Diagnosing arsenic-mediated biochemical responses in rice cultivars using Raman spectroscopy.

Rice (Oryza sativa) is the primary crop for nearly half of the world's population. Groundwater in many rice-growing parts of the world often has elevated levels of arsenite and arsenate. At the same time, rice can accumulate up to 20 times more arsenic compared to other staple crops. This places an enormous amount of people at risk of chronic arsenic poisoning. In this study, we investigated whether Raman spectroscopy (RS) could be used to diagnose arsenic toxicity in rice based on biochemical changes that were induced by arsenic accumulation. We modeled arsenite and arsenate stresses in four different rice cultivars grown in hydroponics over a nine-day window. Our results demonstrate that Raman spectra acquired from rice leaves, coupled with partial least squares-discriminant analysis, enabled accurate detection and identification of arsenic stress with approximately 89% accuracy. We also performed high-performance liquid chromatography (HPLC)-analysis of rice leaves to identify the key molecular analytes sensed by RS in confirming arsenic poisoning. We found that RS primarily detected a decrease in the concentration of lutein and an increase in the concentration of vanillic and ferulic acids due to the accumulation of arsenite and arsenate in rice. This showed that these molecules are detectable indicators of biochemical response to arsenic accumulation. Finally, a cross-correlation of RS with HPLC and ICP-MS demonstrated RS's potential for a label-free, non-invasive, and non-destructive quantification of arsenic accumulation in rice.

Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops.

With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of gene expression. Likewise, new trait improvement approaches can take advantage of targeted editing to improve stress tolerance, disease resistance, and nutritional traits. However, several key steps in the process can prove tricky for researchers who might be new to plant genome editing. Here, we present step-by-step guidelines and best practices for a crop genome editing pipeline that should help to improve the rate of success. Important factors in the process include proper target sequence analysis and single guide RNA (sgRNA) design, sequencing of the target site in the genotypes of interest, performing an in vitro CRISPR/Cas9 ribonucleoprotein (RNP) assay to validate the designed sgRNAs, preparing the transformation constructs, considering a protoplast editing step as further validation, and, finally, stable plant transformation and mutation detection by Sanger and/or next-generation sequencing. With these detailed guidelines, a new user should be able to quickly set up a genome editing pipeline in their crop of interest and start making progress with the different CRISPR/Cas-based editing variants for gene validation and trait improvement purposes.

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Comparative transcriptomic analysis of germinating rice seedlings to individual and combined anaerobic and cold stress.

BACKGROUND: Rice is one of the most important cereals consumed worldwide. Two major abiotic factors affecting rice plants in different growth stages are flooding stress and cold stress. These abiotic stresses can take place independently or simultaneously and significantly affect rice plants during germination and seedling growth. Fortunately, a wide array of phenotypic responses conferring flooding stress and chilling stress tolerance exist within the rice germplasm, indicating the presence of different molecular mechanisms underlying tolerance to these stresses. Understanding these differences may assist in developing improved rice cultivars having higher tolerance to both stresses. In this study, we conducted a comparative global gene expression analysis of two rice genotypes with contrasting phenotypes under cold stress, anaerobic stress, and combined cold and anaerobic stress during germination. RESULTS: The differential gene expression analysis revealed that 5571 differentially expressed genes (DEGs), 7206 DEGs, and 13279 DEGs were identified under anaerobic stress, cold stress, and combined stress, respectively. Genes involved in the carbohydrate metabolic process, glucosyltransferase activity, regulation of nitrogen compound metabolic process, protein metabolic process, lipid metabolic process, cellular nitrogen compound biosynthetic process, lipid biosynthetic process, and a microtubule-based process were enriched across all stresses. Notably, the common Gene Ontology (GO) analysis identified three hub genes, namely Os08g0176800 (similar to mRNA-associated protein mrnp 41), Os11g0454200 (dehydrin), and OS10g0505900 (expressed protein). CONCLUSION: A large number of differentially expressed genes were identified under anaerobic, cold conditions during germination and the combination of the two stress conditions in rice. These results will assist in the identification of promising candidate genes for possible manipulation toward rice crops that are more tolerant under flooding and cold during germination, both independently and concurrently.

