The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA (usually about 20 nucleotides) that is complementary to a target gene or locus and is anchored by a protospacer-adjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks (DSBs), which are subsequently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions, whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.

Although magnesium (Mg) is one of the most important nutrients, involved in many enzyme activities and the structural stabilization of tissues, its importance as a macronutrient ion has been overlooked in recent decades by botanists and agriculturists, who did not regard Mg deficiency (MGD) in plants as a severe health problem. However, recent studies have shown, surprisingly, that Mg contents in historical cereal seeds have markedly declined over time, and two thirds of people surveyed in developed countries received less than their minimum daily Mg requirement. Thus, the mechanisms of response to MGD and ways to increase Mg contents in plants are two urgent practical problems. In this review, we discuss several aspects of MGD in plants, including phenotypic and physiological changes, cell Mg²⁺ homeostasis control by Mg²⁺ transporters, MGD signaling, interactions between Mg²⁺ and other ions, and roles of Mg²⁺ in plant secondary metabolism. Our aim is to improve understanding of the influence of MGD on plant growth and development and to advance crop breeding for Mg enrichment.

Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20) during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11-15 days post-anthesis (DPA) was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15-20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

The type 2 modified augmented design (MAD2) is an efficient unreplicated experimental design used for evaluating large numbers of lines in plant breeding and for assessing genetic variation in a population. Statistical methods and data adjustment for soil heterogeneity have been previously described for this design. In the absence of replicated test genotypes in MAD2, their total variance cannot be partitioned into genetic and error components as required to estimate heritability and genetic correlation of quantitative traits, the two conventional genetic parameters used for breeding selection. We propose a method of estimating the error variance of unreplicated genotypes that uses replicated controls, and then of estimating the genetic parameters. Using the Delta method, we also derived formulas for estimating the sampling variances of the genetic parameters. Computer simulations indicated that the proposed method for estimating genetic parameters and their sampling variances was feasible and the reliability of the estimates was positively associated with the level of heritability of the trait. A case study of estimating the genetic parameters of three quantitative traits, iodine value, oil content, and linolenic acid content, in a biparental recombinant inbred line population of flax with 243 individuals, was conducted using our statistical models. A joint analysis of data over multiple years and sites was suggested for genetic parameter estimation. A pipeline module using SAS and Perl was developed to facilitate data analysis and appended to the previously developed MAD data analysis pipeline (http://probes.pw.usda.gov/bioinformatics_tools/MADPipeline/index.html).

The fungus Pyrenophora tritici-repentis (Died.) Drechs. infects the leaves and kernels of wheat, causing tan spot and red smudge, respectively. Isolates of P. tritici-repentis have been reported to be both phytopathogenic and mycotoxigenic. This research investigates the influence of nitrogen sources on growth and production of mycotoxins by eight different isolates of P. tritici-repentis. A synthetic agar medium (SAM) was used with different nitrogen sources, both inorganic [(NH₄Cl, NH₄NO₃ and (NH₄)₂SO₄)] and organic (l-alanine, l-histidine, and l-lysine), at a concentration of 37.5 mmol L^{− 1}. Individual isolates exhibited different growth rates that varied according to the nitrogen source added to the medium. The choice of nitrogen source also had a major effect on production of the mycotoxins emodin, catenarin and islandicin. The highest concentrations of emodin, 54.40 ± 4.46 μg g^{− 1}, 43.07 ± 23.39 μg g^{− 1} and 28.91 ± 4.64 μg g^{− 1} of growth medium, were produced on the complex medium (V8-potato dextrose agar) by the isolates Alg-H2, 331-2 and TS93-71B, respectively. A relatively high concentration of emodin also was produced by isolates Az35-5 (28.29 ± 4.71 μg g^{− 1} of medium) and TS93-71B (27.03 ± 4.09 μg g^{− 1} of medium) on synthetic medium supplemented with l-alanine. The highest concentrations of catenarin (174.54 ± 14.46 μg g^{− 1} and 104.87 ± 6.13 μg g^{− 1} of medium) were recorded for isolates TS93-71B and Alg-H2 on synthetic medium supplemented with l-alanine and NH₄Cl, respectively. The highest concentration of islandicin (4.64 ± 0.36 μg g^{− 1} medium) was observed for isolate 331-2 in the presence of l-lysine. There was not a close relationship between mycelial growth and mycotoxin production by the fungal isolates. This is the first report on the influence of nitrogen sources on the production of mycotoxins by P. tritici-repentis.

