PG Thesis
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Item High temperature stress on grain phytic acid and mineral bioavaility in rice(Department of plant physiology, College of Agriculture,Vellayani, 2025-11-28) Lishli,D; Beena,RThe study entitled “High temperature stress on grain phytic acid and mineral bioavailability in rice (Oryza sativa L.)” was conducted at the Department of Plant Physiology, College of Agriculture, Vellayani, during 2023–2025. The present study examined the influence of high temperature stress (36 ± 2°C) imposed during the reproductive stage on grain phytic acid accumulation, mineral bioavailability, physiological performance, and yield traits in eleven rice genotypes differing in pericarp colour and stress tolerance. The experiment was conducted during the Rabi 2024 season using a Completely Randomized Design (CRD) with two temperature regimes (ambient and high temperature) and three replications. The plants were maintained under normal conditions until panicle initiation and after which they were exposed to high temperature condition in a polyhouse. Morphophysiological, biochemical, and molecular parameters, including the expression of the SPDT (SULTR-like Phosphorus Distribution Transporter) gene, were analysed to elucidate the molecular basis of genotypic variation in phytic acid content. High temperature stress significantly affected physiological, biochemical, and yield parameters, though the extent of response varied among genotypes. Exposure to elevated temperatures resulted in a 7–18 % reduction in plant height, a 21–51 % decrease in leaf area, and a 15–32 % decline in chlorophyll content. Yield related traits were also adversely affected, with spikelet fertility declining by 2–15 % and grain yield decreasing by 18–50 % due to elevated temperature. Pigmented rice genotypes such as Kalabath and Assam Black maintained higher chlorophyll retention, membrane stability, and yield indicating strong thermotolerance. In contrast, white pericarp genotypes such as Khira, White Jasmine, and Jeerakasala exhibited substantial reductions in photosynthetic efficiency and spikelet fertility, resulting in greater yield losses. Jyothi and Urunikaima demonstrated moderate tolerance, with intermediate stability under stress Under heat stress, a significant biochemical response was evident, reflected by a 2–34 % increase in grain phytic acid compared to plants grown under ambient conditions. This increase reflects a temperature-induced shift in phosphorus metabolism that helps stabilize internal phosphorus reserves. However, it was accompanied by a 20–30% reduction in bioavailable iron and zinc, demonstrating a strong negative correlation between phytic acid accumulation and mineral bioavailability. Molecular analysis revealed that the SPDT gene, responsible for phosphorus transport to grains, was upregulated under stress, thereby promoting enhanced phytic acid biosynthesis. Bran colour exerted a strong influence on stress response and nutrient balance. Pigmented genotypes, particularly those with black and red pericarps, exhibited only a 10–15% reduction in antioxidant activity compared with 25–30% in white rice. Their higher levels of phenolics, anthocyanins, and flavonoids likely mitigated oxidative injury, preserving grain integrity and mineral stability. Conversely, white genotypes, lacking such antioxidant protection, experienced a stronger rise in phytic acid and greater yield decline. In conclusion, high temperature stress during the reproductive stage significantly influenced grain phytic acid content, mineral bioavailability, and yield parameters in rice, with the magnitude and nature of these effects varying among genotypes of different bran colours. The results demonstrate that the increase in phytic acid under heat stress is a protective yet nutritionally disadvantageous response associated with altered phosphorus metabolism and reduced mineral availability. The observed inverse relationship between phytic acid accumulation and mineral bioavailability underscores the importance of developing rice genotypes with optimized phosphorus transport and enhanced thermotolerance. The study thus fulfilled its objective by identifying pigmented genotypes such as Kalabath and Assam Black as promising donor lines combining superior antioxidant potential, balanced phytic acid regulation, and stable mineral bioavailability, providing valuable resources for breeding climate-resilient and nutritionally enriched rice cultivars.Item Compatibility of multinutrient mix sampoorna with herbicides for rice(Department of Agronomy, College of Agriculture , Vellanikkara, 2024) Akhila, P; Prameela, PTank mixing of agrochemicals is a common practise among farmers in order to save labour, time and application cost. However, mixing is done without having a scientific knowledge on effect of tank mixing. Tank mixing of agrochemicals may result in synergistic, antagonistic, or neutral effects on efficacies of chemicals involved. Herbicidal weed control is very common in rice as it is the economically viable