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ThesisItem Open Access Marker trait association studies for resistance to Cercospora Leaf Spot (CLS) in Mung bean [Vigna radiata (L.) Wilczek](Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2022-10-31) Sahoo, Jyoti Prakash; Samal, K.C.; Rout, G.R.; Lenka, D.; Mishra, M.K.n the present study, 90 mung bean genotypes were included for marker-trait association studies for CLS disease resistance using 66 SSR markers. CLS screening of genotypes were carried out using a 1-5 rating scale in natural field conditions during pre-rabi 2018 and pre rabi 2019 and in controlled greenhouse condition during pre-rabi 2019. Total genomic DNA was isolated from the leaf samples of all the genotypes, and SSR marker genotyping was performed. The genotyping and phenotyping data were analysed by using different websites and software packages. GCV was found high in number of clusters per plant, pod length, number of pods per plant, yield, and high for plant height. However, the genetic advance as percent of mean was found high for all the traits except number of seeds per pod, which was medium. PCA differentiated the traits in two major PCs with up to 85.656 % variation. Three promising varieties, i.e., PDM-11, NMK-15-12, and MH-13-23 were also found to be good yielder with other desirable traits and resistance to CLS disease. CLS resistance screening revealed 32 resistant accessions, and 35 moderately resistant accessions. Genetic diversity analysis revealed a moderate molecular diversity in the panel population as a wide variation in alleles showed a range of 80 bp to 300 bp with the average polymorphic information content value of 0.40. Estimation of allele frequencies by population revealed, some alleles of 9 markers (CEDG001, CEDG006, CEDG153, CEDG210, CEDG117, CEDG020, CEDG067, CEDG146, and CEDG071) had linked to CLS resistance. The molecular variance among populations and among individuals were found to be 58% and 42%, respectively. The maximum % of polymorphic loci was 75.76% in the resistance, followed by 56.06% in moderately resistant genotypes, with estimated Shannon diversity 1.308 and 1.399, respectively. The principal coordinate analysis (PCoA) revealed, the explanation of percentage variation by the first 3 axes with up to 71.37%. The archaeopterx tree differentiated the populations into two major clusters, i.e., I, and II, containing 45 number of genotypes each, which were again subdivided into different sub-clusters and sub-sub clusters. Therefore, it is concluded that the panel population used for the study possesses considerable genetic variation for CLS tolerance. The structure analysis categorized the panel genotypes into 2 structure groups. The linkage disequilibrium (LD) patterns of mung bean reflect its long history of domestication. The marker-trait association were detected based on presence of LD in the population. A sharp decline in the decay of disequilibrium was observed at 2-3 base pair for the linked markers, there after observed a very gradual decay for attaining equilibrium. As per the linked markers, 2 novel QTLs (qCls_2 and qCls_8) were detected for CLS resistance in the present investigation. Furthermore, novel QTLs were also identified for some of the quantitative traits, such as for plant height (qPht_8), for number of clusters per plant (qNcl_6 and qNcl_10), for hundred seed weight (qHsw_3 and qHsw_11), and for yield (qYld_2, qYld_7, and qYld_8) under CLS. The novel QTLs obtained from the present investigation could be further validated in diversified mung beangermplasms to understand the stability of the QTLs and then could be introgressed to elite mung bean varieties to develop CLS resistance lines. This study will help the breeding process towards CLS resistance in mung bean using genomic tools with special reference to QTL mapping and genomic assisted breeding.ThesisItem Open Access Molecular Mechanisms Underlying flowering and fruiting ability associated with circadian clock regulation in Vigna radiata under cold stress(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2022-12-06) Manasa S., Lekshmi; Rout, Gyana Ranjan; Panignihi, K.C.; Samal, K.C.; Dash, Abhiram; Dash, ManasiGreen gram or mung bean (Vigna radiata) is the major leguminous crop in tropical countries known for being highly proteinaceous and nitrogen fixing abilities. But, yield loss in mung bean is due to its susceptibility to low temperature is a serious concern for its cultivation and productivity. Low temperature