In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

Microwave Parboiling: Reduction in Process Time, Browning of Rice and Residual Phosphorus Content in the Waste Water.

The conventional prolonged parboiling process results in high operation cost and grain darkening, which may limit consumption. Moreover, residue generation by rice industries is another challenge. The objective of this study was to evaluate the use of microwave irradiation during soaking and gelatinization stages of parboiling rice. Processing time, colorimetric profile, broken and nongelatinized grains, sucrose and glucose content, free 5-hydroxymethyl-2-furfural, and residual phosphorus were evaluated. As the soaking and gelatinization times during microwave treatments increased, the colorimetric parameters increased; however, the values were lower than those with the conventional process. Regardless of soaking time, a decrease in broken and nongelatinized grains was obtained by using the lowest steaming time (5 min). Additionally, lower residual phosphorus content was found in soaking water (10 and 20 min) when using microwave irradiation. Under favorable conditions, a reduction in the levels of broken and nongelatinized grains, residual phosphorus, and color changes was observed, indicating that microwave irradiation may be more beneficial than conventional parboiling. PRACTICAL APPLICATION: Parboiling requires a high volume of water and soaking time, which leads to high costs, underutilization of infrastructures, and high residue in the water after processing. The rapid parboiling process involves the use of microwaves during the soaking and gelatinization stages. The main advantages of the microwave parboiling process include reduced processing time, ranging from 83% to 95%, higher gelatinization, greater yield, reduced darkening, and reduced residual phosphorus in the effluents by 60%. This report can aid industries in streamlining their processes, thereby providing a high-quality, lower cost, and environmentally safe product.

High ammonium supply impairs photosynthetic efficiency in rice exposed to excess light.

Mechanisms involving ammonium toxicity, excess light, and photosynthesis are scarcely known in plants. We tested the hypothesis that high NH4+ supply in presence of high light decreases photosynthetic efficiency of rice plants, an allegedly tolerant species. Mature rice plants were previously supplied with 10 mM NH4+ or 10 mM NO3- and subsequently exposed to 400 µmol m-2 s-1 (moderate light-ML) or 2000 µmol m-2 s-1 (high light-HL) for 8 h. HL greatly stimulated NH4+ accumulation in roots and in a minor extent in leaves. These plants displayed significant delay in D1 protein recovery in the dark, compared to nitrate-supplied plants. These responses were related to reduction of both PSII and PSI quantum efficiencies and induction of non-photochemical quenching. These changes were also associated with higher limitation in the donor side and lower restriction in the acceptor side of PSI. This later response was closely related to prominent decrease in stomatal conductance and net CO2 assimilation that could have strongly affected the energy balance in chloroplast, favoring ATP accumulation and NPQ induction. In parallel, NH4+ induced a strong increase in the electron flux to photorespiration and, inversely, it decreased the flux to Rubisco carboxylation. Overall, ammonium supply negatively interacts with excess light, possibly by enhancing ammonium transport towards leaves, causing negative effects on some photosynthetic steps. We propose that high ammonium supply to rice combined with excess light is capable to induce strong delay in D1 protein turnover and restriction in stomatal conductance, which might have contributed to generalized disturbances on photosynthetic efficiency.

Light-use efficiency and energy partitioning in rice is cultivar dependent.

One of the main limitations of rice yield in regions of high productive performance is the light-use efficiency (LUE). LUE can be determined at the whole-plant level or at the photosynthetic apparatus level (quantum yield). Both vary according to the intensity and spectral quality of light. The aim of this study was to analyze the cultivar dependence regarding LUE at the plant level and quantum yield using four rice cultivars and four light environments. To achieve this, two in-house Light Systems were developed: Light System I which generates white light environments (spectral quality of 400-700 nm band) and Light System II which generates a blue-red light environment (spectral quality of 400-500 nm and 600-700 nm bands). Light environment conditioned the LUE and quantum yield in PSII of all evaluated cultivars. In white environments, LUE decreased when light intensity duplicated, while in blue-red environments no differences on LUE were observed. Energy partition in PSII was determined by the quantum yield of three de-excitation processes using chlorophyll fluorescence parameters. For this purpose, a quenching analysis followed by a relaxation analysis was performed. The damage of PSII was only increased by low levels of energy in white environments, leading to a decrease in photochemical processes due to the closure of the reaction centers. In conclusion, all rice cultivars evaluated in this study were sensible to low levels of radiation, but the response was cultivar dependent. There was not a clear genotypic relation between LUE and quantum yield.

Influence of gamma radiation on the physical and sensory properties of two Brazilian rice cultivars.

