CRISPR/Cas9-mediated knockout of NYC1 gene enhances chlorophyll retention and reduces tillering in Zoysia matrella (L.) Merrill.

KEY MESSAGE: Genome editing by CRISPR/Cas9 can be applied to Z. matrella 'Wakaba', and knockout mutants of ZmNYC1 gene exhibited stay-green phenotype and reduced tillering. Zoysia matrella is a widely used C4 warm-season turfgrass for landscaping, golf courses, and sports fields. Here, we used the CRISPR/Cas9 system to target the Non-Yellow Coloring1 (ZmNYC1) gene in the highly heterozygous allotetraploid Z. matrella 'Wakaba', aiming to generate a novel stay-green variety. Of 441 Agrobacterium-infected calli, 22 (5.0%) were transformed, and 14 of these (63.6%) showed targeted mutations through cleaved amplified polymorphic sequences analysis. Sequencing analysis revealed mutations mostly consisting of 1 or 2 bp indels, occurring 2 to 4 bp upstream of the PAM sequence. Regenerated plants exhibited five ZmNYC1 target locus genotypes, including homozygous mutants with a complete knockout of all four alleles in the T0 generation. Under dark treatment, ZmNYC1-mutated plants displayed suppressed chlorophyll b (Chl b) degradation, leading to higher chlorophyll content and Chl b, with a lower chlorophyll a/chlorophyll b ratio compared to the wild type (WT). However, the ZmNYC1 mutation also inhibited plant growth in homozygous mutant genotypes, exhibiting reduced tillering compared to WT. Additionally, during winter simulation, mutant with a complete knockout retained greenness longer than the WT. This is the first successful use of CRISPR/Cas9 genome editing in zoysiagrass. The mutants of the ZmNYC1 gene would serve as valuable breeding material for developing improved zoysiagrass varieties that can maintain their green color for longer periods, even during winter dormancy.

Unbiased prediction of off-target sites in genome-edited rice using SITE-Seq analysis on a web-based platform.

Genome-editing using the CRISPR-Cas9 system has the potential to substantially accelerate crop breeding. Since off-target editing is one of problems, a reliable method for comprehensively detecting off-target sites is needed. A number of in silico methods based on homology to on-target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq analysis is a comprehensive method that can detect double-strand breaks in vitro. Furthermore, we developed a systematic method using SITE-Seq in combination with web-based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE-Seq analysis of a rice genome targeted by OsFH15 gRNA-Cas9 as a model, and found 41 candidate off-target sites in the annotated regions. Detailed amplicon-sequencing revealed mutations at one off-target site in actual genome-edited rice. Since this off-target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off-target assessment scheme for genome-edited crops that combines the prediction of off-target candidates by SITE-Seq and in silico programs and the validation of off-target sites by amplicon-sequencing.

Tissue Culture and Somatic Embryogenesis in Warm-Season Grasses-Current Status and Its Applications: A Review.

Warm-season grasses are C4 plants and have a high capacity for biomass productivity. These grasses are utilized in many agricultural production systems with their greatest value as feeds for livestock, bioethanol, and turf. However, many important warm-season perennial grasses multiply either by vegetative propagation or form their seeds by an asexual mode of reproduction called apomixis. Therefore, the improvement of these grasses by conventional breeding is difficult and is dependent on the availability of natural genetic variation and its manipulation through breeding and selection. Recent studies have indicated that plant tissue culture system through somatic embryogenesis complements and could further develop conventional breeding programs by micropropagation, somaclonal variation, somatic hybridization, genetic transformation, and genome editing. This review summarizes the tissue culture and somatic embryogenesis in warm-season grasses and focus on current status and above applications including the author's progress.

Quantitative analysis of seven plant hormones in Lotus japonicus using standard addition method.

Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus, which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L. japonicus. The method will be effective, especially when stable isotopes are not available to correct for matrix effects.

Asparaginyl-tRNA synthetase gene (SYNC1) characterized by Lotus corniculatus FOX-superroot lines has effects on plant morphology and amino acid contents of seed in soybean.