causes flower drop, male sterility, impairs anthesis, and grainfilling. In the present study, screening the 204 genotypes (127 Indian varieties, 62 landraces and 15 recombinant inbred lines) of Vigna radiata for cold germination was carried out at National Institute of Science Education and Research. Further, all the analysis was done in two ways, first one using principal component analysis at 10 ͦ C, where PC1 and PC2 explained 57.7% and 18.7%, respectively. The second one was using stress indices like germination percentage, germination stress index, seedling length stress index, resilience capacity index and productive capacity index. The whole of 204 genotypes were categorized into resistant, susceptible and intermediate. So, PAU 911 was selected as the resistant genotype. PUSA 1672 was selected as the intermediate and PUSA 9531 was selected as the susceptible representative for further investigation. PAU 911 was able to show higher values, for leaf area and rootlet number. In case of shoot length and root length, PAU 911 and PUSA 1672 were found to be at par, but significantly higher than susceptible PUSA 9531. Biochemically, PAU 911 showed low membrane leakage, high proline accumulation, low injury index, higher NBT staining and low MDA accumulation, lower SOD activity. The pigments content was found to be on par with PUSA 1672, but was significantly higher than PUSA 9531. Physiologically, PAU911 was having advantage over the intermediate and the susceptible genotypes. PAU911 showed higher RH%, photosynthetic water use efficiency, transpiration rate and stomatal conductance. PAU 911 was also showed lesser temperature, sub-stomatal CO2 conc. and vapour pressure deficit. Photosynthetic assimilation was seen to be highest in PUSA1672. But, the decline in photosynthetic assimilation under cold stress condition was seen to be least in PAU 911, which makes it more desirable. Anatomically, PAU 911 showed an increase in trichome density under stress. The similar trend was seen in all the three genotypes. But, there was higher autofluorescence at 488nm observed in PAU911, when compared to the other genotypes, indicating the presence of higher secondary metabolites in the plant tissues. This accumulation helps in fending of adverse abiotic stress situations. Also, there was higher microtubule immunofluorescence found in PAU 911. Based on multi elemental analysis, there was a higher accumulation of sodium and boron ions were found in PAU911, indicating its innate ability to facilitate membrane stability and pollen tube growth. SDS-PAGE protein profiling showed the appearance of bands at 75 kDa under cold stress, along with changes in Rubisco LSU was also observed. Further, sequencing of the differentially expressed bands helped in understanding the genes associated with it. It was found that TTL1 gene was up-regulated in PAU911 under cold stress. This was further validated using qRT-PCR, which showed a 30-fold rise from control, 4.8-fold increase in PUSA 1672 and insignificant up regulation in PUSA 9531. TTL1 was characterized in silico. It was found that it had TPR and DNA J domains. Further, expression analysis of ICE1 showed a down regulation in all the three genotypes, providing an insight into the temperature range it needs in Vigna radiata to get up-regulated. Further, the expression pattern of evening complex (ELF3-ELF4-LUX) was also indicated a slight insight into that transcriptional-translational feedback loops that is happening in the genotypes under stress condition, during flowering and pod setting time.ThesisItem Open Access Evaluation of bioactive compounds from selected medicinal plant extract as root promoter potential(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Dash, Trupti; Rout, Gyana Ranjan; Samal, K. C.; Palai, S. K.Medicinal plants are used by Indians for various purposes since along. It has pharmaceutical application in drug making by utilizing its active chemical constituents and also some are consumed directly as food. Mankind being more health conscious and environment responsive are eagerly searching for natural substitute than different synthetic chemicals. Most of the active compounds present in the medicinal plants having various chemical properties including root promoting potentials. These compounds may be compared with known commercial growth regulators biochemically as well as using stem cuttings of ornamental plants. The present study investigated the presence of root promoting chemical constituents derived from the