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20202
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Subject: Plant Physiology × End date: 2020 × Start date: 2020 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Physiological biochemical and molecular studies in medicinal rice (Oryza sativa L.) Njavara as influenced by abiotic stresses
    (Department of Plant Physiology, College of Agriculture, Vellayani, 2020) Wagh Yogesh, Sahebrao; KAU; Viji, M M
    The study entitled “Physiological, biochemical and molecular studies in medicinal rice (Oryza sativa L.), Njavara, as influenced by abiotic stresses” was conducted during 2015 to 2019 at the Department of Plant Physiology, College of Agriculture, Vellayani, Thiruvananthapuram. The objective of the study was to elicit information on the physiological, biochemical and molecular attributes associated to secondary metabolites accumulation due to abiotic stresses viz., shade, drought and UV-B stress in medicinal rice Njavara. The study was conducted as three different experiments with black glumed and yellow glumed njavara. Experiment one was a pot culture study laid out in Completely Randomized Design (CRD) with two varieties and five treatments. The study included a combination of two levels of shade stress and two levels of water deficit stress and a control with four replications and with three pots in each replication. The observations were taken at different critical stages of the crop viz., vegetative stage, panicle initiation stage, flowering stage and harvesting stage. The results revealed that the morphological characters such as plant height, leaf area index and specific leaf area were higher under 40% shade whereas under 50% field capacity, they were found to be highly reduced. Number of tillers per plant was higher under control (T5) compared to shaded and water deficit conditions. Leaf gas exchange parameters viz., photosynthetic rate, stomatal conductance and transpiration rate were lesser under both experimental conditions compared to the control. However maximum reduction of leaf gas exchange parameters were recorded at 50% field capacity (T3) followed by 40% shaded condition (T2) compared to the control (T5). Among the biochemical characters, maximum chlorophyll content was found under 40% shaded condition (T2). The biochemical parameters such as flavonoid, phenol and proline content of leaves were found higher under 50% field capacity (T3) at all the growth stages studied. The total flavonoid content in the grains was found higher under 75% field capacity (T4) followed by 20% shade treatment (T1) in both the varieties. The second experiment was carried out in pot culture in CRD with four treatments, three replications and four pots per replication. The crop was subjected to UV-B (280-320 nm) radiation with the help of UV-B fluorescent tubes during the different critical stages of plants i.e. from vegetative stage, from panicle initiation stage and from flowering stage till harvesting in ventilated polyhouse. The UV-B tubes were switched on for 4 hours daily from 10 am to 2 pm and the average intensity of UV-B radiation at the canopy level of plants was maintained at 4 Wm-2. The control was maintained in another compartment of polyhouse without UV-B tubes. The results indicated that the morphological characters viz., plant height, leaf area index, specific leaf area and tiller number reduced significantly under UV-B radiation treatment and the maximum reduction was observed in treatment T1 (UV-B treatment from vegetative stage). Leaf gas exchange parameters as well as chlorophyll content decreased significantly under UV-B radiation treatments compared to the treatment without UV-B radiation. However flavonoid, phenol and proline contents of leaves were found to increase under UV-B treatments (T1, T2 and T3). The accumulation of total flavonoid in grains was found significantly higher in treatment T2 (UV-B radiation treatment given from panicle initiation stage) in both the varieties. The treatments which resulted in the highest accumulation of flavonoid content in grains were selected from experiment 1 (20% shade and 75% field capacity) and experiment 2 (UV-B radiation treatment from panicle initiation stage) for molecular analysis in experiment 3. Protein profiling was done in leaves using SDS-PAGE in which, there was variation in the intensity of large subunit (55 kDa) as well as small subunit (16 kDa) of RuBisCO, between the varieties and the treatments. The intensity of those bands were found higher in 20% shade (T1), 75% field capacity (T2) and control (T4) whereas under UV-B radiation treatment from panicle initiation stage (T3) relatively lesser intensity was exhibited. Gene expression study in grains using qRT-PCR revealed relatively higher expression of chalcone synthase (CHS) and CYP75B4 genes in black glumed njavara (V1) than the yellow glumed njavara (V2) variety. Also the gene expression study revealed that both the genes were over expressed under T3 (UV-B radiation treatment from panicle initiation stage) and T2 (75% field capacity). But both the genes (CHS and CYP75B4) were found down regulated under T1 (20% shade) condition. The present study revealed that the flavonoid content in grains is higher in black njavara (V1) compared to yellow njavara (V2). The study also indicated that the various parameters studied viz., growth, physiological and biochemical were found to have positive influence on the flavonoid accumulation of grains. But the chlorophyll content was found to have negative influence on the flavonoid content of grains. Proline content was less in black njavara (V1) under UV-B radiation compared to yellow njavara (V2). Based on the present study it is concluded that the black glumed njavara performed better than yellow glumed njavara under all the stress conditions studied and can be exploited better for its therapeutic value. Application of mild stress levels viz., water deficit (75% field capacity) or UV-B radiation treatment from panicle initiation stage may be utilized to enhance the medicinal quality of this crop.
