Rice Mutant Lines Research Articles

Rice sucrose non-fermenting related protein kinase (SnRK1) has a limited role in defense against Fall armyworm (Spodoptera frugiperda)

Sucrose non-fermenting (SNF) related protein kinase 1 (SnRK1) is a master regulator of energy deprivation signaling and has also been implicated in abiotic and biotic stress regulation. SnRK1 promotes stress tolerance through metabolic and transcriptional changes and plays important roles in innate immunity against various pathogens. However, whether it plays a role against insect herbivory is not understood. To test this, using the wild type (with SnRK1) and snrk1 mutant lines in rice, we examined the potential role of SnRK1 in rice against the ruinous pest, Fall armyworm (FAW), Spodoptera frugiperda. We also investigated the response of FAW towards these lines at different time intervals after exogenous application of plant hormone, Jasmonic acid (JA), and a JA blocker (Ibuprofen). Additional experiments by feeding FAW with leaf infused diet, fresh leaves, and a short-term exposure of FAW to the lines were also carried out. FAW mass gain, growth and development, and host ecophysiological traits were observed. In addition, we also quantified the major surface defenses- trichomes, and wax before and after herbivory. Our results show that FAW response did not vary between mutants and wild type rice. Meanwhile, we found plant hormonal application influenced the ecophysiological traits regardless of mutants and wild type rice. Collectively, we show that while defense against FAW in rice is JA mediated, SnRK1 has a limited role as observed through manipulative experiments with the wild type and snrk1 mutant rice lines.

Potential of Black Rice Mutants (M4) Through Genetic Parameters to Develop Superior Drought-Resistant Varieties

Rice plays an important role in meeting carbohydrate requirement and calorie needs to ensure food security. However, in recent years, rice productivity has decreased due to land conversion and climate change such as El Niño. The productivity of drought-resistant rice can be increased through breeding. Genetic diversity and heritability are important genetic parameters in the plant breeding process. Information about genetic diversity and heritability helps determine genetic progress through selection. This research aims to examine the potential genetic diversity and heritability of several mutant lines (M4) of black rice (G10) in order to develop superior rice varieties that are adaptive to dry land. In this research, we used experimental methods using a Randomized Block Design (RBD), calculating the value of the Genetic Diversity Coefficient (GDC) and heritability between quantitative characters. The results of narrow genetic research were found in the characteristics of the number of productive tillers and weight of 100 grains, while high heritability was found in the characters of plant height, moderate heritability was found in the total number of tillers, panicle length, number of empty grains per panicle, and weight of 100 grains. Overall, this research succeeded in identifying mutant lines (M4) of black rice (G10) which have the potential to be developed into superior varieties, especially for cultivation in dry land.

Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

The unconventional resistance protein PTR recognizes the Magnaporthe oryzae effector AVR-Pita in an allele-specific manner.

Blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating rice diseases. Disease resistance genes such as Pi-ta or Pi-ta2 are critical in protecting rice production from blast. Published work reports that Pi-ta codes for a nucleotide-binding and leucine-rich repeat domain protein (NLR) that recognizes the fungal protease-like effector AVR-Pita by direct binding. However, this model was challenged by the recent discovery that Pi-ta2 resistance, which also relies on AVR-Pita detection, is conferred by the unconventional resistance gene Ptr, which codes for a membrane protein with a cytoplasmic armadillo repeat domain. Here, using NLR Pi-ta and Ptr RNAi knockdown and CRISPR/Cas9 knockout mutant rice lines, we found that AVR-Pita recognition relies solely on Ptr and that the NLR Pi-ta has no role in it, indicating that it is not the Pi-ta resistance gene. Different alleles of Ptr confer different recognition specificities. The A allele of Ptr (PtrA) detects all natural sequence variants of the effector and confers Pi-ta2 resistance, while the B allele of Ptr (PtrB) recognizes a restricted set of AVR-Pita alleles and, thereby, confers Pi-ta resistance. Analysis of the natural diversity in AVR-Pita and of mutant and transgenic strains identified one specific polymorphism in the effector sequence that controls escape from PtrB-mediated resistance. Taken together, our work establishes that the M. oryzae effector AVR-Pita is detected in an allele-specific manner by the unconventional rice resistance protein Ptr and that the NLR Pi-ta has no function in Pi-ta resistance and the recognition of AVR-Pita.