medicinal plant extract of ten different plant species such as Ocimum sanctum, Azadirachita indica, Nyctanthes, Ayapana triplinervis, Moringa olifera, Plumbago indica, Tinospora cordifolia, Typhonium, Piper betle and Mimosa pudica at 0, 1000ppm, 2000ppm, 3000ppm and 4000ppm concentration. Among the ten different medicinal plant extracts, Moringa olifera and Piper betle leaves extract showed shoot emergence as well as root induction from cuttings of Bougainvillea glabra within two weeks of planting. In control treatments (either without plant extract or growth regulators) did not show any positive response. However, the percentage of rooting is significantly similar with known plant growth regulators (IAA, IBA) with similar concentrations. The best treatments are tested for 24hr and 48 hr duration in three different replications. Further, biochemical analysis was also performed such as TLC, HPTLC and FTIR analysis to identify the specific chemical constituent present in selected leaf extracts of Moringa olifera and Piper betle. On the basis of HPTLC analysis, it has been shown that the chromatographic pattern is closely similar with growth regulator like IBA. On the basis of FTIR analysis, it also showed that the plant extracts derived from Moringa olifera and Piper betle have similar functional groups as compared with IBA and IAA. Further, the plants derived from leave extracts of Moringa olifera and Piper betle were tested different biochemical assay. The chlorophyll and protein content are closely similar with plants derived from cuttings treated with growth regulators. Thus, through multiple analysis and cross match making it was confirmed that leaf extract of Moringa olifera and Piper betle contains auxin or auxin like substances which can be used as a substitute to synthetic plant hormones.ThesisItem Open Access Hydroponics evaluation of iron toxicity tolerance in low land rice (Oryza sativa L.)(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Nargave, Chetan Singh; Mohanty, I. C.; Rout, G. R.; Bastia, D. N.; Tripathy, S. K.The present study entitled “Hydroponics evaluation of iron toxicity tolerance in low land rice (Oryza sativa L.).” was carried out at Department of Agricultural Biotechnology, Odisha university of agriculture and technology. The aim of the study is Screening of lowland rice germplasm for iron toxicity tolerance, and biochemical analysis for validation of iron toxicity tolerance. Rice is the staple food crop which feeds the two third of global population and accounts for more than 20% of the world's calories consumed. Iron toxicity is a serious nutritional problem that affects rice productivity in both irrigated and rainfed lowland soils. It arises as a result of the conversion of insoluble Fe3+ to soluble Fe2+ under both anaerobic and low pH circumstances. Excess iron uptake in the rice plant shows the yellowing and bronzing effects first on the older leaves. Leaf symptoms are evaluated by the leaf bronzing index. There is need to screen the diverse rice germplasm to study the level of reaction towards tolerance to iron toxicity as success in breeding crops for yield and other quantitative traits depends on the use of methods to evaluate genotypes accurately. Field evaluation suffers from large fluctuations in soil iron concentration in a particular research field, level of water content and variation in micro-environment due to which a large degree of heterogeneity is seen in soil iron uptake and its interaction that impact different plants in the same field. This heterogeneity, in turn, can affect plant performance and yield. Present study was based on the visual symptoms to assess the iron toxicity of the 130 rice genotypes in the hydroponic culture. In Pilot experiment it was observed that as the iron concentration increase (above 250ppm), the yellowing and reddish browning of the leaves became visible from the tip of older leaves. The different concentrations of Fe used in screening are 0ppm (controlled), 150ppm, 250ppm and 400ppm, the pH of the media was maintained at 5. In the screening 39 resistance, 56 moderately resistance and 35 susceptible genotypes were reported. In which Dhusura, Kendrajhali, Basapatri and Tikimahsuri genotypes are found highly resistance. And genotypes Hatipanjara, Sebati, Budidhan and Urbashi are found highly susceptible. Due to Corona pandemic the objective wise experiments could not be completed. According to previous research on the biochemical changes due to iron toxicity in rice, it is expected to increase in the phenolic content, proline content and SOD activity and decrease of the protein content differently in the tolerant and non-tolerant genotypes that are grown in toxic iron concentrations. Hydroponics screening is done under controlled condition which is efficient and fast for screening of large number of germplasm than the field screening. The leaf bronzing score is considered as most convincing trait for the screening of tolerance to the iron toxicity, and it can be used in further crop improvement programs.ThesisItem Open Access Studies on in vitro plant regeneration and phytochemical analysis of Chia (Salvia hispanica sL.)