BACKGROUND: Little has been reported about the effects of gamma radiation on the physical and sensory characteristics of rice. Samples were irradiated with 60 Co in doses 0, 1, 2 and 5 kGy, on a rate of 0.4 kGy h-1 . RESULTS: The evaluation of instrumental color showed that increasing doses promoted the yellowing (higher b* value) of the rice grains. Hardness was decreased and stickiness was increased in cooked rice with increasing doses. Sensory evaluation using quantitative descriptive analysis showed that increasing doses promoted higher occurrence of yellowish appearance, emergence of burnt aroma, and an increase of bitter and burnt taste. The sensory acceptance test by 9-point hedonic scale showed alteration of the samples irradiated with 5 kGy in all evaluated attributes (appearance, color, aroma, taste and texture). The lowest dose (1 kGy), however, had good acceptability by the panelists. CONCLUSION: Rice may be subjected to 1 kGy dose of gamma radiation without changes or with small changes in physical properties and with good overall acceptability. © 2016 Society of Chemical Industry.

Mitochondrial GPX1 silencing triggers differential photosynthesis impairment in response to salinity in rice plants.

The physiological role of plant mitochondrial glutathione peroxidases is scarcely known. This study attempted to elucidate the role of a rice mitochondrial isoform (GPX1) in photosynthesis under normal growth and salinity conditions. GPX1 knockdown rice lines (GPX1s) were tested in absence and presence of 100 mM NaCl for 6 d. Growth reduction of GPX1s line under non-stressful conditions, compared with non-transformed (NT) plants occurred in parallel to increased H2 O2 and decreased GSH contents. These changes occurred concurrently with photosynthesis impairment, particularly in Calvin cycle's reactions, since photochemical efficiency did not change. Thus, GPX1 silencing and downstream molecular/metabolic changes modulated photosynthesis differentially. In contrast, salinity induced reduction in both phases of photosynthesis, which were more impaired in silenced plants. These changes were associated with root morphology alterations but not shoot growth. Both studied lines displayed increased GPX activity but H2 O2 content did not change in response to salinity. Transformed plants exhibited lower photorespiration, water use efficiency and root growth, indicating that GPX1 could be important to salt tolerance. Growth reduction of GPX1s line might be related to photosynthesis impairment, which in turn could have involved a cross talk mechanism between mitochondria and chloroplast originated from redox changes due to GPX1 deficiency.

Comparative genomic hybridization analysis of rice dwarf mutants induced by gamma irradiation.

Comparative genomic hybridization (CGH) is a powerful tool used to analyze changes in copy number, polymorphisms, and structural variations in the genome. Gene copy number variation (CNV) is a common form of natural diversity in the genome, which can create new genes and alter gene structure. Thus, CNVs may influence phenotypic variation and gene expression. In this study, to detect CNVs, we irradiated rice seeds with gamma rays (300 Gy) and selected two dwarf mutagenized plants, GA-III-189 and -1052, in the M3 generation. These plants were subjected to CGH analysis using Agilent's RICE CGH array. Most of the CNVs identified were less than 10 kb in length. We detected 90 amplified and 18 deleted regions in GA-III-189, and 99 amplified and 11 deleted regions in GA-III-1052. Of note, CNVs were located on chromosome 12 in both GA-III-189 and -1052, which contained 39 commonly amplified regions in 29 genes. The commonly amplified genes included six genes encoding F-box domain-containing proteins. Alterations in these F-box domain-containing genes were confirmed by quantitative RT-PCR. Integration of CGH and gene expression data identified copy number aberrations and novel genes potentially involved in the dwarf phenotype. These CGH and gene expression data may be useful for uncovering the mechanisms underlying the dwarf phenotype.

Genetic diversity in mutated and non-mutated rice varieties.

We studied the genetic variability due to mutation induced by γ-rays (10, 15, and 20 Kr) on various traits of twelve rice genotypes. Mutated and non-mutated seeds were sown in the field between July 2013 and 2014 using a split plot design. Yield and yield-related trait data was recorded, which showed significant (P < 0.05) genotypic and irradiation effects. Gamma radiation exerted non-significant effects on the panicle length of all plants, indicating the uniformity of performance of this character at different radiation levels. The plant height, grains per main panicle, panicle length, 1000-grain weight, grain weight per main panicle, and fertility percentage was minimum in Basmati-198, Basmati-Pak, Shaheen-2000, Super Basmati, Basmati-385, and Super Basmati, respectively, when exposed to radiation at 20 Kr. However, Basmati-370 attained maximum flag leaf area at this level of radiation. Broad sense heritability ranged from 72.0 to 97.7%, indicating the possibility of selection during earlier generations. Phenotypic correlation was positive and significant between grain weight per main panicle with panicle length and flag leaf area. Number of grains per main panicle and genotypic correlation were positive among grain weight per main panicle, panicle length, fertility percentage, 1000-grain weight, and plant height, indicating the effectiveness of these traits in the selection for yield. Phylogenetic analyses revealed a pair-wise similarity ranging from 0.51-0.76 before mutation and 0.39-0.89 after mutation. This study proved that γ-rays at 20 Kr would be most effective in creating genetic variability in the existing germplasm.