Screening of Some 10th Generation of Rice (Oryza sativa L.) Mutant Lines Using Agronomic and Biochemical Evaluations in Saline Conditions

Screening of Some 10th Generation of Rice (Oryza sativa L.) Mutant Lines Using Agronomic and Biochemical Evaluations in Saline Conditions

Genetic variability, correlation and path analysis studies for yield traits in stable mutant lines of black aromatic rice of Manipur

Genetic variability, correlation and path analysis studies for yield traits in stable mutant lines of black aromatic rice of Manipur

Generation of major glutelin-deficient (GluA, GluB, and GluC) semi-dwarf Koshihikari rice line.

We generated a new Koshihikari rice line with a drastically reduced content of glutelin proteins and higher lodging resistance by using new and conventional plant breeding techniques. Using CRISPR/Cas9-mediated genome editing, we generated mutant rice with drastically decreased contents of major glutelins. A Koshihikari rice mutant line, a123, lacking four glutelins (GluA1, GluA2, GluB4, and GluB5) was used as a host, and another five major glutelin genes (GluA3, GluB1a, GluB1b, GluB2, and GluC) were knocked out through two iterations of Agrobacterium-mediated transformation. Mutant seeds were deficient in the GluA family, GluB family, and GluC, and the line obtained was named GluABC KO. Glutelin content was much lower in GluABC KO than in the existing low-glutelin rice mutant LGC-1. A null segregant of GluABC KO was selected using new-generation sequencing and backcrossing, and the sd-1 allele for the semi-dwarf trait was introduced to increase lodging resistance.

Association Studies in Red Rice Mutant Lines of IRGA-318-11-6-9-2B

Rice is the important cereal food crop. It provides 20% of the world's dietary energy and predominant dietary energy source. It is estimated that the demand for rice will be 137.30 million tonnes by 2050. In order to achieve this, the production per unit area needs to be increased by mutation in turn creating variation. Induced mutants were grown in the field during Kharif 2022 at the Zonal Agricultural and Horticultural Research Station, Brahmavar, Udupi District, Karnataka the results showed grain yield had positive correlations with several traits including number of productive tillers, number of grains per panicle, panicle weight, spikelet fertility, flag leaf length and width but negative correlations with days to fifty per cent flowering, days to maturity, and plant height. Days to maturity, flag leaf length, plant height, panicle weight, number of productive tillers per plant per plant, number of grains per panicle, spikelet fertility and grain length showed a direct positive effect on grain yield. This indicated the effectiveness of direct selection for these traits in improvement of grain yield per plant. Days to fifty per cent flowering, flag leaf width, panicle length, test weight, number of filled grains per panicle, panicle length, and grain breadth had negative direct effects on grain yield per plant.

Field screening for blast resistance gene donors, among rice mutant lines of Mira-1

Rice blast disease, caused by Pyricularia grisea was one of the most destructive fungal diseases in rice worldwide. Therefore, resistance breeding requires continuous efforts to enrich the reservoir of resistance rice lines to effectively tackle the disease. Mutation induction of the Mira-1 rice variety by gamma rays has been conducted, and M6 generation of rice mutant lines was used for this experiment. The rice mutant lines were selected from a total 112 number of rice mutant lines and it had been planted at Cikembar, Sukabumi Village. Cikembar is a hot spot area of blast disease which was used by researchers for the identification of rice lines resistant to blast, and plants were naturally infected. Disease progress was recorded as leaf and neck blast from 0 to 9 ( highly resistant to highly susceptible ). The results showed that disease severity was significantly different in the rice mutant lines studied and it was consistent and not affected by the plant arrangement in the field. Five rice mutant lines showed high resistance to blast disease, and it found 25 numbers of rice mutant lines scored 1 or resistant to blast disease, meanwhile, their wild type of Mira-1 rice variety showed susceptibility to leaf blast and neck blast diseases respectively. The agronomic traits of rice mutant lines were better than their wild type of Mira-1 rice variety. It was found that Mi-37, Mi-77, the Mi-82 rice mutant lines were the best lines for the gene donors of rice-resistant mutant line.

Multi-omics analysis identifies EcCS4 is negatively regulated in response to phytotoxin isovaleric acid stress in Echinochloa crus-galli.