The application of useful genes from model plants to crops is an important step to verify its agricultural usefulness. SYNC1, an asparaginyl-tRNA synthetase gene, was previously identified through the Full-length cDNA Over-eXpressor gene (FOX gene-hunting system) of Arabidopsis cDNA by using super-growing root (SR) culture of Lotus corniculatus, and was suggested to have a potential in increasing some amino acid contents and plant biomass. To identify the functionality of SYNC1 gene in a typical legume crop soybean, the effects of its overexpression in transgenic plants to agricultural traits and free amino acid contents were evaluated. The transgenic soybean plants were produced from infected half-seed explants of 1 day old seedlings with the suspension of Agrobacterium tumefaciens harboring expression vector pB7WG2D-SYNC1. The transgenic plants that overexpressed SYNC1 gene had increased asparagine and lysine contents in matured seeds, and increased aspartate, lysine, alanine and histidine contents in germinated seeds. The changes in those free amino acid contents affected plant morphology and led to significant increase in plant length, number of branches and number of branch nodes as yield components of soybean. The transgenic plants also showed a tendency of higher number of pods, seeds and total seed weight per plant. These results showed that the overexpression of SYNC1 gene contributes on the increase of plant free amino acid contents and biomass, and this approach is expected to be applicable in other legumes, grain and forage crops.

Field trial of insect-resistant and herbicide-tolerant genetically modified cotton (Gossypium hirsutum L.) for environmental risk assessment in Japan.

Japan imports cottonseed mainly from Australia and the USA where more than 96% of all cotton varieties grown are genetically modified (GM). GM crops undergo an environmental risk assessment (ERA) under the Law Concerning the Conservation and Sustainable Use of Biological Diversity before import into Japan. Potential adverse effects on biodiversity are comprehensively assessed based on competitiveness, production of harmful substances and outcrossing ability. Even though imported cottonseed is intended for food and feed uses and not for cultivation, the potential risks from seed spillage during transport must be evaluated. In most cases, the ERA requires data collected from in-country field trials to demonstrate how the GM crop behaves in Japan's environment. Confined field trials in Japan were conducted for the ERA of Lepidoptera-resistant and glufosinate-tolerant GM cotton (Gossypium hirsutum L.) lines GHB119 and T304-40. These lines were compared with conventional varieties for growth habit, morphological characteristics, seed dormancy, and allelopathic activity associated with competitiveness and production of harmful substances. Outcrossing ability was not a concern due to the absence of sexually compatible wild relatives in Japan. Although slight statistical differences were observed between the GM line and its conventional comparator for some morphological characteristics, transgenes or transformation were not considered to be responsible for these differences. The trial demonstrated that competitiveness and production of harmful substances by these GM cotton lines were equivalent to conventional cotton varieties that have a long history of safe use, and no potential adverse effects to biosafety in Japan were observed.

Plant regeneration from embryogenic callus derived from shoot apices and production of transgenic plants by particle inflow gun in dwarf napier grass (Pennisetum purpureum Schumach.).

Napier grass (Pennisetum purpureum Schumach.) is a highly productive C4 tropical forage grass that has been targeted as a potential bioenergy crop. To further increase the efficiency of bioethanol production by molecular breeding, a reliable protocol for genetically transforming napier grass is essential. In this study, we report the creation of transgenic napier grass plants derived from embryogenic callus cultures of shoot apices. Embryogenic callus was initiated in three accessions of napier grass and a napier grass×pearl millet hybrid using Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L-1 6-benzylaminopurine (BAP) and 50 µM copper sulfate (CuSO4). Of the accessions tested, a dwarf type with late-heading (DL line) had the best response for embryogenic callus formation. Highly regenerative calli that formed dense polyembryogenic clusters were selected as target tissues for transformation. A plasmid vector, pAHC25, containing an herbicide-resistance gene (bar) and the ß-glucuronidase (GUS) reporter gene was used in particle bombardment experiments. Target tissues treated with 0.6 M osmoticum were bombarded, and transgenic plants were selected under 5.0 mg L-1 bialaphos selection. Although a total of 1400 target tissues yielded nine GUS-positive bialaphos-resistant calli, only one transgenic line that was derived from target tissue with the shortest culture term produced four transgenic plants. Thus, the length of time that the target tissue is in callus culture was one of the most important factors for acquiring transgenic plants in napier grass. This is the first report of successfully producing transgenic napier grass plants.

Sequencing and comparative analyses of the genomes of zoysiagrasses.

