PhD Thesis
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Item Evaluation and characterisation of selected landraces of rice for bioactive compounds and validation of its nutraceutical potential through in silico analysis(Department of Plant Physiology, College of Agriculture ,Vellanikkara, 2024-05-21) Mikhina,M S.; Abida, P SEvaluation and characterization of selected landraces of rice for bioactive compounds and validation of its nutraceutical potential through in silico analysis Abstract Nearly ninety per cent of the world's production of rice comes from Asian nations, where it is an often-cultivated crop. The world produced 513.68 million tonnes of rice in 2022–2023, with China producing the most at 149 million tonnes, followed by India with 132 million tonnes. Rice is consumed by nearly one-third of the global population accounting for about 80 per cent of their energy requirements. In addition to being the staple grain of Asia, rice is quickly taking the lead as the main diet in Latin America and Africa. Oryza sativa is the most widely grown species of the available approximately 11,000 rice types and 100,000 landraces as well as improved cultivars. Existence of such a huge genetic diversity enables humans to improve the crop’s performance in spite of changing climatic scenarios and burgeoning population. Traditional rices are enriched with amino acids, antioxidants, phytochemicals, vitamin E and other nutrients. In addition to their antioxidant activity, traditional rice varieties offer anti-diabetic and anti-inflammatory benefits. Epidemiological studies have shown that regular consumption of whole grain is associated with reduced risks of chronic diseases such as cardiovascular diseases, type 2 diabetes and some cancers. Ayurveda considers traditional whole rice grains as nourishing food and medicine. Brown rice and red rice are great sources of fiber, B vitamins, calcium, zinc, iron, manganese, selenium, magnesium and other nutrients. Due to its higher digestibility, nutritional quality, potential health benefits and biological activity, rice is categorized as the queen of cereals. But these rices are underexploited and requires a boost in yielding potential, for their commercialization. In this background, the present study was envisaged to delve into the nutraceutical superiority of a few selected speciality rice and highlight the significance of conserving and protecting them. The study was undertaken as four experiments spread across College of Agriculture, Vellanikkara, RARS, Pattambi and CFTRI, Mysore. As first part of the study, a pot culture experiment was conducted for morpho-physio and biochemical evaluation of the selected twenty-five rice landraces, laid in completely randomised design. Among the selected landraces, Kavunginpoothala recorded significantly highest plant height (109.56 cm), followed by Chenkayama (101.51 cm), while Ithikandapan was the shortest (93.93 cm). The local check Jyothi and Uma showed a plant height of 96.35 cm and 103.35 cm respectively. Maximum number of tillers were observed in Palthondi (6) which was on par with Chenkayama, Chuvannavattan, Thavalakkannan, Rakthasali and Japan Violet. Black jasmine (4) possessed the least number of tillers and was on par with Kala namak and Krishnakaumudh. Among the selected landraces, the number of panicles of Palthondi (6) Chenkayama, Thavalakkannan, Rakthasali, Japan Violet and Chuvanna Vattan were on par with each other. The local check Jyothi possessed significantly higher number of panicles per plant. The genotype Kavunginpoothala showed maximum days for 50% flowering (132) followed by Chenkayama (121), which was on par with Kavuni (120). The study on physiological aspects showed that the highest net photosynthetic rate at reproductive stage was exhibited by Kala Namak (28.73 µmol CO2 m-2 s -1 ) followed by Kothambalarikayama (28.33 µmol CO2 m-2 s -1 ) and the lowest was shown by Japan Violet (20.87 µmol CO2 m-2 s -1 ). Chempav possessed highest chlorophyll content (3.25 mg g-1 ) which was on par with Chenkayama (3.20 mg g-1 ) and the lowest chlorophyll content was observed in Japan Violet (1.00 mg g-1 ). Highest IAA content at reproductive stage was observed for Kavunginpoothala (873.60 µg of unoxidized auxin g-1 h-1 ) and Ithikandapan (354.87 µg of unoxidized auxin g-1 h1 ) exhibited the lowest IAA content. Irrespective of growth stages, Kavunginpoothala recorded maximum GA content (18.99 mg/g, 20.82 mg/g, 20.87 mg/g). In maturity stage, Ithikandapan (12.69 mg/g) had lowest GA content. Thavalakkannan and Chenkayama, showed a purple coloured stem during reproductive stage. At maturity, Black Jasmine, Ramli, Kala Namak, and Karutha Modan exhibited a black coloured husk while remaining all other genotypes were in brown