Knowledge of herbicidal targets is critical for weed management and food safety. The phytotoxin isovaleric acid (ISA) is effective against weeds with a broad spectrum, carries low environmental risks, and is thus an excellent herbicide lead. However, the biochemical and molecular mechanisms underlying the action of ISA remain unclear. Multi-omics data showed that acetyl coenzyme A (acetyl-CoA) was the key affected metabolite, and that citrate synthase (CS) 4 was substantially down-regulated under ISA treatment in Echinochloa crus-galli leaves. In particular, the transcript level of EcCS4 was the most significantly regulated among the six genes involved in the top 10 different pathways. The EcCS4 encodes a protein of 472 amino acids and is localized to the cell membrane and mitochondria, similar to the CS4s of other plants. The protein content of EcCS4 was down-regulated after ISA treatment at 0.5 h. ISA markedly inhibited the CS4 activity in vitro in a concentration-dependent manner (IC50 = 41.35 μM). In addition, the transgenic rice plants overexpressing EcCS4 (IC50 = 111.8 mM for OECS4-8 line) were more sensitive, whereas loss-of-function rice mutant lines (IC50 = 746.5 mM for oscs4-19) were more resistant to ISA, compared to wild type (WT) plants (IC50 = 355.6 mM). CS4 was first reported as a negative regulator of plant responses to ISA. These results highlight that CS4 is a candidate target gene for the development of novel herbicides and for breeding herbicide-resistant crops. © 2023 Society of Chemical Industry.

Assessment of Grain Zinc, Iron and Protein Content in Selected Red Rice (Oryza Sativa L.) Mutant Lines

Traditional red rice cultivars grown in coastal regions of Karnataka are popular as food and medicine and specifically in promoting lactation. Effect of zinc, iron and protein deficiency is more in children and lactating women. Analysis of variance evidenced that treatment with gamma rays created significant variability and all the mutant lines exhibited wide range of values for zinc (20-41.5ppm), iron (10-35 ppm), protein content (8-11.9g/100 g) and yield traits. Grain zinc exhibited negative significant association with grain yield per plant, while protein and iron content showed non-significant and negative association with grain yield per plant and very low residual effect of 0.2721 indicated that the traits included in this study explained high percentage of variation in the grain yield and grain zinc, iron and protein content. BMRM15 and BMRM13 mutants showed reduced plant height, early maturity, recorded one percentage increase of protein (9.5% and 10.0%), higher zinc (28 and 25 ppm) and iron content (17 and 13 ppm). These mutant lines can supplement the micronutrient requirement of consumers and also help prevent the micronutrient deficiency

2-DE-based and shotgun proteomics approach in the analysis of the seed proteome of a low phytic acid rice (Oryza sativa, ssp. japonica) mutant

A low phytic acid (lpa) rice mutant line, denoted as Os-lpa-XS110-2, was developed through gamma irradiation (Liu et al., 2007). Subsequently, this mutant line was cultivated alongside its parental wild-type counterpart, Xiushui 110, in four distinct field trials conducted in China. The primary objective of this research was to conduct a comparative proteomic analysis between the parental and mutant lines, aiming to detect and compare protein species with varying levels of relative abundance and their potential involvement in phytic acid biosynthesis. To elucidate the molecular underpinnings of this alteration, proteome analyses were conducted on both the parental and mutant lines, employing LC-MS/MS in conjunction with 2-dimensional gel electrophoresis. Only, 7 proteins consistently exhibited significant differential abundance across all trial locations, suggesting that the regulation of these proteins is less susceptible to environmental influences. In a broader context, among the 700 detected proteins, 56 displayed noteworthy up- or down-regulation across diverse geographical regions. Notably, storage and housekeeping proteins emerged as the most frequently identified protein categories, with globulin and glutelin demonstrating prominent differences in abundance. However, based on our current understanding, none of the identified proteins appear to have direct roles in phytic acid biosynthesis.

Evaluation of some Varieties and Mutant Lines of Rice in Response to Thermal Conditions in the Double Cropping System

Evaluation of some Varieties and Mutant Lines of Rice in Response to Thermal Conditions in the Double Cropping System

Biochemical Analysis of SBEIIb Mutant Rice Lines Generated through CRISPR/Cas-mediated Targeted Mutagenesis

Knock out of the Starch branching Enzyme [SBE] encoding genes lead to increase in the Apparent Amylose Content in cereals. Reduced SBE activity decreases the frequency of branch points in the Amylopectin fraction, while increasing the Amylose Content [AC]. AC is positively correlated to the Resistant Starch [RS], which acts as dietary fibre leading to lowering Glycemic Index [GI]. Among the 4 Starch branching enzyme encoding genes, SBEIIb is exclusively expressed in Rice Endosperm. Therefore, in this study, the homozygous OsSBEIIb mutant lines of CO51 rice cultivar generated through CRISPR/cas9 were selected and biochemical analysis was carried out to determine the AC. Among the events studied, all the events showed a relatively increased AC as compared to the wild type. These findings highlight the need of identifying and developing rice genotypes with high RS and Amylose, which can be suitable for consumption by people suffering from diabetes, obesity, and other colon related illnesses.

