Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

CsBZIP40 confers resistance against citrus bacterial canker by repressing CsWRKY43-CsPrx53/CsSOD13 cascade mediated ROS scavenging

As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri subsp.citri(Xcc)seriously impacts citrus plantation and fruit production globally.In an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated.Here,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing CsBZIP40.CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity.CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges.In contrast,its inhibition produces strong resistance to CBC.CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants.CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance.Overall,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance.These mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.

CitGVD:a comprehensive database of citrus genomic variations

Citrus is one of the most important commercial fruit crops worldwide.With the vast genomic data currently available for citrus fruit,genetic relationships,and molecular markers can be assessed for the development of molecular breeding and genomic selection strategies.In this study,to permit the ease of access to these data,a web-based database,the citrus genomic variation database(CitGVD,http://citgvd.cric.cn/home)was developed as the first citrusspecific comprehensive database dedicated to genome-wide variations including single nucleotide polymorphisms(SNPs)and insertions/deletions(INDELs).The current version(V1.0.0)of CitGVD is an open-access resource centered on 1,493,258,964 high-quality genomic variations and 84 phenotypes of 346 organisms curated from in-house projects and public resources.CitGVD integrates closely related information on genomic variation annotations,related gene annotations,and details regarding the organisms,incorporating a variety of built-in tools for data accession and analysis.As an example,CitGWAS can be used for genome-wide association studies(GWASs)with SNPs and phenotypic data,while CitEVOL can be used for genetic structure analysis.These features make CitGVD a comprehensive web portal and bioinformatics platform for citrus-related studies.It also provides a model for analyzing genome-wide variations for a wide range of crop varieties.