colour. Among the entries, grains for nearly nineteen are red pigmented; five are white pigmented and one being black pigmented. The highest average panicle weight was possessed by Chuvanna Vattan (18.51 g) followed by Rakthasali (18.35 g). The lowest average panicle weight was for Chenkayama (10.36 g), which was on par with Kavunginpoothala. The local check Uma and Jyothi exhibited an average panicle weight of 27.46 g and 25.64 g, respectively. Chuvanna Vattan (26.68 g) exhibited the maximum 1000 grain weight compared to other genotypes, followed by Njavara (26.26 g). The lowest 1000 seed weight was observed in Rakthasali (16.69 g). The local check Uma and Jyothi had 1000 weight of 27.46 g and 28.49 g, respectively. Black Jasmine possessed the significantly high iron (39.65, 24.71 mg/kg) and zinc (19.21, 17.67 mg/kg) contents in unpolished and polished grains. The anthocyanin content was found high in Black Jasmine (1110.05 µg/g), followed by red variety Rakthasali (1058.49 µg/g) and Njavara (1001.51 µg/g). The nutraceutical components such as gamma oryzanol, tocopherol, total polyphenols and flavonoids was significantly high in Black Jasmine (72.85 mg/100 g, 5.32 µg/g, 13.13 mg/g and 6.46 mg/g, respectively) followed by Rakthasali (63.12 mg/100 g, 4.33 µg/g, 8.89 mg/g, and 6.25 mg/g, respectively) and Njavara (61.80 mg/100 g, 4.26 µg/g, 8.77 mg/g and 6.17 mg/g, respectively). The cooking and eating quality of rice was determined by the proximate content and carbohydrate digestibility. Among the studied genotypes, significantly highest ash (2.53%), fibre (2.43%), fat (2.64%), lowest digestibility (60%) and high antioxidant activity were observed in Black Jasmine. It was also observed that, polishing caused a reduction in bioactive contents. Due to polishing, nearly 80 and 50 per cent reduction was found in mineral content and antioxidant activity. Among the selected genotypes, it could be noted that, black pigmented landraces exhibited significantly high bioactive compounds followed by red and white. Thus, based on the consistency in bioactive compounds, HPLC characterization was also carried out at CSIR-CFTRI, Mysore for a subset of the major compounds to identify and quantify them in nine selected representatives from the red, black and white rice types. The HPLC analyses indicated that Rakthasali had the highest chlorogenic acid (14.27 mg/100 g) content, while Black jasmine had high gallic acid (3.56 mg/100 g). Njavara showed highest t-cinnamic acid (1.47 mg/100 g) content compared to other varieties. These sub compounds may be the reason for high nutraceutical potential these pigmented genotypes. It was also noted that white genotypes had the presence of many sub compounds, even though white rice is considered inferior to black and red. In the next phase of the study, the three best promising genotypes (Rakthasali, Black Jasmine and Njavara) were grown in field during Kharif and Rabi. The field experiments were laid in randomised block design at RARS, Pattambi, with an aim to analyse the phenolic content, anthocyanin content and antioxidant activity. The results indicated that, the grains harvested from the Kharif season exhibited high phenolic and anthocyanin contents along with high antioxidant activity compared to the Rabi season crop. Subsequently, all the grains were subjected to storage (ageing) study to check the bioactive compound activity during storage. The grains were stored under ambient conditions and bioactive profiling (total phenolics, anthocyanin, antioxidant activity) was carried out in grains at harvest, 6, 9 and 12 months after storage. A tremendous reduction was observed in all the grains stored. Among the genotypes, Rakthasali, Thavalakkannan and Kattamodan exhibited lowest reduction in phenolic content (~30 %). Black Jasmine, Japan Violet and Chenkayama had lowest reduction in anthocyanin content (~ 15 %). Rakthasali, Karutha Modan, Krishnakaumudh and Njavara showed lowest reduction in antioxidant activity (~ 30 %) among the selected entries. Simultaneously, an in silico study was conducted on CB dock platform to find out the best bioactive compound against receptors of major lifestyle diseases such as cancer, diabetes and inflammation, based on the interaction dynamics. The major sub-compounds of phenolics, flavonoids, oryzanols, anthocyanins and tocols were selected for the docking study. The disease specific target proteins/receptors were selected for performing the docking assay. Chlorogenic acid was identified as best phenolic sub-compound against cancer, diabetes and inflammation. Among the oryzanol sub-compounds, stigmasteryl ferulate, sitostanyl ferulate and cycloartenyl ferulate exhibited best interaction with receptors, evidenced by superior docking scores. Among flavonoids, luteolin, myrcetin