Peroxisomal APX knockdown triggers antioxidant mechanisms favourable for coping with high photorespiratory H2 O2 induced by CAT deficiency in rice.

The physiological role of peroxisomal ascorbate peroxidases (pAPX) is unknown; therefore, we utilized pAPX4 knockdown rice and catalase (CAT) inhibition to assess its role in CAT compensation under high photorespiration. pAPX4 knockdown induced co-suppression in the expression of pAPX3. The rice mutants exhibited metabolic changes such as lower CAT and glycolate oxidase (GO) activities and reduced glyoxylate content; however, APX activity was not altered. CAT inhibition triggered different changes in the expression of CAT, APX and glutathione peroxidase (GPX) isoforms between non-transformed (NT) and silenced plants. These responses were associated with alterations in APX, GPX and GO activities, suggesting redox homeostasis differences. The glutathione oxidation-reduction states were modulated differently in mutants, and the ascorbate redox state was greatly affected in both genotypes. The pAPX suffered less oxidative stress and photosystem II (PSII) damage and displayed higher photosynthesis than the NT plants. The improved acclimation exhibited by the pAPX plants was indicated by lower H2 O2 accumulation, which was associated with lower GO activity and glyoxylate content. The suppression of both pAPXs and/or its downstream metabolic and molecular effects may trigger favourable antioxidant and compensatory mechanisms to cope with CAT deficiency. This physiological acclimation may involve signalling by peroxisomal H2 O2 , which minimized the photorespiration.

Gene expression characteristics of growth-inhibited rice seedlings induced by low-energy N⁺-beam implantation.

Using the RNA sequencing, transcriptomes of growth-inhibited rice seedlings induced by low-energy N+-beam implantation were analyzed. The results showed that rice genes related to binding, electron carrier activity, transcription factor, auxin signal transduction, photosynthesis, and oxidoreductase activity were involved in the response to ion-beam implantation. Moreover, low-energy N+-beam implantation can induce the reactivation of the repressed rice transposable elements. These data are a useful resource for plant ion-beam implantation research.

Cloning and expression analysis of the Lonicera japonica Thunb. chlorogenic acid synthetase gene (LjCCoAOMT1) in rice.

Complete coding DNA sequences of a closely related chlorogenic acid synthetase gene (LjCCoAOMT1) were isolated from Lonicera japonica Thunb. by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). LjCCoAOMT1 was subsequently overexpressed in Escherichia coli and a 25-kD protein was detected by electrophoresis and western blot analysis. High-performance liquid chromatography (HPLC) analysis showed that recombinant LjCCoAOMT1 methylates the caffeic acid substrate to generate ferulic acid. Further analysis showed that the chlorogenic acid content was significantly correlated with the expression level of LjCCoAOMT1 in various tissues of L. japonica Thunb. at different developmental stages. A plant expression vector containing LjCCoAOMT1 was constructed and Agrobacterium-mediated transgenic rice was successfully obtained. Light treatment analysis showed that LjCCoAOMT1 transgenic rice was more sensitive than wild-type rice in responding to the changes in lighting conditions. Although gibberellic acid (GA3) could promote the growth of both wild-type and LjCCoAOMT1 transgenic rice, LjCCoAOMT1 transgenic rice appeared to be more sensitive to GA3. Furthermore, high concentrations of GA3 significantly facilitated the growth of LjCCoAOMT1 transgenic rice.

RNA-seq reveals the downregulated proteins related to photosynthesis in growth-inhibited rice seedlings induced by low-energy N+ beam implantation.

Transcriptomic research based on RNA sequencing (RNA-seq) technology is innovative and will provide new opportunities and biological perspectives for the use of ion-beam implantation in plants. Using RNA-seq, transcriptomes of whole rice seedlings generated from seeds implanted with a low-energy N+ beam were analyzed 96 h after planting. We identified 544 transcripts that were differentially expressed genes (DEGs) in the non-growth-inhibited sample implanted by an N+ beam, including 262 upregulated transcripts and 282 downregulated transcripts. Next, 776 transcripts were identified as DEGs in significantly damaged and growth-inhibited rice seedlings induced by N+ beam implantation, including 283 upregulated transcripts and 493 downregulated transcripts. The Gene Ontology (GO) analysis showed that 19 downregulated DEGs in the injured rice seedlings are enriched for the biological process GO term photosynthesis. The Kyoto Encyclopedia of Genes and Genomes pathway analysis also showed that 11 downregulated DEGs related to chlorophyll a/b-binding proteins are statistically significantly enriched in the photosynthesis-antenna protein pathway (Pathwayid: map00196). This result suggested that the downregulated and injured photosynthesis system contributed to the growth inhibition of rice seedlings induced by low-energy N+ beam implantation.