Contemplation of protein–protein interactions for salt tolerance in rice mutant lines

Contemplation of protein–protein interactions for salt tolerance in rice mutant lines

Changing biosynthesis of terpenoid percursors in rice through synthetic biology.

Many highly valued chemicals in the pharmaceutical, biotechnological, cosmetic, and biomedical industries belong to the terpenoid family. Biosynthesis of these chemicals relies on polymerization of Isopentenyl di-phosphate (IPP) and/or dimethylallyl diphosphate (DMAPP) monomers, which plants synthesize using two alternative pathways: a cytosolic mevalonic acid (MVA) pathway and a plastidic methyleritritol-4-phosphate (MEP) pathway. As such, developing plants for use as a platform to use IPP/DMAPP and produce high value terpenoids is an important biotechnological goal. Still, IPP/DMAPP are the precursors to many plant developmental hormones. This creates severe challenges in redirecting IPP/DMAPP towards production of non-cognate plant metabolites. A potential solution to this problem is increasing the IPP/DMAPP production flux in planta. Here, we aimed at discovering, understanding, and predicting the effects of increasing IPP/DMAPP production in plants through modelling. We used synthetic biology to create rice lines containing an additional ectopic MVA biosynthetic pathway for producing IPP/DMAPP. The rice lines express three alternative versions of the additional MVA pathway in the plastid, in addition to the normal endogenous pathways. We collected data for changes in macroscopic and molecular phenotypes, gene expression, isoprenoid content, and hormone abundance in those lines. To integrate the molecular and macroscopic data and develop a more in depth understanding of the effects of engineering the exogenous pathway in the mutant rice lines, we developed and analyzed data-centric, line-specific, multilevel mathematical models. These models connect the effects of variations in hormones and gene expression to changes in macroscopic plant phenotype and metabolite concentrations within the MVA and MEP pathways of WT and mutant rice lines. Our models allow us to predict how an exogenous IPP/DMAPP biosynthetic pathway affects the flux of terpenoid precursors. We also quantify the long-term effect of plant hormones on the dynamic behavior of IPP/DMAPP biosynthetic pathways in seeds, and predict plant characteristics, such as plant height, leaf size, and chlorophyll content from molecular data. In addition, our models are a tool that can be used in the future to help in prioritizing re-engineering strategies for the exogenous pathway in order to achieve specific metabolic goals.

The potential of irradiated inoculants consortium for zinc accumulation in rice mutant lines

Some of the microorganisms in soil play important roles in plant performance by improving mineral nutrition. Zinc (Zn) is an essential micronutrient that has various important functions and is only obtained by the intake of foods. Rice is a staple food for Indonesian people. Increasing Zn concentration in rice through fertilization and gamma irradiated inoculants consortium is an effective way to accumulate Zn in rice grains. The objective of this research was to utilize gamma-irradiated inoculants and enhancement of Zn content in rice mutant lines. This study used inoculants consortium from potential microorganisms consisting of Aspergillus niger, Azotobacter sp, Bacillus circulants, and Trichoderma harzianum. They can be used to solubilize metals or minerals that are accumulated in part of the plants. The irradiation of the inoculant was conducted by 250 Gy gamma rays. Seven Mira-1 rice mutant lines together with their wild type, Inpari Nutri Zinc rice variety, were used. Treatment combinations, i.e., Control, T1, T2, T3, T4, and T5 were applied to the rice materials randomized block experimental design with three replications. Agronomic characteristics at the vegetative phase were observed and Zn content in rice grain was measured by atomic absorption spectrophotometry. The result shows significant differences in agronomic characteristics from different treatments. Zn content in rice mutant lines was higher than their wild type, and the irradiated inoculants consortium was very effective in enhancing Zn content in rice grains. The best treatment for accumulation of Zn in rice grain was irradiated inoculants consortium combined with 10 kg/ha zinc sulfate (treatment T4), resulting in a grain Zn content of 72.19 ppm in the 82 rice mutant line.