Increasing the level of resistant starch in 'Presidio' rice through multiplex CRISPR-Cas9 gene editing of starch branching enzyme genes.

Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR-Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.

Identifying the Genetic Basis of Mineral Elements in Rice Grain Using Genome-Wide Association Mapping.

Mineral malnutrition is a major problem in many rice-consuming countries. It is essential to know the genetic mechanisms of accumulation of mineral elements in the rice grain to provide future solutions for this issue. This study was conducted to identify the genetic basis of six mineral elements (Cu, Fe, K, Mg, Mn, and Zn) by using three models for single-locus and six models for multi-locus analysis of a genome-wide association study (GWAS) using 174 diverse rice accessions and 6565 SNP markers. To declare a SNP as significant, -log10(P) ≥ 3.0 and 15% FDR significance cut-off values were used for single-locus models, while LOD ≥ 3.0 was used for multi-locus models. Using these criteria, 147 SNPs were detected by one or two GWAS methods at -log10(P) ≥ 3.0, 48 of which met the 15% FDR significance cut-off value. Single-locus models outperformed multi-locus models before applying multi-test correction, but once applied, multi-locus models performed better. While 14 (~29%) of the identified quantitative trait loci (QTLs) after multiple test correction co-located with previously reported genes/QTLs and marker associations, another 34 trait-associated SNPs were novel. After mining genes within 250 kb of the 48 significant SNP loci, in silico and gene enrichment analyses were conducted to predict their potential functions. These shortlisted genes with their functions could guide future experimental validation, helping us to understand the complex molecular mechanisms controlling rice grain mineral elements.

Mapping QTLs for Reproductive Stage Salinity Tolerance in Rice Using a Cross between Hasawi and BRRI dhan28.

Salinity stress is a major constraint to rice production in many coastal regions due to saline groundwater and river sources, especially during the dry season in coastal areas when seawater intrudes further inland due to reduced river flows. Since salinity tolerance is a complex trait, breeding efforts can be assisted by mapping quantitative trait loci (QTLs) for complementary salt tolerance mechanisms, which can then be combined to provide higher levels of tolerance. While an abundance of seedling stage salinity tolerance QTLs have been mapped, few studies have investigated reproductive stage tolerance in rice due to the difficulty of achieving reliable stage-specific phenotyping techniques. In the current study, a BC1F2 mapping population consisting of 435 individuals derived from a cross between a salt-tolerant Saudi Arabian variety, Hasawi, and a salt-sensitive Bangladeshi variety, BRRI dhan28, was evaluated for yield components after exposure to EC 10 dS/m salinity stress during the reproductive stage. After selecting tolerant and sensitive progeny, 190 individuals were genotyped by skim sequencing, resulting in 6209 high quality single nucleotide polymorphic (SNP) markers. Subsequently, a total of 40 QTLs were identified, of which 24 were for key traits, including productive tillers, number and percent filled spikelets, and grain yield under stress. Importantly, three yield-related QTLs, one each for productive tillers (qPT3.1), number of filled spikelets (qNFS3.1) and grain yield (qGY3.1) under salinity stress, were mapped at the same position (6.7 Mb or 26.1 cM) on chromosome 3, which had not previously been associated with grain yield under salinity stress. These QTLs can be investigated further to dissect the molecular mechanisms underlying reproductive stage salinity tolerance in rice.

Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity.

Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement.

Genetic factors underlying anaerobic germination in rice: Genome-wide association study and transcriptomic analysis.

The success of rice (Oryza sativa L.) germination and survival under submerged conditions is mainly determined by the rapid growth of the coleoptile to reach the water surface. Previous reports have shown the presence of genetic variability within rice accessions in the levels of flooding tolerance during germination or anaerobic germination (AG). Although many studies have focused on the physiological mechanisms of oxygen stress, few studies have explored the breadth of natural variation in AG tolerance-related traits in rice. In this study, we evaluated the coleoptile lengths of a geographically diverse rice panel of 241 accessions, including global accessions along with elite breeding lines and released cultivars from the United States, under the normal and flooded conditions in laboratory and greenhouse environments. A genome-wide association study (GWAS) was performed using a 7K single-nucleotide polymorphism (SNP) array and the phenotypic data of normal coleoptile length, flooded coleoptile length, flooding tolerance index, and survival at 14 d after seeding (DAS). Out of the 30 significant GWAS quantitative trait loci (QTL) regions identified, 14 colocalized with previously identified candidate genes of AG tolerance, whereas 16 were potentially novel. Two rice accessions showing contrasting phenotypic responses to AG stress were selected for the transcriptomics study. The combined approach of GWAS and transcriptomics analysis identified 77 potential candidate genes related to AG tolerance. The findings of our study may assist rice improvement programs in developing rice cultivars with robust tolerance under flooding stress during germination and the early seedling stage.