(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Yadav, N. Subhash; Samal, K. C.; Rout, G. R.; Mishra, Dayanidhi; Nayak, R. K.The present study entitled “Studies on in vitro plant regeneration and phytochemical analysis of Chia (Salvia hispanicaL.).” was carried out with prime objective of assessing morphological, biochemical and nutritional characterization of Chia. The field experiment was conducted in the experimental station of AICRN on Potential Crops. Chia (Salvia hispanica L.) belongs to the mint family Lamiaceae, the genus Salvia. The genus Salvia is derived from the Latin word "Salvare," in latin means "to heal" referring to the medicinal properties of the genus. Chia seeds have been used as a food source since 3500 B.C, and it was grown as a commercial crop in the regions of central Mexico between 1500 and 900 B.C. Chia is one of the most highly nutritious crops in the world. It has a high economic value in both national and international markets. Chia, which was once lost to history, is now emerging as a new "superfood" that provides a good source of anti-oxidants, dietary fiber, and omega-3-fatty acids to supplement human nutritional needs. Other than contributing to human nutrition needs, it also aids in the prevention of disorders related to nervous system, inflammatory, cardiovascular, and diabetes, among others. Field experiment showed an average yield of 784 kg/ha.In the present study, the biochemical studies showed the presence of higher amounts of carbohydrates, phenols, flavanols and anti-oxidants. The seed protein content of Chia was evaluated and was found to be 183mg/g which is greater than other major crops like wheat, rice and maize. Carbohydrate content (371 mg/g) is also high in Chia seeds. The biochemical analysis showed the presence of high amounts of phenols and flavanoids in the seeds of Chia. The results had shown the content of 1.29 mgGAE/g phenol in the Chia seeds and 0.48mg/g. The mineral content estimated by ICP-OES shows presence of micronutrients like Fe (11.7mg/100g), Mg (335mg/100g), Mn(5.97mg/100g), Zn (12.01mg/100g), Cu (1.94mg/100g), Ca (397.78mg/100g), Na (42.15mg/100g) and K (605.83mg/100g). The FTIR analysis showed the presence of the functional groups and peak banding analyzed and found high peaks related to protein, pectin (polysaccharides), PUFA (fatty acids) and lipids etc. HPTLC analysis shows the presence of Gallic acid a phenol. Thus the present study unveils that the seeds of Chia crop are rich source of different essential elements such as Fe, Mg, Ca, K etc. and other biochemicals. The Chia seed extract has higher anti-oxidant activity as well. Hence this psuedo-cereal Chia can be used to provide good food supplement. As this is a newly introduced crop in india there is very less study on the crop and to utilize the benefits of the crop, further research in various aspects to increase the environment adaptability and yield should be done.ThesisItem Open Access Assessment of Aluminium Phytotoxicity in greengram (Vigna radiata L.Wilczek)(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Padhi, Rajalaxmi; Rout, Gayana Ranjan; Samal, D. K.; Nayak, R. K.Mungbean [Vigna radiata (L.) Wilczek] is a major pulse crop that is also known as greengram or green bean. Mungbean belongs to Leguminosae family cultivated primarily for its protein-rich edible seeds. After chickpea and pigeon pea, it is the third most widely grown pulse crop in India. India is the world's largest producer and consumer of mung bean. When the soil gets more acidic, the potentially hazardous ionic forms of Al dissolve into the soil solution, limiting root development and its function. Aluminum is slowly released from crystal structures in the environment; however, in acidic circumstances, this process speeds up. Acidic oxides (CO2, SO2, and NO2) in the air are the major source of soil acidification. The