and fisetin are the promising drug candidates against these diseases. In case of tocols, strong interaction with target proteins was noted for gamma toco-trienol, beta toco-trienol and delta toco-trienol. delphinidin-3-O-galactoside, and cyanidin-3-O-glucoside are the major sub compounds of anthocyanin which showed best docking scores with selected target proteins. From the present study, based on the docking scores and ADMET properties, the above selected sub compounds which were also pronounced in Rakthasali, Black Jasmine and Njavara as per their HPLC profiles, were found promising to combat lifestyle disorders Further validation by wet lab studies is required to reinforce their promising roles. The study describes rice bran is the main source of minerals, vitamin E and other bioactive compounds known to possess medicinal properties. The study highlights the nutraceutical potential of Rakthasali, Black Jasmine and Njavara and the possibilities for exploiting them by selecting the apt growing season, ageing stage and targeted extraction of the relevant bioactive compound (s), for reaping maximum benefits. The relevance of further examination of these varieties for their stress resilience and marginal yield increase potential also is a future thrust.Item Marker assisted backcross breeding in rice variety jyothi for drought tolerance(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2024-01-03) Shankar, C.; Abida, P SDrought is one of the important abiotic stressors that can substantially reduce rice production by 35 - 40 per cent. Water stress will significantly impact the growth and development of rice crops, but drought at the reproductive stage will directly affect the grain yield. In 2018, the State Disaster Management Authority, Kerala declared major rice growing districts including Palakkad and Alappuzha as drought affected. Jyothi is a high-yielding popular rice variety, widely cultivated variety across Kerala. Despite its widespread popularity, it exhibits poor spikelet fertility per cent under water deficit conditions, indicating that it is susceptible to drought. Considering the negative impact of drought stress on rice production and the losses incurred by farmers, efforts were initiated at the College of Agriculture, Vellanikara to pyramid QTLs (qDTY4.1, qDTY6.1, and qDTY9.1) for yield under drought into the rice variety Jyothi (recurrent parent) from the drought tolerant variety Chuvannamodan (donor parent). The present work was conducted during 2021-2023 with the objective of introgressing the target QTLs of interest into variety Jyothi through marker assisted backcross breeding. Genomic DNA was isolated from both parents and subjected to PCR amplification for parental polymorphism using a set of 208 microsatellites. Out of which, 85 markers were found to be polymorphic between the parents. Parental varieties were hybridized and resultant F1 hybrids were confirmed using foreground markers (RM518, RM587 and RM566). A total of 27 F1 hybrids showed heterozygous banding pattern for all foreground markers. The F1 hybrids were backcrossed with recurrent parent for the production of the BC1F1 population. Also, F1 hybrids were allowed to selfing for the production of F2 and subsequently F3 generation. Gene annotation and enrichment analysis were done using Simple Sequence Repeat (SSR) markers associated with the target QTLs of interest. It was found that 23 genes were associated with drought tolerance, spikelet fertility and root related traits at three different QTL regions. Foreground selection in 200 BC1F1 progenies along with the parents was done to identify the plants with the target QTLs. SSR markers RM518, RM587 and RM566 closely linked with qDTY4.1, qDTY6.1 and qDTY9.1, respectively were used as foreground markers. Screening of BC1F1 population revealed that 60 progenies possessed heterozygous alleles for at least one QTL. A total of 27 BC1F1 progenies including 10 with qDTY4.1 + qDTY6.1, 9 with qDTY6.1 + qDTY9.1 and 8 with qDTY9.1 + qDTY4.1 were identified to possess two QTLs pyramided. Background selection of 27 BC1F1 foreground selected progenies using 76 SSR markers, polymorphic between the parents, revealed the presence of wide variability among these progenies with respect to recurrent parent genome recovery (RPGR). The results of graphical genotyping revealed a maximum RPGR of 57.7 per cent in the BC1F1 progeny 24.165, followed by 56 per cent in progeny 1.2. Drought tolerance of 15 BC1F1 with two QTL combination along with the parental varieties was characterized under reproductive stage drought stress using thirteen traits as per standard evaluation system (IRRI, 2002), including water mining traits. All the backcrossed progenies exhibited better performance under water deficit