The major hindrance to multidetector CT imaging of the left extraperitoneal space (LES), and the detailed spatial relationships to its related spaces, is that there is no obvious density difference between them. Traditional gross anatomy and thick-slice sectional anatomy imagery are also insufficient to show the anatomic features of this narrow space in three-dimensions (3D). To overcome these obstacles, we used a new method to visualize the anatomic features of the LES and its spatial associations with related spaces, in random sections and in 3D.

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.

Cuticular wax composition greatly impacts plant responses to dehydration. Two parallel pathways exist in Triticeae for manipulating wax composition: the acyl elongation, reduction, and decarbonylation pathway that is active at the vegetative stage and yields primary alcohols and alkanes, and the β-diketone pathway that predominates at the reproductive stage and synthesizes β-diketones. Variation in glaucousness during the reproductive stage of wheat is mainly controlled by the wax production genes, W1 and W2, and wax inhibitor genes, Iw1 and Iw2. Little is known about the metabolic and physiological effects of the genetic interactions among these genes and their roles in shifting wax composition during plant development. We characterized the effect of W1, W2, Iw1, and Iw2 and analyzed their interaction using a set of six near-isogenic lines (NILs) by metabolic, molecular and physiological approaches. Loss of functional alleles of both W genes or the presence of either Iw gene depletes β-diketones and results in the nonglaucous phenotype. Elimination of β-diketones is compensated for by an increase in aldehydes and primary alcohols in the Iw NILs. Accordingly, transcription of CER4-6, which encodes an alcohol-forming fatty acyl-CoA reductase, was elevated 120-fold in iw1Iw2. CER4-6 was transcribed at much higher levels in seedlings than in adult plants, and showed little difference between the glaucous and nonglaucous NILs, suggesting that Iw2 counteracts the developmental repression of CER4-6 at the reproductive stage. While W1 and W2 redundantly function in β-diketone biosynthesis, a combination of both functional alleles led to the β-diketone hydroxylation. Consistent with this, transcription of MAH1-9, which encodes a mid-chain alkane hydroxylase, increased seven-fold only in W1W2. In parallel with the hydroxyl-β-diketone production patterns, glaucousness was intensified and cuticle permeability was reduced significantly in W1W2 compared to the other NILs. This suggests that both W1 and W2 are required for enhancing drought tolerance.

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co-transferred plasmid backbone and low edit rate in wheat, a large T0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium-delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain-regulatory genes, TaCKX2-1, TaGLW7, TaGW2, and TaGW8, in T0 , T1 , and T2 generation plants at an average edit rate of 10% without detecting off-target mutations in the most Cas9-active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160-bp deletion in TaCKX2-D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium-delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.

Lycoris aurea is a medicine-valuable and ornamental herb widely distributed in China. Former studied have showed that methyl jasmonate (MJ) treatment could increase the content of glanthamine-a worldwide medicine for symptomatic treatment of Alzheimer's disease in genus Lycoris plants. To explore the possible role of miRNAs in the regulation of jasmonic acid signaling pathway and uncover their potential correlations, we investigated the expression profiles of small RNAs (sRNAs) and their targets in Lycoris aurea, with MJ treatment by using next-generation deep sequencing.

Increase in grain yield is always a major objective of wheat genetic improvement. The SQUAMOSA promoter-binding protein-like (SPL) genes, coding for a small family of diverse plant-specific transcription factors, represent important targets for improving grain yield and other major agronomic traits in rice. The function of the SPL genes in wheat remains to be investigated in this respect. In this study, we identified 56 wheat orthologues of rice SPL genes belonging to 19 homoeologous groups. Like in rice, nine orthologous TaSPL genes harbour the microRNA156 recognition elements (MRE) in their last exons except for TaSPL13, which harbour the MRE in its 3'-untranslated region (3'UTR). We modified the MRE of TaSPL13 using CRISPR-Cas9 and generated 12 mutations in the three homoeologous genes. As expected, the MRE mutations led to an approximately two-fold increase in the TaSPL13 mutant transcripts. The phenotypic evaluation showed that the MRE mutations in TaSPL13 resulted in a decrease in flowering time, tiller number, and plant height, and a concomitantly increase in grain size and number. The results show that the TaSPL13 mutants exhibit a combination of different phenotypes observed in Arabidopsis AtSPL3/4/5 mutants and rice OsSPL13/14/16 mutants and hold great potential in improving wheat yield by simultaneously increasing grain size and number and by refining plant architecture. The novel TaSPL13 mutations generated can be utilized in wheat breeding programmes to improve these agronomic traits.

Normally, a salt amount greater than 3.5% (w/v) is defined as hypersaline. Large amounts of hypersaline wastewater containing organic pollutants need to be treated before it can be discharged into the environment. The most critical aspect of the biological treatment of saline wastewater is the inhibitory/toxic effect exerted on bacterial metabolism by high salt concentrations. Although efforts have been dedicated to improving the performance through the use of salt-tolerant or halophilic bacteria, the diversities of the strains and the range of substrate spectrum remain limited, especially in chlorophenol wastewater treatment.