Raman Spectroscopy Enables Non-invasive and Confirmatory Diagnostics of Aluminum and Iron Toxicities in Rice.

Metal toxicities can be detrimental to a plant health, as well as to the health of animals and humans that consume such plants. Metal content of plants can be analyzed using colorimetric, atomic absorption- or mass spectroscopy-based methods. However, these techniques are destructive, costly and laborious. In the current study, we investigate the potential of Raman spectroscopy (RS), a modern spectroscopic technique, for detection and identification of metal toxicities in rice. We modeled medium and high levels of iron and aluminum toxicities in hydroponically grown plants. Spectroscopic analyses of their leaves showed that both iron and aluminum toxicities can be detected and identified with ∼100% accuracy as early as day 2 after the stress initiation. We also showed that diagnostics accuracy was very high not only on early, but also on middle (day 4-day 8) and late (day 10-day 14) stages of the stress development. Importantly this approach only requires an acquisition time of 1 s; it is non-invasive and non-destructive to plants. Our findings suggest that if implemented in farming, RS can enable pre-symptomatic detection and identification of metallic toxins that would lead to faster recovery of crops and prevent further damage.

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement.

Advances in molecular technologies over the past few decades, such as high-throughput DNA marker genotyping, have provided more powerful plant breeding approaches, including marker-assisted selection and genomic selection. At the same time, massive investments in plant genetics and genomics, led by whole genome sequencing, have led to greater knowledge of genes and genetic pathways across plant genomes. However, there remains a gap between approaches focused on forward genetics, which start with a phenotype to map a mutant locus or QTL with the goal of cloning the causal gene, and approaches using reverse genetics, which start with large-scale sequence data and work back to the gene function. The recent establishment of efficient CRISPR-Cas-based gene editing promises to bridge this gap and provide a rapid method to functionally validate genes and alleles identified through studies of natural variation. CRISPR-Cas techniques can be used to knock out single or multiple genes, precisely modify genes through base and prime editing, and replace alleles. Moreover, technologies such as protoplast isolation, in planta transformation, and the use of developmental regulatory genes promise to enable high-throughput gene editing to accelerate crop improvement.

Phenotypic variation and genome-wide association studies of main culm panicle node number, maximum node production rate, and degree-days to heading in rice.

BACKGROUND: Grain yield is a complex trait that results from interaction between underlying phenotypic traits and climatic, edaphic, and biotic variables. In rice, main culm panicle node number (MCPNN; the node number on which the panicle is borne) and maximum node production rate (MNPR; the number of leaves that emerge per degree-day > 10°C) are primary phenotypic plant traits that have significant positive direct effects on yield-related traits. Degree-days to heading (DDTH), which has a significant positive effect on grain yield, is influenced by the interaction between MCPNN and MNPR. The objective of this research is to assess the phenotypic variation of MCPNN, MNPR, and DDTH in a panel of diverse rice accessions, determine regions in the rice genome associated with these traits using genome-wide association studies (GWAS), and identify putative candidate genes that control these traits. RESULTS: Considerable variation was observed for the three traits in a 220-genotype diverse rice population. MCPNN ranged from 8.1 to 20.9 nodes in 2018 and from 9.9 to 21.0 nodes in 2019. MNPR ranged from 0.0097 to 0.0214 nodes/degree day > 10°C in 2018 and from 0.0108 to 0.0193 nodes/degree-day > 10°C in 2019. DDTH ranged from 713 to 2,345 degree-days > 10°C in 2018 and from 778 to 2,404 degree-days > 10°C in 2019. Thirteen significant (P < 2.91 x 10-7) trait-single nucleotide polymorphism (SNP) associations were identified using the multilocus mixed linear model for GWAS. Significant associations between MCPNN and three SNPs in chromosome 2 (S02_12032235, S02_11971745, and S02_12030176) were detected with both the 2018 and best linear unbiased prediction (BLUP) datasets. Nine SNPs in chromosome 6 (S06_1970442, S06_2310856, S06_2550351, S06_1968653, S06_2296852, S06_1968680, S06_1968681, S06_1970597, and S06_1970602) were significantly associated with MNPR in the 2019 dataset. One SNP in chromosome 11 (S11_29358169) was significantly associated with the DDTH in the BLUP dataset. CONCLUSIONS: This study identifies SNP markers that are putatively associated with MCPNN, MNPR, and DDTH. Some of these SNPs were located within or near gene models, which identify possible candidate genes involved in these traits. Validation of the putative candidate genes through expression and gene editing analyses are necessary to confirm their roles in regulating MCPNN, MNPR, and DDTH. Identifying the underlying genetic basis for primary phenotypic traits MCPNN and MNPR could lead to the development of fast and efficient approaches for their estimation, such as marker-assisted selection and gene editing, which is essential in increasing breeding efficiency and enhancing grain yield in rice. On the other hand, DDTH is a resultant variable that is highly affected by nitrogen and water management, plant density, and several other factors.

Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds.

CRISPR-Cas gene editing technologies offer the potential to modify crops precisely; however, in vitro plant transformation and regeneration techniques present a bottleneck due to the lengthy and genotype-specific tissue culture process. Ideally, in planta transformation can bypass tissue culture and directly lead to transformed plants, but efficient in planta delivery and transformation remains a challenge. This study investigates transformation methods that have the potential to directly alter germline cells, eliminating the challenge of in vitro plant regeneration. Recent studies have demonstrated that carbon nanotubes (CNTs) loaded with plasmid DNA can diffuse through plant cell walls, facilitating transient expression of foreign genetic elements in plant tissues. To test if this approach is a viable technique for in planta transformation, CNT-mediated plasmid DNA delivery into rice tissues was performed using leaf and excised-embryo infiltration with reporter genes. Quantitative and qualitative data indicate that CNTs facilitate plasmid DNA delivery in rice leaf and embryo tissues, resulting in transient GFP, YFP, and GUS expression. Experiments were also initiated with CRISPR-Cas vectors targeting the phytoene desaturase (PDS) gene for CNT delivery into mature embryos to create heritable genetic edits. Overall, the results suggest that CNT-based delivery of plasmid DNA appears promising for in planta transformation, and further optimization can enable high-throughput gene editing to accelerate functional genomics and crop improvement activities.

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Reference-Guided De Novo Genome Assembly to Dissect a QTL Region for Submergence Tolerance Derived from Ciherang-Sub1.

Global climate change has increased the number of severe flooding events that affect agriculture, including rice production in the U.S. and internationally. Heavy rainfall can cause rice plants to be completely submerged, which can significantly affect grain yield or completely destroy the plants. Recently, a major effect submergence tolerance QTL during the vegetative stage, qSub8.1, which originated from Ciherang-Sub1, was identified in a mapping population derived from a cross between Ciherang-Sub1 and IR10F365. Ciherang-Sub1 was, in turn, derived from a cross between Ciherang and IR64-Sub1. Here, we characterize the qSub8.1 region by analyzing the sequence information of Ciherang-Sub1 and its two parents (Ciherang and IR64-Sub1) and compare the whole genome profile of these varieties with the Nipponbare and Minghui 63 (MH63) reference genomes. The three rice varieties were sequenced with 150 bp pair-end whole-genome shotgun sequencing (Illumina HiSeq4000), followed by performing the Trimmomatic-SOAPdenovo2-MUMmer3 pipeline for genome assembly, resulting in approximate genome sizes of 354.4, 343.7, and 344.7 Mb, with N50 values of 25.1, 25.4, and 26.1 kb, respectively. The results showed that the Ciherang-Sub1 genome is composed of 59-63% Ciherang, 22-24% of IR64-Sub1, and 15-17% of unknown sources. The genome profile revealed a more detailed genomic composition than previous marker-assisted breeding and showed that the qSub8.1 region is mostly from Ciherang, with some introgressed segments from IR64-Sub1 and currently unknown source(s).

Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.

Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.

Transcriptome profiling of two rice genotypes under mild field drought stress during grain-filling stage.

Drought is one of the most critical abiotic stresses that threaten crop production worldwide. This stress affects the rice crop in all stages of rice development; however, the occurrence during reproductive and grain-filling stages has the most impact on grain yield. Although many global transcriptomic studies have been performed during the reproductive stage in rice, very limited information is available for the grain-filling stage. Hence, we intend to investigate how the rice plant responds to drought stress during the grain-filling stage and how the responses change over time under field conditions. Two rice genotypes were selected for RNA-seq analysis: '4610', previously reported as a moderately tolerant breeding line, and Rondo, an elite indica rice cultivar susceptible to drought conditions. Additionally, 10 agronomic traits were evaluated under normal irrigated and drought conditions. Leaf tissues were collected during grain-filling stages at two time points, 14 and 21 days after the drought treatment, from both the drought field and normal irrigated field conditions. Based on agronomic performances, '4610' was less negatively affected than Rondo under mild drought conditions, and expression profiling largely aligned with the phenotypic data. The transcriptomic data indicated that, in general, '4610' had much earlier responses than its counterpart in mitigating the impact of drought stress. Several key genes and gene families related to drought stress or stress-related conditions were found differentially expressed in this study, including transcription factors, drought tolerance genes and reactive oxygen species scavengers. Furthermore, this study identified novel differentially expressed genes (DEGs) without function annotations that may play roles in drought tolerance-related functions. Some of the important DEGs detected in this study can be targeted for future research.

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

Non-Invasive Identification of Nutrient Components in Grain.

Digital farming is a modern agricultural concept that aims to maximize the crop yield while simultaneously minimizing the environmental impact of farming. Successful implementation of digital farming requires development of sensors to detect and identify diseases and abiotic stresses in plants, as well as to probe the nutrient content of seeds and identify plant varieties. Experimental evidence of the suitability of Raman spectroscopy (RS) for confirmatory diagnostics of plant diseases was previously provided by our team and other research groups. In this study, we investigate the potential use of RS as a label-free, non-invasive and non-destructive analytical technique for the fast and accurate identification of nutrient components in the grains from 15 different rice genotypes. We demonstrate that spectroscopic analysis of intact rice seeds provides the accurate rice variety identification in ~86% of samples. These results suggest that RS can be used for fully automated, fast and accurate identification of seeds nutrient components.

OsCOP1 regulates embryo development and flavonoid biosynthesis in rice (Oryza sativa L.).

KEY MESSAGE: Novel mutations of OsCOP1 were identified to be responsible for yellowish pericarp and embryo lethal phenotype, which revealed that OsCOP1 plays a crucial role in flavonoid biosynthesis and embryogenesis in rice seed. Successful production of viable seeds is a major component of plant life cycles, and seed development is a complex, highly regulated process that affects characteristics such as seed viability and color. In this study, three yellowish-pericarp embryo lethal (yel) mutants, yel-hc, yel-sk, and yel-cc, were produced from three different japonica cultivars of rice (Oryza sativa L). Mutant seeds had yellowish pericarps and exhibited embryonic lethality, with significantly reduced grain size and weight. Morphological aberrations were apparent by 5 days after pollination, with abnormal embryo development and increased flavonoid accumulation observed in the yel mutants. Genetic analysis and mapping revealed that the phenotype of the three yel mutants was controlled by a single recessive gene, LOC_Os02g53140, an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). The yel-hc, yel-sk, and yel-cc mutants carried mutations in the RING finger, coiled-coil, and WD40 repeat domains, respectively, of OsCOP1. CRISPR/Cas9-targeted mutagenesis was used to knock out OsCOP1 by targeting its functional domains, and transgenic seed displayed the yel mutant phenotype. Overexpression of OsCOP1 in a homozygous yel-hc mutant background restored pericarp color, and the aberrant flavonoid accumulation observed in yel-hc mutant was significantly reduced in the embryo and endosperm. These results demonstrate that OsCOP1 is associated with embryo development and flavonoid biosynthesis in rice grains. This study will facilitate a better understanding of the functional roles of OsCOP1 involved in early embryogenesis and flavonoid biosynthesis in rice seeds.