The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice.

The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.

Effect of ultraviolet-B radiation in laboratory on morphological and ultrastructural characteristics and physiological parameters of selected cultivar of Oryza sativa L.

Ultraviolet-B radiation (UVBR) affects plants in many important ways, including reduction of growth rate and primary productivity, and changes in ultrastructures. Rice (Oryza sativa) is one of the most cultivated cereals in the world, along with corn and wheat, representing over 50% of agricultural production. In this study, we examined O. sativa plants exposed to ambient outdoor radiation and laboratory-controlled photosynthetically active radiation (PAR) and PAR + UVBR conditions for 2 h/day during 30 days of cultivation. The samples were studied for morphological and ultrastructural characteristics, and physiological parameters. PAR + UVBR caused changes in the ultrastructure of leaf of O. sativa and leaf morphology (leaf index, leaf area and specific leaf area, trichomes, and papillae), plant biomass (dry and fresh weight), photosynthetic pigments, phenolic compounds, and protein content. As a photoprotective acclimation strategy against PAR + UVBR damage, an increase of 66.24% in phenolic compounds was observed. Furthermore, PAR + UVBR treatment altering the levels of chlorophylls a and b, and total chlorophyll. In addition, total carotenoid contents decreased after PAR + UVBR treatment. The results strongly suggested that PAR + UVBR negatively affects the ultrastructure, morphology, photosynthetic pigments, and growth rates of leaf of O. sativa and, in the long term, it could affect the viability of this economically important plant.

Comparación de los efectos provocados por el calor y la irradiación sobre Aspergillus parasiticus / Comparison of effects resulting from the application of physical treatments to Aspergillus parasiticus

El propósito de este trabajo fue estudiar la inhibición de la produción de aflatoxinas asociadas al crecimiento del hongo Aspergillus parasiticus NRRL 2999, mediante el empleo de radiaciones ionizantes. El hongo fue cultivado en arroz de tipo comercial en condiciones de temperatura y humedad ideales para la producción de toxinas. Los cultivos así obtenidos fueron irradiados con dosis de 1,5 kGy* (2D10) al cabo de distintos tiempos de desarrollo, observándose que la máxima inhibición de la producción de toxinas se alcanzaba al irradiar cultivos de 20 horas de desarrollo. Asimismo, se estudió el efecto del calor y su combinación con la irradiación sobre la producción de aflatoxinas, la cual fue seguida a lo largo de 11 días de desarrollo de los cultivos. El contenido de toxinas de los distintos cultivos se dosó por dilución a estinción en cromatografía de capa delgada. Los resultados obtenidos mostraron que, tanto un calentamiento de 15 min a 55-C, como la irradiación de los cultivos con una dosis de 1,5kGy, disminuyen el contenido de toxinas, siendo este último processo el más efectivo. Más aún, la combinación de estos dos agentes físicos, irradiando los cultivos inmediatamente después de ser sometidos al calentamiento, reduce los niveles de toxinas por debajo de los límites de detección sugeridos por las organizaciones mundiales de la salud

Comparación de los efectos provocados por el calor y la irradiación sobre Aspergillus parasiticus / Comparison of effects resulting from the application of physical treatments to Aspergillus parasiticus

El propósito de este trabajo fue estudiar la inhibición de la produción de aflatoxinas asociadas al crecimiento del hongo Aspergillus parasiticus NRRL 2999, mediante el empleo de radiaciones ionizantes. El hongo fue cultivado en arroz de tipo comercial en condiciones de temperatura y humedad ideales para la producción de toxinas. Los cultivos así obtenidos fueron irradiados con dosis de 1,5 kGy* (2D10) al cabo de distintos tiempos de desarrollo, observándose que la máxima inhibición de la producción de toxinas se alcanzaba al irradiar cultivos de 20 horas de desarrollo. Asimismo, se estudió el efecto del calor y su combinación con la irradiación sobre la producción de aflatoxinas, la cual fue seguida a lo largo de 11 días de desarrollo de los cultivos. El contenido de toxinas de los distintos cultivos se dosó por dilución a estinción en cromatografía de capa delgada. Los resultados obtenidos mostraron que, tanto un calentamiento de 15 min a 55-C, como la irradiación de los cultivos con una dosis de 1,5kGy, disminuyen el contenido de toxinas, siendo este último processo el más efectivo. Más aún, la combinación de estos dos agentes físicos, irradiando los cultivos inmediatamente después de ser sometidos al calentamiento, reduce los niveles de toxinas por debajo de los límites de detección sugeridos por las organizaciones mundiales de la salud (AU)