Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

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Paddy-upland rotation is an unavoidable cropping system for Asia to meet the increasing demand for food. The reduction in grain yields has increased the research interest on the soil properties of rice-based cropping systems. Paddy-upland rotation fields are unique from other wetland or upland soils, because they are associated with frequent cycling between wetting and drying under anaerobic and aerobic conditions; such rotations affect the soil C and N cycles, make the chemical speciation and biological effectiveness of soil nutrient elements varied with seasons, increase the diversity of soil organisms, and make the soil physical properties more difficult to analyze. Consequently, maintaining or improving soil quality at a desirable level has become a complicated issue. Therefore, fully understanding the soil characteristics of paddy-upland rotation is necessary for the sustainable development of the system. In this paper, we offer helpful insight into the effect of rice-upland combinations on the soil chemical, physical, and biological properties, which could provide guidance for reasonable cultivation management measures and contribute to the improvement of soil quality and crop yield.

Rice is an important staple crop throughout the world, but environmental stress like low-light conditions can negatively impact crop yield and quality. Using pot experiments and field experiments, we studied the effects of shading on starch pasting viscosity and starch content with six rice varieties for three years, using the Rapid Visco Analyser to measure starch pasting viscosity. Shading at different growth stages and in different rice varieties all affected the starch pasting characteristics of rice. The effects of shading on starch pasting viscosity at middle and later growth stages were greater than those at earlier stages. Shading enhanced breakdown but reduced hold viscosity and setback at tillering-elongation stage. Most pasting parameters changed significantly with shading after elongation stage. Furthermore, the responses of different varieties to shading differed markedly. The change scope of starch pasting viscosity in Dexiang 4103 was rather small after heading, while that in IIyou 498 and Gangyou 906 was small before heading. We observed clear tendencies in peak viscosity, breakdown, and pasting temperature of the five rice varieties with shading in 2010 and 2011. Correlation analysis indicated that the rice amylose content was negatively correlated with breakdown, but was positively correlated with setback. Based on our results, IIyou 498, Gangyou 906, and Dexiang 4103 had higher shade endurance, making these varieties most suitable for high-quality rice cultivation in low-light regions.

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