Zoysiais a warm-season turfgrass, which comprises 11 allotetraploid species (2n= 4x= 40), each possessing different morphological and physiological traits. To characterize the genetic systems of Zoysia plants and to analyse their structural and functional differences in individual species and accessions, we sequenced the genomes of Zoysia species using HiSeq and MiSeq platforms. As a reference sequence of Zoysia species, we generated a high-quality draft sequence of the genome of Z. japonica accession 'Nagirizaki' (334 Mb) in which 59,271 protein-coding genes were predicted. In parallel, draft genome sequences of Z. matrella 'Wakaba' and Z. pacifica 'Zanpa' were also generated for comparative analyses. To investigate the genetic diversity among the Zoysia species, genome sequence reads of three additional accessions, Z. japonica'Kyoto', Z. japonica'Miyagi' and Z. matrella'Chiba Fair Green', were accumulated, and aligned against the reference genome of 'Nagirizaki' along with those from 'Wakaba' and 'Zanpa'. As a result, we detected 7,424,163 single-nucleotide polymorphisms and 852,488 short indels among these species. The information obtained in this study will be valuable for basic studies on zoysiagrass evolution and genetics as well as for the breeding of zoysiagrasses, and is made available in the 'Zoysia Genome Database' at http://zoysia.kazusa.or.jp.

Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon.

Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus.

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the ß-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.

Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume Lotus japonicus.

Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20 × Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14 days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac-), shoot dry weight without inoculation (SWbac-), root length without inoculation (RLbac-), and root dry weight (RWbac-), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.

Assays of the production of harmful substances by genetically modified oilseed rape (Brassica napus L.) plants in accordance with regulations for evaluating the impact on biodiversity in Japan.

Environmental risk assessment of transgenic crops is implemented under the Cartagena Protocol domestic law in accordance with guidelines for implementing the assessment established by the Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Ministry of Environment (MOE) in Japan. Environmental risk assessments of transgenic crops are implemented based on the concept of 'substantial equivalence' to conventional crops. A unique requirement in Japan to monitor the production of harmful substances, or allelochemicals, is unparalleled in other countries. The potential for allelochemicals to be secreted from the roots of transgenic crops to affect other plants or soil microflora or for substances in the plant body to affect other plants after dying out must be evaluated. We evaluated the allelopathic potential of seven transgenic oilseed rape (Brassica napus L.) lines that express glufosinate tolerance in terms of substantial equivalence to conventional oilseed rape lines, and established evaluation methods. Our results indicate no potential production of allelochemicals for any of the seven transgenic oilseed rape lines compared with conventional oilseed rape lines.

Particle inflow gun-mediated transformation of multiple-shoot clumps in rhodes grass (Chloris gayana).

Rhodes grass (Chloris gayana) is one of the most important warm-season forage grasses. It is cultivated in tropical and subtropical parts of the world and is mostly used for grazing and hay production. We have established a particle-bombardment transformation protocol for rhodes grass using multiple-shoot clumps (MSCs) as the target tissue. A vector pAHC25 containing a herbicide-resistance gene (bar) together with the beta-glucuronidase (GUS) gene was used in transformation experiments. The most efficient recovery of bialaphos-resistant tissue was achieved when the bombarded MSCs were first cultured for 15 d on bialaphos-free medium before being subjected to selection pressure. The resistant tissues regenerated transgenic plants that displayed GUS gene expression. Under optimized conditions, 251 target pieces yielded 46 transgenic plants from 4 independent transgenic lines.

Quantitative trait locus analysis of multiple agronomic traits in the model legume Lotus japonicus.

The first quantitative trait locus (QTL) analysis of multiple agronomic traits in the model legume Lotus japonicus was performed with a population of recombinant inbred lines derived from Miyakojima MG-20 x Gifu B-129. Thirteen agronomic traits were evaluated in 2004 and 2005: traits of vegetative parts (plant height, stem thickness, leaf length, leaf width, plant regrowth, plant shape, and stem color), flowering traits (flowering time and degree), and pod and seed traits (pod length, pod width, seeds per pod, and seed mass). A total of 40 QTLs were detected that explained 5%-69% of total variation. The QTL that explained the most variation was that for stem color, which was detected in the same region of chromosome 2 in both years. Some QTLs were colocated, especially those for pod and seed traits. Seed mass QTLs were located at 5 locations that mapped to the corresponding genomic positions of equivalent QTLs in soybean, pea, chickpea, and mung bean. This study provides fundamental information for breeding of agronomically important legume crops.

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum).

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.