and easy method. Usually post emergence herbicides are applied at 15-20 days of sowing or transplanting. There is a possibility of tank mixed spraying if the herbicides are compatible with Sampoorna. Hence a research programme entitled “Compatibility of multinutrient mix Sampoorna with herbicides for rice” was conducted during July to November 2022 to evaluate the weed control efficiency and compatibility of few post emergence herbicides with Sampoorna KAU multimix in transplanted wetland rice. Experiment consisted of thirteen treatments which included application of five herbicides at recommended rates (bispyribac sodium @ 0.03 kg/ ha, cyhalofop butyl @ 0.08 kg/ ha, 2,4-D @ 0.8 kg/ ha and premix herbicides AlmixⓇ (premix of metsulfuron methyl and chlorimuron ethyl) @ 0.004 kg/ ha and Vivaya Ⓡ (premix of cyhalofop butyl and penoxsulam) @ 0.15 kg/ ha. Respective tank mixed applications with Sampoorna KAU multimix @ 10 g/L were also included along with hand weeded plots with or without Sampoorna and unweeded check. The design was RBD replicated thrice and the variety was Uma. Spraying was given 50 days after transplanting. Phytotoxicity to crop was observed third and seventh day after spraying. Growth and yield attributes of crop were studied and weed parameters like weed count and weed dry matter production were also recorded. Phytotoxicity to rice was observed with tank mix application of 2,4- D+ Sampoorna. Brown spots on leaves and yellowing were noticed by third day itself. The symptoms reduced by 7 days after spraying as new leaves were free of toxicity. This also led to reduction in chlorophyll content at panicle initiation and heading stages. The chlorophyll content at PI stage in 2,4-D + Sampoorna was 2.94 mg/g fresh weight where s in 2,4-D alone spray it was 3.42 mg/. In all other treatments Sampoorna application resulted in slight increase in chlorophyll content. In the case of AlmixⓇ + Sampoorna though visual phytotoxicity symptoms were not present, reduction in plant height was noticed. Reduction in plant height in 2, 4-D+ Sampoorna was 17 % and 10 % at 30 and 60 days after transplanting respectively, compared to its sole application. While AlmixⓇ+ Sampoorna resulted in 13 % and 6 % reduction at 30 DAT and 60 DAT respectively. Individual application of herbicides resulted in comparable tiller counts with respect to their Sampoorna- tank mixed applications except in the case of 2, 4-D and AlmixⓇ at 60 DAT. Similar trend was noticed in number of productive tillers, grain yield and straw yield where all herbicides except 2, 4-D and AlmixⓇ registered higher values with tank mixed application with Sampoorna. Grain yield in 2,4-D+ Sampoorna was 15 percent lower than 2,4-D alone and corresponding reduction in AlmixⓇ+ Sampoorna was 8 percent. In the case of bispyribac sodium, cyhalofop butyl and premix cyhalofop+ penoxsulam, the sole application and tank mix registered comparable grain and straw yields and the grain yield ranged from 5444 to 5647 kg/ ha. Hand weeded plots registered the lowest weed dry matter at all stages. Sole application as well as tank mixed application of all herbicides were comparable in weed dry matter production and weed control efficiency. The weed dry matter in unweeded plot was 1373 kg/ ha whereas the values ranged from 150 kg/ha to 203 kg/ ha in all other treatments, indicating that tank mixing herbicides with Sampoorna has not adversely affected efficiency of herbicides. With respect to effect of Sampoorna application on grain yield of rice, the effect was not very conspicuous as the soil experimental field was not deficient with respect to micro nutrient status. The grain yields in hand weeded plots as well as well as hand weeded+ Sampoorna were comparable with grain yield of 5686 kg/ ha and 5722 kg/ ha respectively.2,4-D+ Sampoorna followed by AlmixⓇ with Sampoorna registered yield reduction of 21 percent and 14 percent compared to hand weeding+ Sampoorna spray which registered higher yield of 5722 kg/ ha. Among tank mixed combinations of herbicides and Sampoorna, 2, 4-D registered the lowest N and K uptake on tank mixing with Sampoorna followed by tank mix application of AlmixⓇ with Sampoorna. All other treatments registered comparable P uptake values except unweeded control, which was the inferior to others. It can be concluded that few post emergence herbicides for rice weed control ie; bispyribac sodium, cyhalofop butyl and VivayaⓇ (premix of cyhalofop butyl and penoxsulam) can be recommended for tank mix application with KAU Sampoorna as foliar spray at 15-20 days after sowing or transplanting. 