present investigation entitled “Assessment of aluminium phytotoxicity in green gram (Vigna radiata L. Wilczek)” was carried out. The screening was conducted by using a genotype (IPM-02-14) with different concentration for aluminium toxicity. Different levels of aluminium concentration i.e. 0, 50 µM, 75 µM, 100 µM, 125 µM were tested on the basis of morphophysiological, biochemical, and yield attribute traits. In comparison to control, the treatment 125 µM Al showed the greatest reduction in height, root length, root dry weight, shoot dry weight followed by treatment 100 µM, 75 µM and 50 µM. The biochemical parameters like proline, chlorophyll content, SOD, catalase, ROS, and aluminium uptake are not able to conducted due to COVID pandemic situation but it has been discussed with the published literatures. It has been reported that the activity was increased with increase of aluminium concentration. Higher levels of aluminum are associated with plant growth, higher photosynthetic rate, higher chlorophyll content, better membrane stability, higher NR activity, higher accumulation of proline and SOD, less lipid peroxidation in leaves, and less accumulation of aluminium in root biomass, among other things, as observed in our study.ThesisItem Open Access Breaking the yield barriers in black rice using mutagenesis(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Sudhabindu, Kintala; Tripathy, Swapan Kumar; Rout, G. R.; Panda, SomanathRice (Oryza sativa) is the most widely grown food crop on the planet. There are various types of rice available, including white rice, red rice, brown rice, and black rice, which is high in iron, zinc, manganese, and phosphorus. A tiny amount of black rice consumed daily can protect us from a variety of ailments. However, tall plant type, shy tillering, low fertilizer response, low yield potential and unacceptable taste are the major hinderances for large scale cultivation. So, mutation breeding can be tried for genetic improvement of black rice land races to induce genetic variation and isolation of desirable plant types. In this context, a set of eight black rice varieties were assessed for agronomic and quality traits in Kharif, 2020. The cv. “Chak Hao-Poreiton revealed long bold grain type and higher panicle length, grain number, seed weight, fertility percentage and hulling percentage as compared to other available black rice varieties. The said elite popular Manipuri black rice cv “Chak Hao-Poreiton” was chosen for seed mutagenesis with 0.5% EMS and in vitro mutagenesis with 0.005-0.015% EMS. The M1 generation was grown in the field during the Rabi 202021. A few valuable high tillering semi-dwarf mutants have been recovered in M2 (Kharif, 2021) which can be a starting point towards breaking yield barriers and genetic correction. Further, the recovery of chlorophyll mutations in M2 generation through seed mutagenesis reflects the onset of genetic changes due to EMS. For in vitro mutagenesis, the callus induction was optimized at MS + 2mg/l Picloram + 0.5mg/l Kinetin followed by efficient plant regeneration at MS + 2mg/l BAP + 0.5mg/l NAA + 1.0mg/l Thidiazuron(TDZ). Further, EMS at varying concentration from 0.0025- 0.015% was maintained in the subculture and regeneration medium (R). Addition 0.0075% EMS to the R-medium reduced the plant regeneration response to almost 50% (43.6%) as compared to the control (86.0%) and hence, routinely used to induce genetic variation. Only 75 plants were successfully acclimatised in pots in partial shade after being shifted to a pot mixture (peatmoss: perlite 2:1). Plants grown from callus cultures will be used to investigate phenotypic variation, if any, in order to improve agroeconomic attributes in subsequent generations. The high throughput plant regeneration system optimized in the present research can be utilised to perform in vitro mutagenesis with 0.0075% EMS to induce genetic variability in black rice.ThesisItem Open Access Exploring rice germplasm resources for natural resistance to glyphosate(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Sahu, Rajesh Kumar; Tripathy, Swapan Kumar; Rout, G. R.; Mishra, ManmohanProductivity of rice is threatened by a number of biotic and abiotic factors. Among these weed accounts as a major constraint for rice production. It competes with the accompanying crop for water, soil, nutrients, space, and light resulting drastic reduction in crop yield. Manual and mechanical weeding incurs huge cost and often becomes impracticable especially