To characterize differences in soybean resistance to salt stress, two soybean species, the wild salt-tolerant soybean Glycine cyrtoloba (serial number ACC547) and the cultivated salt-sensitive soybean G. max (cv. Melrose) were treated with 0, 50, 100, or 150 mmol L^{− 1} NaCl for 5 days. A series of physiological parameters were determined in both shoots and roots, including content of chlorophyll (Chl) and malondialdehyde (MDA); electrolyte leakage (EL); hydrogen peroxide (H₂O₂) concentration; superoxide oxygen radical (O₂⁻) production rate; activities of several enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD); and selective ion (Na⁺ and K⁺) accumulation. Our results showed that the relative salt tolerance of ACC547 was associated with lower loss of Chl content; lower MDA content, EL, H₂O₂ concentration, and O₂⁻ production rate in both shoots and roots; higher POD activity caused by new isoforms in roots; and higher K⁺ concentration and K⁺/Na⁺ ratio in shoots. These results suggested that relative lower membrane injury, efficient K⁺ vs. Na⁺ selective accumulation, and newly induced POD isoenzymes are mechanisms of salt tolerance in soybean.

Understanding the responses of field crops such as soybean to climate warming is critical for economic development and adaptive management of food security. A field warming experiment was conducted using infrared heaters to investigate the responses of soybean phenology, photosynthetic characteristics, and yield to climate warming in the North China Plain. The results showed that 0.4 °C and 0.7 °C increases in soybean canopy air and soil temperature advanced anthesis stage by 3.8 days and shortened the length of entire growth stage by 4.5 days. Warming also decreased the leaf photosynthetic rate by 6.6% and 10.3% at the anthesis and seed filling stages, respectively, but increased the leaf vapor pressure deficit by 9.4%, 15.7%, and 14.1% at the anthesis, pod setting, and seed filling stages, respectively. However, leaf soluble sugar and starch were decreased by 25.6% and 20.5%, respectively, whereas stem soluble sugar was reduced by 12.2% at the anthesis stage under experimental warming. The transportation amount of leaf soluble sugar and contribution rate of transportation amount to seed weight were reduced by 58.2% and 7.7%, respectively, under warming. As a result, warming significantly decreased 100-seed weight and soybean yield by 20.8% and 45.0%, respectively. Our findings provide better mechanistic understanding of soybean yield response to climate warming and could be helpful for forecasting soybean yield under future climate warming conditions.

Spring frost can severely damage or even kill rapeseed/canola (Brassica napus L.) seedlings. A protocol for large scale screening of rapeseed germplasm under frost-simulating conditions has not yet been developed. Accordingly, the present study was conducted to develop a protocol for screening rapeseed germplasm under artificial frost-simulation conditions in a plant growth chamber and in a greenhouse. Nine rapeseed varieties, including three commercial hybrids, three spring types, and three winter types were used. Cold acclimation at 4°C was applied for 0, 7, or 14 days to two-week old seedlings. The seedlings were treated with four freezing temperatures (−4°C, −8°C, −12°C, and −16°C). The length of the freezing period was 16 h, including the ramping of temperature down from 4°C and up from the respective freezing temperature to 4°C. Plants were allowed to recover at 4°C for 24 h before they were moved back to the greenhouse. Frost damage was scored on a 0-5 scale, where 0 denotes completely dead and 5 denotes no damage. Seedling survival from the freezing treatment increased from the non-acclimation to the cold acclimation treatment. However, no significant differences (P<0.05) were found between 7 and 14 days of acclimation. Frost treatment at −4°C resulted in significant differences in seedling damage relative to the other three temperatures, with the −16°C treatment resulting in the highest overall seedling damage. Significant differences were found between the spring type and the other two types (hybrid and winter). However, no significant differences were found between the hybrid and winter types. The suggested protocol for the assessment of frost tolerance is acclimation of two-week old seedlings for 7 days at 4°C followed by frost treatment at −4°C for 16h.