2,4-D and AlmixⓇ are not compatible with Sampoorna and hence are not suitable for tank mix application.Item Targeted editing of rice micro RNA osa-miR396b through CRISPR/Cas9 system(Department of Plant Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture , Vellanikkara, 2022-10-15) Sanjay Sathian; Rehna AugustineRice (Oryza sativa L.) is one of the most produced and consumed food crops in the world. There is an urgent need to increase rice production to feed the increasing population. Rice yield is determined by several components like grain size, grain weight, number and architecture of panicles, number of spikelets per panicle and grain filling. The microRNA family osa-miR396 is known to suppress the expression of rice growth regulating factors (OsGRFs) resulting in reduced growth and yield. The miRNA osa-miR396b is reported to be a negative regulator of spikelet number and inflorescence development. CRISPR/Cas9 mediated knockout of osa-miR396b gene can thus possibly result in an enhanced yield in rice. Hence, the current study ‘Targeted editing of rice microRNA, osa-miR396b through CRISPR/Cas9 system’ was conducted during the period from 2019 to 2022 at the Department of Plant Biotechnology, College of Agriculture, Kerala Agricultural University, Vellanikkara, Thrissur. Oryza sativa ssp. japonica cultivar Nipponbare was selected for the study due to well established transformation protocols and higher transformation efficiency. Initially, the sequence information of the rice microRNA gene osa-miR396b was retrieved from ‘miRbase’. The stem loop sequence obtained was used to design guide RNAs (gRNAs) using the software CRISPR-P v2.0 and CRISPR-PLANT v2. The gRNAs were selected for further studies mainly based on GC content and number of off-target sites. The target osa-miR396b gene sequence was confirmed by amplifying the genomic region flanking the target using gene specific primers followed by sequencing and the sequence analysis using Clustal Omega and BLASTn showed 100% similarity with reported sequences. The osa-miR396b G1 CRISPR/Cas9 construct was generated by restriction digestion of CRISPR/Cas9 binary vector pRGEB32 using BsaI restriction enzyme followed by ligation with annealed and phosphorylated gRNA. The osa-miR396b G1 construct was cloned to E. coli strain DH5α. The positive clones were confirmed by Sanger sequencing of the plasmid DNA isolated from the colonies and sequence analysis using Clustal Omega. Three (osa-miR396b G1 #2, osa-miR396b G1 #3 and osa-miR396b G1 #4) out of four plasmids sequenced were having gRNA insertion. The osa-miR396b G1 #4 CRISPR/Cas9 construct was mobilized to A. tumefaciens strain EHA105. Positive clones were confirmed by PCR amplification of hygromycin resistance gene (hptII) using specific primers. Colony #1 of A. tumefaciens with osamiR396b G1 CRISPR/Cas9 construct out of the two positive colonies was used for rice transformation. Genetic transformation of rice was achieved through Agrobacterium-mediated transformation. The first step was induction of calli from dehusked and sterilized Nipponbare seeds. Five-day-old calli were infected with Agrobacterium harbouring osa-miR396b G1 CRISPR/Cas9 construct for 1.5-2 min. and co-cultivated for 48 hours. An empty vector was also transformed as control. After washing off excess Agrobacterium growth with sterile distilled water containing Augmentin (300 mgL-1 ) or carbenicillin (250 mgL-1 ), the calli were kept for selection in selection medium supplemented with hygromycin (50 mgL-1 ) and Augmentin (300 mgL-1 ) or carbenicillin (400 mgL-1 ). The calli showing proliferation of microcalli were transferred to regeneration medium supplemented with NAA (0.02 mgL-1 ) and kinetin (2.0 mgL-1 ) for inducing somatic embryogenesis. The somatic embryos were allowed to develop into small plantlets which were transferred to rooting medium for root development. The rooted plantlets were initially maintained in sterile distilled water and then hardened in sterile soil-cocopeat mixture in paper cups and transferred to pots with soilsand-cow dung mixture. A total of 94 putative transformed plants for osa-miR396b G1 CRISPR/Cas9 construct and four vector control plants were obtained. For confirming successful transformation, PCR amplification of hptII gene using hygromycin gene specific primers was done. DNA extracted from 35 plants and two vector control were used as PCR template. A total of 16 out of 35 transformed and two vector control plants were hygromycin positive, indicating successful transformation. The osa-mir396b partial gene sequence was amplified using gene specific primers and sequenced by Sanger sequencing for detecting mutation. Detection of mutation was carried out using ‘Inference of CRISPR Edits (ICE)’ software by Synthego. Analysis using ‘ICE’ detected indel mutations in seven plants. Five plants (71.42%) had deletions and two (28.57%) had insertions around the cut site. Four plants (57.14%) had heterozygous mutations (mutation in one allele) and three (42.86%) had chimeric (more than two) mutations. The mutation efficiency was calculated to be 43.75%. The mutations obtained could lead to a non-functional osa-miR396b gene in these plants. The study successfully demonstrated application of CRISPR/Cas9 system to mutate rice microRNA gene. The knockout of osa-miR396b gene will likely promote the expression of rice GRF genes improving the grain yield. Further studies should be conducted to study inheritance pattern of mutations in the subsequent generations. Genotypic and phenotypic analyses is to be