摘要：
The development of genetically modified (GM) crops speeds up the obtainment of novel varieties with improved agronomic characteristics. However, the risk evaluation of the use of GMs is mandatory before their release in the market. In this paper, an untargeted and comprehensive nuclear magnetic resonance-based metabolomic study was carried out on the peel and flesh of a transgenic lemon clone (E23) expressing the chit42 gene and exhibiting an increased tolerance to some pathogenic fungi and on its wild type. Results highlighted a substantial equivalence of the metabolomics profile of the transgenic clone compared to the wild type. In addition, an enhanced response of the E23 clone toward fungal pathogens affecting the postharvest management in lemon was evidenced. These results confirm the potential of genetic engineering for the punctual modification of specific agronomic traits without altering the whole pattern of metabolites and open new perspectives for a more sustainable and effective management of specific postharvest diseases in citrus.
关键词：
Genetic engineering;High-throughput screening;Molecular engineering in plants;Mutation;Next-generation sequencing;Gene editing
摘要：
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.
关键词：
citrus;Agrobacterium;transient expression efficiency;yellow fluorescent protein (YFP)
摘要：
Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple, easy to perform, and achieve gene expression rapidly. This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope. These factors included the composition of the infiltration buffer, the Agrobacterium cell density, the leaf development stage, the incubation temperature, and plant genotype. The highest transient expression level of yellow fluorescent protein (YFP) was detected in Mexican lime (Citrus aurantifolia) on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1 (15 mmol L~(-1) 2-(N-morpholino) ethanesulfonic acid, 10 mmol L~(-1) MgCl_2, and 200 μmol L~(-1) acetosyringone), which had an optical density of 0.8 and was incubated at 22°C. Additionally, this transient expression assay was applied to other citrus genotypes. Of note, trifoliate orange (Poncirus trifoliata) and kumquat (Fortunella obovate) had higher expression efficiency than other six genotypes of the Citrus genus. Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.
摘要：
Storage of individual seal-packaged citrus fruit at room temperature in China is a very common practice that requires a pre-storage treatment with a high concentration of an effective fungicide as imazalil (IMZ) to prevent decay. In this study, lemons were washed with NaOCl (200 mg L-1) or not, treated with IMZ (50 or 1000 mg L-1) at 20 or 50 degrees C and individually wrapped with a 16-mu m thick extensible polyvinylchloride film (Film A) or two heat shrinkable polyolefinic films, thick 15 (Film B) or 19-mm (Film C). The sequential treatment with NaOCl and IMZ at 50 mg L-1 at 50 degrees C, was as effective as IMZ at 1000 mg L-1 at 20 degrees C in controlling Penicillium decay. Losses for decay in fruit wrapped with the two more permeable films (Film A and Film B) never exceeded 10%, while in those wrapped with Film C (the least permeable) peaked to 41%. All films reduced weight losses, which at the end of storage were 11% in fruit wrapped with Film A and below 4% in those wrapped with the other two films, while were 41% in unwrapped ones. After one week of storage, only 50% of unwrapped fruit were marketable whereas all wrapped fruit were still marketable after 8 weeks. Respiration as well as juice acetaldehyde and ethanol were slightly affected by the two more permeable films, while an abnormal production of CO2, acetaldehyde and ethanol occurred in those wrapped with film C. Changes in chemical parameters were relevant in fruit sealed with Film C and minor in those with Film A and Film B. Decay control and quality preservation of lemons stored at room temperature can be achieved for several weeks by a sequential treatment with NaOCl and a heated water emulsion of IMZ at 50 mg L (1), when fruit are wrapped with plastic films highly permeable to gases. (C) 2016 Elsevier B.V. All rights reserved.
摘要：
Endogenous auxin is an important regulator of in vivo organ development, but its role in in vitro organogenesis is unclear. It has been observed that the basal end of epicotyl cuttings of juvenile citrus seedlings produces fewer shoots than the apical end. Here, we report that elevated endogenous auxin levels in the basal end of citrus epicotyl cuttings are inhibitory for in vitro shoot organogenesis. Using transgenic citrus plants expressing an auxin-inducible GUS reporter gene, we have observed elevated levels of auxin at the basal end of stem cuttings that are mediated by polar auxin transport. Depleting endogenous auxin or blocking polar auxin transport enhances shoot organogenesis. An auxin transport inhibitor, N-1-naphthylphthalamic acid (NPA), can also enhance shoot organogenesis independent of its action on polar auxin transport. Finally, we demonstrate that the promotional effects of depleting endogenous auxin or blocking polar auxin transport on shoot organogenesis are cytokinin-dependent. Our study thus provides meaningful insights into possible roles of endogenous auxin and polar auxin transport, as well as auxin–cytokinin interactions, in in vitro shoot organogenesis. Meanwhile, our results may also provide practical strategies for improving in vitro shoot organogenesis for citrus and many other plant species.
摘要：
Stem-pitting (SP) is the main type of citrus tristeza virus (CTV) that causes severe damage to citrus trees, especially those of sweet orange, in Hunan province, China. Understanding the local CTV population structure should provide clues for effective mild strain cross-protection (MSCP) of the SP strain of CTV. In this study, markers for the p23 gene, multiple molecular markers (MMMs), and sequence analysis of the three silencing suppressor genes (p20, p23 and p25) were employed to analyze the genetic diversity and genotype composition of the CTV population based on 51 CTV-positive samples collected from 14 citrus orchards scattered around six major citrus-growing areas of Hunan. The results indicated that the CTV population structure was extremely complex and that infection was highly mixed. In total, p23 gene markers resulted in six profiles, and MMMs demonstrated 25 profiles. The severe VT and T3 types appeared to be predominantly associated with SP, while the mild T30 and RB types were related to asymptomatic samples. Based on phylogenetic analysis of the amino acid sequences of p20, p23 and p25, 19 representative CTV samples were classified into seven recently established CTV groups and a potentially novel one. A high level of genetic diversity, as well as potential recombination, was revealed among different CTV isolates. Five pure SP severe and two pure mild strains were identified by genotype composition analysis. Taken together, the results update the genetic diversity of CTV in Hunan with the detection of one possible novel strain, and this information might be applicable for the selection of appropriate mild CTV strains for controlling citrus SP disease through cross-protection.