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    ThesisItemOpen Access
    Identification of molecular markers and quantitative trait loci(QTLs)associated with drought tolerant and plant production traits in rice(Oryza sativa L.)using association genetic analysis
    (Department Of Plant physiology, College Of Agriculture, Vellayani, 2020) Nithya, N; KAU; Beena, R
    The present investigation entitled “Identification of molecular markers and Quantitative Trait Loci (QTLs) associated with drought tolerant and plant production traits in rice (Oryza sativa L.) using association genetic analysis” was conducted at Department of Plant Physiology, College of Agriculture, Vellayani and RARS, Pattambi during 2016-18 . The objective of the study was to identify molecular markers, Quantitative trait loci (QTLs) associated with drought tolerance and plant production traits in rice under drought condition. The extend of variation for water stress indicators for morpho-physiological and plant production traits were assessed by evaluating 81 rice genotypes collected from RARS, Pattambi and NRRI, Cuttack under water stress and irrigated conditions in the open field. Each genotype was raised in four rows of 2m length and was exposed to water stress at panicle initiation stage for a period of 25 consecutive days by withdrawing irrigation along with irrigated control and replicated twice. The morpho- physiological, and plant production traits were recorded after imposing water stress. Significant variation was observed for these traits under water stress condition. Genotyping of 81 rice genotypes were done using 100 SSR primers. Quality and quantity of DNA was checked. Amplification pattern of 100 primers were scored as per standard procedure. Population structure was calculated using the software “STRUCTURE” with genotypic score value. The molecular markers/ QTLs linked to drought tolerance and plant production traits were identified using the software, TASSEL 5. The result of the study revealed that morphological parameters such as the plant height at flowering was observed to be highest in PTB1 and lowest in ASD-16 under water stress condition in both experiments. The number of tillers were maximum for PTB7 (10.5 plant-1) and minimum for Kuttithekkan under water stress condition in both experiments. Delayed days to 50% flowering (7 days) was observed for the genotypes under water stress in two experiments compared to irrigated control. Physiological parameters such as Relative Water Content (RWC) decreased where as leaf temperature increased significantly in most of the genotypes under water stress condition. Highest leaf rolling (score – 7.78) was observed in Prathyasha in both experiment I (7.78) and experiment II (7.84). But the genotypes PTB55 (1.78) & PTB 29 (1.11) showed least leaf rolling symptoms in both experiment I and II respectively. Among the genotypes, the RWC was recorded to be highest in PTB15 (78.72%) while the lowest was recorded in Prathyasha (57.34%) under water stress condition in experiment I and in experiment II maximum relative water content was observed in PTB27 (72.98%) and minimum in ADT37 (50.25%). Membrane stability index was highest in PTB29 in experiment I (94.36 %) and in PTB27 in the experiment II (92.24%). Maximum leaf temperature was observed in PTB1 and minimum in Prathyasha under water stress condition in both experiments. Among the genotypes, chlorophyll stability index was recorded to be highest in PTB27 (95.59%) while the lowest in Pandichempan (80.36%) in experiment I, and in experiment II maximum was recorded in variety PTB7 (92.85%) and minimum in Pandichempan (75.52 %). The grain yield per plant under water stress condition was positively correlated with parameters such as tiller numbers, days to 50% flowering, relative water content, membrane stability index, chlorophyll stability index , spikelet fertility percentage and 1000 grain weight where as negatively correlated with leaf temperature, leaf rolling score, leaf drying score and percentage relative yield reduction. Under water stress the first principal component showed 41.77% variation, while second component exhibited 16.57% variation. Among the 14 morpho-physiological and plant production traits, relative water content, cell membrane stability index and chlorophyll stability index contributed towards maximum diversity. Under irrigated control condition the first principal component revealed 48.9 % variation, while the second component showed 16.57% variation. Among the 8 morpho-physiological and plant production traits studied, the relative water content and spikelet fertility percentage contributed towards maximum diversity. Clustering by Ward method was done to establish a relationship among the 81 rice genotypes. Similar types of genotypes were clustered together based on the phenotypic data. All the genotypes were clustered mainly into 5 clusters under water stress as well as irrigated control condition. Genotyping of eighty one rice genotypes were done using 100 SSR markers. Among them, 40 primers which demonstrated polymorphism among the genotypes were used to classify the genomic regions (QTLs) associated with drought tolerance and plant production traits through association genetic analysis. The marker trait association for all the traits under water stress and irrigated control condition were calculated using GLM and MLM model of TASSEL5 software. A total of 136 marker trait associations were observed while performing GLM analysis and MLM analysis resulted in 48 marker trait association based on the P and r2values in water stress as well as irrigated control condition. LD was distributed unequally on each chromosome and more concentrated on chromosomes 1 and 7. LD analysis in the whole population showed that there were significant LD pairs (P<0.05). A total of 52 LD pairs were observed under water stress and irrigated control conditions and out of these, there were 46 inter chromosomal LD pairs and 6 intra chromosomal LD pairs. The markers RM3825, RM455, RM490, RM259 and RM1026 showed significant associations with many phenotypic traits in water stress as well as irrigated control conditions. The Q-Q (Quantile-Quantile) plot also confirmed the association of these markers with phenotypic traits. In summary, there was significant variation for morpho-physiological and plant production traits among rice genotypes under water stress condition. Genotypes having higher relative water content, cell membrane stability index and chlorophyll stability index were found to be tolerant to drought. In the present study, the genotypes viz PTB28, PTB29, PTB30, PTB15, PTB7, PTB55, N-22 and Chomala identified as drought tolerant can be used in breeding programmes to improve drought tolerance in rice. From this study, 29 significant (P<0.05) marker trait associations were detected using mixed linear model (MLM). The identified most significant markers and their corresponding QTLs are, RM455 (Chromosome7) was linked to tiller number, leaf temperature, relative water content, yield per plant and spikelet fertility percentage. RM490 (Chromosome1) was associated with leaf temperature and chlorophyll stability index. Marker RM259 (Chromosome1) was associated with leaf temperature, chlorophyll stability index, leaf drying score, yield per plant and spikelet fertility percentage. RM1026 (Chromosome 9) was associated with leaf rolling score and drying score. Among them, RM 490 and RM259 showed co-location of QTLs for leaf temperature and chlorophyll stability index and RM259 & RM1026 showed co-location of QTLs for leaf drying score.