done to study effect of mutated osamiR396b gene on its target genes and on yield and other agronomically important traits.Item CRISPR/Cas9 mediated editing of microRNA osa-miR1432 for enhanced grain filling in rice(Department of Plant Biotechnology, Centre for Plant Biotechnology and molecular Biology, College of Agriculture , Vellanikkara, 2022-09-26) Varsha, K; Rehna AugustineRice (Oryza sativa L.) is the predominant food crop for almost half of the world population. In light of the rising world population, it has become imperative to increase rice grain yield per unit area to meet the global food demand. The CRISPR/Cas9 technology has recently emerged as a powerful tool for genetically modifying crops due to its simplicity, precision and genome editing capabilities like knock-in, knock-out, knock-down and expression activation. Recent research revealed that knock-down of rice microRNA, osa-miR1432 can simultaneously enhance resistance towards blast disease and overall yield in rice. Hence the current study, ‘CRISPR/Cas9 mediated editing of microRNA osa-miR1432 for enhanced grain filling in rice’ was undertaken at the Department of Plant Biotechnology, College of Agriculture, Vellanikkara, Kerala Agricultural University, Thrissur during the period from 2019-2022 with an objective to improve overall yield in rice by enhancing grain filling rate through targeted editing of microRNA osa-miR1432 using CRISPR/Cas9 system. The genomic sequence information of the osa-miR1432 gene was retrieved from rice genome annotation project. The stem-loop sequence of osa-miR1432 available in the microRNA database, miRBase was used for guide RNA (gRNA) designing. The gRNA was designed using the online design tool CRISPR-P v2.0. The best gRNA (osamiR1432 G1) was selected based on its features like location in the genome, on-score value, GC content and no. of off-target sites. Genomic region of osa-miR1432 gene, flanking the gRNA target was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and Clustal Omega analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the gRNA. The development of CRISPR/Cas9 construct was accomplished by annealing of the gRNA strands followed by ligating them into the digested and purified pRGEB32 vector using high fidelity BsaI restriction enzyme. The pRGEB32 vector containing the gRNA construct was cloned into E. coli strain DH5α by transformation using the heat shock method. Plasmid DNA was isolated from the colonies obtained on the transformed plate and were Sanger sequenced using universal M13 reverse primer. The third colony (osamiR1432 G1 #3) was confirmed to be a positive clone after analysing the sequence data. The CRISPR/Cas9 construct was then mobilized into Agrobacterium tumefaciens strain EHA105 using freeze-thaw method. The positive clones were confirmed by plasmid PCR using hygromycin gene specific primers as well as guide sequence specific primers. The positive colony in EHA105 (pRGEB32: osa-miR1432 G1#1) was then used for rice genetic transformation experiments. The rice cultivar Nipponbare (Oryza sativa ssp. japonica cv. Nipponbare) was chosen for the study. Nipponbare seeds were inoculated on to N6 medium supplemented with 2, 4- D (3.0 mg/L) for callus induction. Five days old calli were co-cultivated with Agrobacterium cultures harboring the desired gRNA construct. After 1.5 to 2 days of co-cultivation, the calli were washed thoroughly using bacteriostatic agents augmentin or carbenicillin to remove the excess Agrobacterium load. The calli were then placed on selection medium containing hygromycin and augmentin or carbenicillin. The hygromycin resistant calli showed proliferation after 14 days of incubation. These proliferating microcalli were then transferred to regeneration medium containing NAA (0.02 mg/L) and Kinetin (2.0 mg/L). The regenerated shoots were placed in rooting medium which developed proper roots after one week. The plantlets developed were properly hardened in containment polyhouse. Rice genetic transformation protocol was optimized to suit our laboratory and culture conditions. A total of 25 putative transformant T0 plants were examined for the presence of hygromycin resistance gene and 17 plants were found to be hygromycin positive. PCR amplification of the partial osa-miR1432 gene, flanking the gRNA target region was performed in the hygromycin positive plants and the PCR products were Sanger sequenced to detect mutation. Sequence data analysis using the software Sequencher 5.4.6 detected mutation in seven plants. The detected mutations were single base insertions of A (85.7%) or T (14.3%) exactly at 3 bp upstream to the PAM sequence. The mutation efficiency was found to be 41.18% and the mutations detected were classified as homozygous (71.43%) or heterozygous (28.57%). In the current study, using the CRISPR/Cas9 technology rice plants with mutation in the osa-miR1432 gene were successfully developed which can lead to enhanced grain filling in riceItem Impact of biofertilizers on iron homeostasis under elevated CO2 condition in rice, Oryza sativa(Department