condition than the susceptible parent, Jyothi. It was found that progeny 20.118 (qDTY4.1 + qDTY6.1) had a higher number of filled grains (FG) per panicle (41) and spikelet sterility (SS) per cent (49.38 %) compared to Jyothi with 1 FG and 99.29 % SS under drought. The progeny 1.10 (qDTY9.1 + qDTY4.1) possessed higher root length (97 cm), root volume (100 cc), root to shoot ratio (1.70), root dry weight (25.9 g) than Jyothi (82.2 cm, 76 cc, 1.4, 14.64 g). Also, progenies 23.153 (qDTY4.1 + qDTY6.1) and 3.30 (qDTY6.1 + qDTY9.1) exhibited delay of 8 days to show the leaf rolling symptoms than Jyothi with 4.2 days. The BC1F1 progenies positive in foreground selection was backcrossed with the recurrent parent and 87 BC2F1 seeds were harvested. The resultant BC2F1, BC1F2 and F3 lines with potential drought tolerant QTLs can serve as the base population for enhancing the drought tolerance in Jyothi. Further, genotypic and phenotypic evaluation of the QTL pyramided progeny in subsequent generations will lead to the development of drought tolerant cultivar in the background of popular rice variety Jyothi.Item Physiological and molecular breeding approaches to enhance high temperature stress tolerance in high yielding short duration rice varieties suited to kole tracts of Kerala(Department of Plant Physiology, College of Agriculture, Vellanikkara, 2025-05-19) Lakshmi, G Ajay; Abida, P SRice cultivation in the Kole tracts, a unique wetland ecosystem of Kerala, is being drastically affected by the rising temperatures driven by climate change. The higher temperature during the second cropping season coincides with the reproductive stage of the crop leading to significant yield reduction, thereby affecting the overall productivity of the crop. To sustain rice production and harness the full potential of this fertile ecosystem in double cropping scenario, it is essential to understand the physiological and molecular mechanisms underlying the heat induced productivity decline which will facilitate the development of high yielding rice varieties having high temperature resilience. In this context, the current study employs physiological and molecular breeding approaches to improve resilience of rice to heat stress. The study consisted of three experiments using five rice varieties namely Nagina 22 (90-95 days), KAU Manu Ratna (95-100 days), MO 16 (Uma) (115-120 days), KAU Manu Varna (128-138 days) and Ptb 39 (Jyothi) (115-120 days). Variety Nagina 22 served as the heat tolerant check while the other four are popular high yielding rice varieties cultivated in Kerala. In the first experiment, the acquired thermotolerance of the above five rice varieties were evaluated through Temperature Induction Response Technique (TIRT). The experiment was laid out in a completely randomized design (CRD) with three replications in the laboratory. Two-day old seedlings were exposed to a gradual temperature rise (induction range: 32°C to 42°C for 5 h by increasing each degree from 32°C to 42°C every 30 minutes), followed by exposure to a challenging temperature (lethal temperature: 49°C for 3 h) and a subsequent recovery period of three days at ambient conditions. Nagina 22 registered a higher survival per cent (91.67), recovery growth (47.83 mm) and heat-stable protein levels (0.059 µg g -1). Jyothi recorded higher survival per cent (100%) and heat stable protein (0.040 µg g -1) indicating strong innate tolerance. KAU Manu Ratna, KAU Manu Varna, and Uma exhibited moderate to poor tolerance, particularly with respect to recovery growth and heat-stable protein levels, indicating their susceptibility to heat stress during seedling stage. The second experiment was a pot culture study using the five rice genotypes to evaluate the comparative potential of ameliorant sprays viz. salicylic acid (400ppm), Hoagland solution (1/4x) and water spray over unsprayed control in high temperature stress mitigation and overall growth promotion. The varieties were evaluated simultaneously both under ambient and high temperature conditions between January to April, 2023, each following a completely randomized design (CRD) with three replications. The high temperature chamber builds up temperature 5-8oC higher than ambient condition, which was regularly monitored using data loggers. The rice genotypes grown under both the conditions, were subjected to four different foliar sprays viz. T1-salicylic acid (400 ppm), T2-Hoagland solution (1/4x), along with water sprayed (T3) and unsprayed (T4) controls, administered at maximum tillering stage and booting stage. The observations were taken one week after the second spray corresponding to panicle initiation in each variety. The day/night temperature regime in high temperature chamber (44oC/32oC) and ambient condition (38oC/25oC) was