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Central Agricultural University, College of Post Graduate Studies in Agricultural Sciences, Umiam

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2016 - 2019142020 - 20227
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Subject: Biotechnology and molecular Biology ×
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    ThesisItemOpen 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.
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    ThesisItemOpen 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, Wricha
    Rice (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.
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    ThesisItemOpen 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, Mayank
    Rice 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.
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    ThesisItemOpen 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, Wricha
    Rice (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.
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    ThesisItemOpen 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.
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    ThesisItemOpen 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, Wricha
    Rice (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.
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    ThesisItemEmbargo
    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 Ali
    The 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.
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    ThesisItemOpen Access
    Standardization of in vitro regeneration protocol and genetic diversity analysis in blackgram using RAPD markers /
    (College of Post Graduate Studies in Agricultural Sciences, CAU-Imphal, Umiam, 2019) Vedulla, Usha Tejaswini; Khanna, V. K.
    The productivity of blackgram (Vigna mungo (L.) Hepper) has become a limiting factor since the crop is adversely affected by various biotic and abiotic factors. As it is a self-pollinated crop having narrow genetic base, breeding is however difficult. Hence, immediate attention is required to improve the crop through transgenic technologies. In this study, a regeneration protocol was standardized using plant growth hormones such as cytokinins (BAP) and auxins (NAA and IBA) in three accessions namely KU-16-41, GP-115-IC-10703 and PANT-U-40. A total of 20 different MS media were used, of which MS media containing BAP 1.0 mg/l took less number of days for explant establishment in all the accessions with an average of 5.8, 5.8 and 6.6 days in KU-16-41, GP-115-IC-10703 and PANT-U-40, respectively. MS media containing BAP 4.0 mg/l showed best average percent of responding explants with 97.1% in KU-16-41, 98.4% in GP-115-IC-10703 and 95.8% in PANT-U-40. Shoot number was highest in media containing BAP 4.0 mg/l with an average of 9.8, 14.2 and 9.6 in KU-16-41, GP-115-IC-10703 and PANT-U-40, respectively. Length of the shoot was highest in media with BAP 1.0 mg/l and NAA 1.5 mg/l and this media was considered as the best since subsequent shooting and rooting was observed with an average root number of 10.4, 14.5 and 10.4 and average root length 5.4, 5.8 and 5.4 cm in KU-16-41, GP-115-IC-10703 and PANT-U-40, respectively. Those explants which did not show rooting were then transferred to rooting medium (BAP + IBA) where media with BAP 1.0 mg/l and IBA 1.0 mg/l was regarded as best since highest no. of roots and root length was recorded. The major limitation for low productivity in blackgram is lack of genetic variability. Hence, diversity analysis was carried out with 20 RAPD markers in 31 accessions of blackgram. All the 20 RAPD primers showed polymorphism by generating 271 polymorphic bands among a total of 278 bands with band size ranging from 0.13 to 3.0 Kb. The percent polymorphism was found to be 97.48%. PIC value ranged from 0.504 (OPG-04) to 0.861 (OPG-03). The primers OPG-03 and OPH-04 were found to be more informative as they generated highest PIC value. The dissimilarity coefficient generated among the 31 accesions ranged from 0.162 to 0.399. The dissimilarity value was found to be the highest (0.399) among the genotypes PANT-U-6 and GP-52-NO-5/31. Hence these two accessions are more diverse as they were genetically less similar. Least dissimilarity value (0.162) was found among the genotypes KU-34 and GP-46-99-213. In accordance with dissimilarity values, clustering was done using neighbor-joining UPGMA and three major clusters (I, II and III) were obtained with 13, 12 and 6 genotypes, respectively.
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    ThesisItemOpen 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. Tombisana
    Capsicum 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.