A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

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.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp.citri to achieve resistance to citrus canker disease

Citrus canker,caused by Xanthomonas citri subsp.citri(Xcc),is a serious bacterial disease that affects citrus production worldwide.Citron C-05(Citrus medica)is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc.However,it has not been determined when,where,and how Xcc is restricted in the tissues of Citron C-05 during the infection process.In the present study,we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc(eGFP-Xcc)strain in Citron C-05 along with five susceptible biotypes(i.e.,lemon,pummelo,sour orange,sweet orange,and ponkan mandarin)upon inoculation via the spraying or leaf infiltration of a bacterial suspension.The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes,Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection.Not surprisingly,resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death,as well as greater upregulation of several defense-related genes,including a pathogenesis-related gene(PR1)and a glutathione S-transferase gene(GST1),compared with sweet orange as a susceptible control.Taken together,our results not only provide further valuable details of the spatiotemporal dynamics of the host entry,propagation,and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

Endogenous auxin and its manipulation influence in vitro shoot organogenesis of citrus epicotyl explants

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.