also monitored at the time of taking observations. Pollen viability was higher in salicylic acid (400 ppm) spray (T1) under high temperature, especially in Nagina 22 (98.07%) and KAU Manu Ratna (97.13%) suggesting better pollen function, successful fertilization and partitioning. In KAU Manu Ratna, salicylic acid (400 ppm) spray (T1) displayed significantly higher increase in spikelet fertility (8%) under ambient conditions while in the high temperature chamber, water spray (T3) displayed significantly higher increase in spikelet fertility (70%), compared to the unsprayed control in the respective conditions. Physiological parameters like membrane stability index, photosynthetic rate, transpiration rate, stomatal conductance, etc. were measured in the flag leaf at panicle initiation stage. Salicylic acid (400 ppm) spray improved photosynthetic rate and contributed to the grain yield significantly irrespective of variety in the ambient condition. Among varieties, the photosynthetic rate was significantly higher for KAU Manu Ratna (9.09 µmol CO₂ m⁻² s⁻¹). Under ambient condition, KAU Manu Varna (16.2 g/plant) effectively used salicylic acid (400 ppm) spray to improve yield, next to Nagina 22 (19.08 g/plant). Similarly, KAU Manu Varna displayed highest yield when sprayed with water (1.67g/plant) followed by salicylic acid (400 ppm) (1.64 g/plant) among all treatments across genotypes. Visible varietal difference in response to different ameliorants was noted owing to their contributory significance. Thousand grain weight data indicated that the grains from salicylic acid (400 ppm) treated plants were heavier (by 16 % in KAU Manu Ratna, 35% in Uma ,25% in KAU Manu Varna, and 21% in Jyothi) as compared to the respective unsprayed controls in ambient condition confirming better grain filling. Salicylic acid (400 ppm) emerged as the best treatment to enhance heat tolerance in Manu Ratna and Manu Varna while Hoagland (1/4x) worked better in Nagina 22. It can be inferred from the study that the ameliorative advantage of each foliar supplement used in the study is variety dependent and their dose needs to be standardized for each variety to improve heat tolerance without penalizing primary growth. The third experiment involved marker assisted breeding for improvement of heat tolerance of high yielding rice variety KAU Manu Ratna by hybridization with heat tolerant donor parent Nagina 22 employing single sequence repeats (SSRs) marker based genotyping. Parental polymorphism was ascertained by genotyping the parents using previously identified 44 rice markers associated to heat tolerance (Waghmare et al., 2018). Out of these 43 SSR markers, 21 exhibited parental polymorphism thereby showing a 48.83 per cent polymorphism rate between the two parents. The size of polymorphic markers identified ranged from 97 bp (RM473a) to 292 bp (RM10346). F1 plants were selfed to derive F2 population. Phenotyping of F2 population was done to identify heat tolerant progenies in a high temperature chamber with an average of 5oC higher than ambient temperature. Spikelet fertility was used to identify tolerant and susceptible bulks and was confirmed using the already identified polymorphic markers that differentiated the two bulks. Markers RM13 and RM495 were able to differentiate both the bulks, clearly showing co-segregation. Therefore, these markers, RM13 and RM495 may be putatively linked with spikelet fertility in the F2 population generated by crossing N22 and KAU Manu Ratna. Genotyping and phenotyping of the F2 population provided insights about the presence or absence of the heat tolerant marker signatures in the F2 progeny and their potential to maintain yield characters. The current study provides insights into assessing as well as enhancing high temperature tolerance in rice genotypes by integrating physiological screening through TIRT, exogenous amelioration and molecular breeding techniques. Further research could focus on refining the dosages of ameliorants and their possible combinations which may improve the overall performance of rice, irrespective of varieties. Unravelling the defective mechanisms of source-sink partitioning in susceptible varieties need to be focussed in the path ahead. The mapping population derived from the F2 could be effectively used for variety development and to study closely linked markers associated to heat tolerant QTLs. The F2 population could be effectively used to develop multiple mapping population targeting different heat tolerance mechanisms. Thus, the results of the current study offer a strong foundation for development of climate resilient rice involving mitigative amelioration and marker assisted breeding so as to address the global food security challenges consequential of climate change.