Molecular dissection of a rice plant with mutation of the leucine carboxyl methyl transferase gene

Drought is a major environmental factor that negatively affects crop growth and productivity. Rice is a drought-sensitive crop, and its development and yield are greatly affected by stress. Using a forward genetics approach, a drought-insensitive TILLING line, ditl2, induced using gamma-ray irradiation, was selected from 150 rice mutant lines. Under drought treatment conditions, the ditl2 mutant showed a high survival rate, fresh weight, relative water content, soluble sugar content, and low hydrogen peroxide (H2O2) content. Whole-genome re-sequencing of ditl2 showed a single-base adenine deletion of the OsLCMT (LOC_Os03g56370) gene, annotated as leucine carboxyl methyl transferase, resulting in a frameshift (OsLCMTp.Thr163fs) with a short peptide of 163 amino acids. The substrate gene OsSnRK1A of OsLCMT was identified through an experiment using yeast two-hybrid and BiFC assays. The subcellular localization of the two genes was confirmed to be in the cytosol. The methyltransferase activities of OsLCMT and OsLCMT.Thr163fs were examined using the target protein OsSnRK1A in an in vitro assay. Consequently, the methyltransferase activity of OsLCMT.Thr163fs was lower than that of the wild-type gene. High expression of starch hydrolysis pathway genes, such as amylases, amylomaltases, and plastidic sugar transporters, was found in ditl2 compared to wild-type (WT) plants. The ditl2 mutant is useful as a genetic resource for understanding the relationship between protein methylation and drought insensitivity and improving drought tolerance in rice mutant breeding.

Spectrum and Density of Gamma and X-ray Induced Mutations in a Non-Model Rice Cultivar.

Physical mutagens are a powerful tool used for genetic research and breeding for over eight decades. Yet, when compared to chemical mutagens, data sets on the effect of different mutagens and dosages on the spectrum and density of induced mutations remain lacking. To address this, we investigated the landscape of mutations induced by gamma and X-ray radiation in the most widely cultivated crop species: rice. A mutant population of a tropical upland rice, Oryza sativa L., was generated and propagated via self-fertilization for seven generations. Five dosages ranging from 75 Gy to 600 Gy in both X-ray and gamma-irradiated material were applied. In the process of a forward genetic screens, 11 unique rice mutant lines showing phenotypic variation were selected for mutation analysis via whole-genome sequencing. Thousands of candidate mutations were recovered in each mutant with single base substitutions being the most common, followed by small indels and structural variants. Higher dosages resulted in a higher accumulation of mutations in gamma-irradiated material, but not in X-ray-treated plants. The in vivo role of all annotated rice genes is yet to be directly investigated. The ability to induce a high density of single nucleotide and structural variants through mutagenesis will likely remain an important approach for functional genomics and breeding.

A Novel RHS1 Locus in Rice Attributes Seed-Pod Shattering by the Regulation of Endogenous S-Nitrosothiols.

Seed or pod shattering in rice (Oryza sativa) is considered to be one of the major factors involved in the domestication of rice as a crop. High seed shattering results in significant yield losses. In this study, we characterize the RICEHIGHSHATTERING 1 (RHS1) that corresponds to the locus LOC_Os04g41250 from a greenhouse screen, involving 145 Ac/Ds transposon mutant rice lines. The knockout mutant line rhs1 exhibited a significantly high shattering of grains in comparison to the wild-type plants. The exogenous application of nitric oxide (NO) resulted in a significant reduction in the expression of RHS1 in wild-type rice plants. The absence of RHS1, which encodes a putative armadillo/beta-catenin repeat family protein, resulted in high sensitivity of the rhs1 plants to nitrosative stress. Interestingly, the basal expression levels of QSH1 and SHAT1 genes (transcription factors that regulate seed-pod shattering in rice) were significantly lower in these plants than in wild-type plants; however, nitrosative stress negatively regulated the expression of QSH1 and SHAT1 in both WT and rhs1 plants, but positively regulated QSH4 expression in rhs1 plants alone. The expression levels of genes responsible for NO production (OsNIA1, OsNIA2, and OsNOA1) were lower in rhs1 plants than in WT plants under normal conditions. However, under nitrosative stress, the expression of OsNIA2 significantly increased in rhs1 plants. The expression of CPL1 (a negative regulator of seed shattering in rice) was significantly lower in rhs1 plants, and we found that CPL1 expression was correlated with S-nitrosothiol (SNO) alteration in rhs1. Interestingly noe1, a rice mutant with high SNO levels, exhibited low seed shattering, whereas rhs1 resulted in low SNO levels with high seed shattering. Therefore, RHS1 is a novel gene that negatively regulates the shattering trait in rice via regulation of endogenous SNO levels. However, the molecular mechanisms involved in the control of RHS1-mediated regulation of seed shattering and its interaction with nitric oxide and involvement in plant defense need to be investigated further.