in areas with heavy weed infestation. However, non-selective herbicide particularly glyphosate proved to be a feasible solution to eradicate weed infestation. Therefore, development of herbicide resistance in rice turns to be the major focus in rice breeding. In the present pursuit, LD50 of glyphosate application in rice was determined at 0.10% which was also shown to be effective for control of broad spectrum weeds. There was significant genotype x concentration (of glyphosate) interaction which indicated scope for exploring natural resistance of rice germplasm to glyphosate. A total 416 rice diverse germplasm lines were screened for herbicide tolerance at 0.10% glyphosate and promising 19 glyphosate tolerant genotypes Basmati 564, Debadhan, HH212Sal-2-Y3- Y2, HL 19WS33A-16, HL19WS33A-367, HURZ 1, IET 12150, IET 1244, IR 82475- 110-2-2-1-2, IR 128773-4-3-1-4B, IR 128773-4-3-2-5B, Kalajeera Sel., Kandhamal local, Kandhamal local Sel., ORCZ 59, Pusa Basmati-1, Pusa Sugandha 3, SR 9 and Thapur with score 1.0 were selected using herbicide tolerance score (0-9 scale) which was primarily based on seedling mortality. The validation of glyphosate tolerance of these test genotypes based on tolerance score, seedling growth parameters and SPAD meter value varied among test genotypes. However, Kandhamal local sel, ORCZ59 and SR9 revealed highest level of glyphosate tolerance followed by HL19WS33A-367, HURZ1 based on seedling mortality and HH212Sal-2-Y3-Y2 based on leaf drying score.ThesisItem Open Access Studies on in vitro plant regeneration and phytochemical analysis of Grain amaranth (Amaranthus hypochondriacus L.)(Department of Agricultural Biotechnology, OUAT, Bhubaneswar, 2021) Sahoo, Asit Ranjan; Samal, Kailash Chandra; Rout, G. R.; Mishra, Dayanidhi; Mohanty, AnitaThe present study entitled, “Studies on in vitro plant regeneration and phytochemical analysis of Grain amaranth (Amaranthus hypochondriacus L.)” was carried out with the prime objective of assessing morphological, biochemical, and nutritional characterization of four number of Grain Amaranth varieties. The field experiment was conducted in the experimental station of All India Coordinated Research Network on Potential Crops (AICRN on Potential Crops), OUAT, Bhubaneswar. Grain amaranth is popularly known as pseudo cereal due to its edible starchy seeds nature, also recognized as baby food. In recent past year it’s attracting increased interest, both from the agricultural point of view and from aspects regarding processing. In this study we have taken four varieties of grain amaranth in five different replication. The phenotypic study of four varieties of grain amaranth was found different on the basis of agronomic characterization. The highest yield was recorded by Suvarna (0.606Kg/plot and 11.23 q/ha), whereas the lowest yield was found in BGA2 (0.586Kg/plot and 10.86 q/ha). In GA2 varieties 50 % flowering commenced within 45.68 days, but in case of BGA2 variety it took 51.52 days to come 50 % flowering. RMA 7 took 102.5 days for maturities, whereas Suvarna took more days for maturity among four varieties i.e. 104.2 days. Length of panicle found highest in RMA7 38.9 cm whereas lowest panicle length was found in Suvarna 35.68 cm. The highest number plant population was observed in the case of Suvarna variety (74.3) followed by BGA2 (73.5). The lowest plant population was found in GA2 variety. The biochemical analysis study revealed the phenol content of Grain Amaranth BGA 2 (0.72 mg/g), GA2 (0.76 mg/g), SUVARNA (0.68 mg/g) and RMA 7 (0.71 mg/g). Chlorophyll content was found highest in the variety BGA2 (1.537 mg/g). Total carbohydrates content in the variety RMA7 (319mg/g) was found highest. The FTIR analysis revealed that Grain amaranth is having a wider range of different functional groups such as alkyls, ethers, carboxylic acid, ester, alkanes, alkyl halides and aliphatic amines and higher amount of aromatic group of compound. The mineral element profile analysis by ICP- OES revealed that Iron content of grain amaranth (66mg/100g), Magnesium (284.50 mg/100g), Manganese (5.71 mg/100g), Zinc (11.30mg/100g), Calcium (178.7 mg/100g), Potassium (400.50 mg/100g). Thus the present study unveils that seeds of Grain Amaranthus varieties are rich source of different essential elements such as Ca, Fe, Mg, K, Zn, Mn etc. and other biochemical. The seeds have higher antioxidant activity as well. Hence this pseudocereal Grain Amaranth can be used to provide good food supplements to the infants as well as adults.
