and 20 aphid releases on random leaf foliage in B-9, B-54, Pusa Bold, Rohini, RGN-384 and RMM-09-10 revealed that ten aphids enhanced host susceptibility as compared to other aphid doses except in RMM-09-10. It was also recorded that aphid inoculum at 6 AM enhanced host susceptibility in comparison to 6 PM aphid release. Moreover, Relative humidity was a modulating factor in enhancing host susceptibility and 95% RH was the most effective over 70% and 50% RH. Among temperature, 160C and 220C promoted aphid clonal proliferation in comparison to 280C and 340C. The age of the plant was also a factor in modulating host susceptibility. From the present study, an initial aphid inoculum (10), timing of aphid release (6 AM), humidity (95% RH), temperature (220C) and age of plant enhanced host susceptibility towards aphid clonal proliferation. The maximum absorbance of phloem exudate was evidenced from 4 number of leaves, 0.5mM EDTA concentration and 12 hours dark mediated phloem sap isolation without compromising secondary induced stress. The absorbance of aphid herbivore phloem sap at 215 nm is significantly higher in Rohini, RGN-384 and RMM-09-10 but remains same in B-9, B-54 and Pusa Bold. The absorbance of aphid herbivore phloem sap at 260nm is significantly higher in B-9, Rohini and RGN-384 and significantly lower in B-54, Pusa Bold and RMM-09-10. The absorbance of aphid herbivore phloem sap at 280nm is significantly higher in B-9, Rohini and RGN-384 and significantly lower in Pusa Bold and RMM 09-10 but remains same in B-54. The GC-MS analysis of aphid herbivore phloem exudates isolated at the earliest and significant time point identified several reported antimicrobial compounds which shows that aphid herbivore targets on the in-built anti-microbial defense in the phloem sap and upon aphid herbivore, the anti-microbial metabolites concentration was reduced. This finding initiated extended curiosity about the microbiota enrichment from the aphid herbivore phloem exudates. The further study indicated that aphid herbivore enriched phloem microbiota at the earliest and significant host susceptible phase. One of the identified metabolites, dodecanoic acid (DA) showed an anti-biotic effect on aphid herbivore mediated phloem microbiota. The crucial findings from the present study indicated that aphid herbivore reduce the anti-microbial concentration in the phloem sap at the earliest and significant time point which coincided to the enhanced host susceptibility. Therefore, aphid herbivore mediated microbiota inoculum in the phloem sap is a novel phenomenon being reported for the first time in plant-aphid interaction biology.ThesisItem Open Access Production of virus free quality planting materials in chilli var. dalle khursani by in vitro meristem tip culture /(College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2019) Bhattacharjee, Saumika; Meetei, Ng. TombisanaCapsicum spp. is curtailed as one of the wider known spice crops hailing around the world with at least twenty five known cultivated varieties. Dalle Khursani (Capsicum annum) an indigenous variety of the foothills and slopes of Sikkim and Darjeeling is famously used for its powder and pickles owing to its high degree of pungency through the chemical Capsaicinoids. Capsicum like its counter crops is to a larger extent susceptible to the second largest destructible pathogens i.e. viruses. Among which the name of Cucumber mosaic virus draws remarkable attention as being a source of extensive yield losses and deteriorated fruit quality. The present study was taken with the following objectives: i) Molecular characterization of virus infecting Dalle Khursani based on coat protein gene sequence ii) Production of virus free plants by in-vitro meristem tip culture Cucumber mosaic virus (CMV) was visually detected in the field based on its symptoms and later confirmed through molecular techniques. RT-PCR using CMV coat protein gene specific primers CPTALL 5’ and CPTALL 3’ were utilized to confirm the presence of the virus. The PCR product with the expected size of 950 bp was sent for sequencing and the sequence obtained on database search using BLAST confirmed to belong to coat protein of CMV. The phylogenetic study with other CP gene reference sequences showed the isolate to cluster with other CMV belonging to subgroup IB whereas the reference sequence of CMV subgroup IA and II clustered separately thus indicating the present CMV isolate to belong to subgroup IB. For the production of virus free plants, shoot tips from confirmed infected plants were used as source of explants for meristem tip culture. Meristems of 0.1- 0.5 mm were dissected from the infected shoot tips and cultured in 29 media concentrations all set with five different plant growth regulators i.e. BAP, KIN, TDZ , NAA and IBA in addition to the presence of a control. Of the 29 combinations, 20 showed signs of regeneration wherein the concentrations of TDZ particularly 5 μM TDZ gave the best performance in terms of shoot length (2.82 ± 0.09 cm), number of shoots (2.81 ± 0.31) and percentage of response to survivability (88.83%). The in vitro explants bearing roots were transferred to artificial soil for hardening and 75% were recorded to survive. Leaf samples from the in vitro generated plants along with the control were once again subjected to the norms of molecular methods to confirm the presence/absence of CMV. Absence of bands confirmed the regeneration of virus free plantlets.