of Plant Physiology, College of Agriculture , Vellayani, 2023-01-25) Musti Sri Phani Bhavya; Manju, R VThe study entitled “Impact of biofertilizers on iron homeostasis under elevated CO2 condition in rice, Oryza sativa” was proposed with an objective of assessing the impact of biofertilizers on iron uptake, translocation and grain iron content in rice variety Uma, under CO2 enriched condition. The experiment was conducted using open top chamber (OTC) facility at the Department of Plant Physiology, College of Agriculture, Vellayani, during the period 2021-2022. In this experiment, growth, yield, quality and iron uptake characteristics of rice variety Uma under elevated CO2 condition as influenced by the application of different biofertilizers were evaluated. The experiment was laid out in CRD with 4 treatments [(KAU-POP (control), POP+Azolla, POP+PGPR and POP+AMF)] under 2 conditions [(Ambient CO2 (aCO2) and Elevated CO2 (eCO2)-500 ppm)] with 3 replications. The experiment results revealed that elevated CO2 had a significant positive influence on growth of the plants which was further enhanced by biofertilizer application. When yield parameters were considered, increased CO2 was found to have an adverse effect. There was 58 per cent and 39.28 per cent reduction in filled grains per panicle and grain yield when plants were grown under eCO2 condition. The increase in temperature associated with CO2 enhancement can affect the pollen viability resulting in increased grain chaffiness and yield reduction. Application of biofertilizers had significantly increased grain yield, among which AMF treatment had shown highest impact under both the CO2 conditions. Among the physiological parameters, total chlorophyll content, total reducing sugars and photosynthetic rate increased in the plants grown inside OTC. However, a reduction was noted in total soluble proteins and transpiration rate under elevated CO2 condition. In all these physiological parameters, the biofertilizers treated plants showed higher response when compared to control plants. Grain carbohydrate content was significantly increased by 10.6 per cent in control plants under eCO2 while protein and amylose contents of grains decreased by 14 per cent and 13 per cent indicating reduced grain quality under eCO2. Even under eCO2 condition, the protein content in PGPR treated plants increased by 7.1 per cent and amylose content in AMF treated plants increased by 4.86 per cent when compared to control plants (aCO2). 115 CO2 enrichment and biofertilizers application were found to influence iron uptake and translocation significantly. The pre-anthesis Fe uptake in control plants was dropped by 11.76 per cent and Fe-accumulation by 4.4 per cent under eCO2 condition. But application of PGPR could bring 57.89 per cent increase in iron uptake under ambient condition and 64.7 per cent increase under eCO2 condition. During post-anthesis stage, Fe-uptake was found to increase compared to pre-anthesis stage. Fe-accumulation in the shoot decreased during post-anthesis stage due to re-mobilization of accumulated Fe into grains. There was a drop in Fe-uptake in control plants under eCO2 by 12.5 per cent at post-anthesis stage. At pre-anthesis and post-anthesis stages, plants treated with PGPR (eCO2) have shown increase in Fe uptake by 47.36 per cent and 40.7 per cent, respectively, than control plants. All the biofertilizers including Azolla, PGPR and AMF had significant impact on grain Fe-content under both the CO2 conditions. The Fe content of grains in control plants was dropped by 21 per cent under eCO2 condition. Application of PGPR was found to be most effective resulting in 80.5per cent and by 72.8 per cent increase in Fe-content under eCO2 and aCO2 conditions, respectively. The Fe content in grains was partitioned more into bran than into the polished rice. Control plants when exposed to eCO2 reduced Fe-content in bran and polished rice by 12 per cent and 23.4 per cent, respectively. PGPR treatment increased the Fe-content by 28.6 per cent and 61.7 per cent in bran and polished rice under elevated CO2 condition and by 22.3 per cent and 46.2 per cent under ambient condition when compared to control plants. Diminishing quality of rice with the increasing concentration of CO2 is a global concern today. Elevated CO2 had a positive impact on growth but negatively influenced grain yield due to eCO2 associated high temperature. By the response of experimental plants to AMF and PGPR treatments, these bio-fertilizers can be recommended in overcoming the impacts of elevated CO2 associated high temperature and thereby improving the plant performance. In the present programme, iron uptake and translocation were found to be modified in an unfavourable manner under eCO2 which reflected in the lower iron content of rice grains. The response of iron homeostasis of the experimental plants to the application of biofertilizers, especially PGPR, under eCO2 strongly suggests the possibility of utilizing them for designing iron management strategies to achieve higher yield and quality in rice.