Worldwide, citrus is one of the most important fruit crops and is grown in more than 130 countries, predominantly in tropical and subtropical areas. The healthy progress of the citrus industry has been seriously affected by biotic and abiotic stresses. Several diseases, such as canker and huanglongbing, etc., rigorously affect citrus plant growth, fruit quality, and yield. Genetic engineering technologies, such as genetic transformation and genome editing, represent successful and attractive approaches for developing disease-resistant crops. These genetic engineering technologies have been widely used to develop citrus disease-resistant varieties against canker, huanglongbing, and many other fungal and viral diseases. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based systems have made genome editing an indispensable genetic manipulation tool that has been applied to many crops, including citrus. The improved CRISPR systems, such as CRISPR/CRISPR-associated protein (Cas)9 and CRISPR/Cpf1 systems, can provide a promising new corridor for generating citrus varieties that are resistant to different pathogens. The advances in biotechnological tools and the complete genome sequence of several citrus species will undoubtedly improve the breeding for citrus disease resistance with a much greater degree of precision. Here, we attempt to summarize the recent successful progress that has been achieved in the effective application of genetic engineering and genome editing technologies to obtain citrus disease-resistant (bacterial, fungal, and virus) crops. Furthermore, we also discuss the opportunities and challenges of genetic engineering and genome editing technologies for citrus disease resistance.

BackgroundBud mutation is a vital method of citrus. ‘Wuzi Ougan’ (mutant type, MT) as a bud variant of ‘Ougan’ (wild type, WT) was first found in 1996 and has become popular because of its male sterility and seedless character. Previous analysis of its cytological sections and transcriptome revealed that the abnormal microsporogenesis that occurs before the tetrad stage of anther development might be the result of down-regulated oxidation-reduction biological processes in MT. To reveal the mechanism behind the male sterility in MT at the post-transcriptional stage, proteome profiling and integrative analysis on previously obtained transcriptome and proteome data were performed in two strains.ResultsThe proteome profiling was performed by iTRAQ (isobaric Tags for relative and absolute quantitation) analysis and 6201 high-confidence proteins were identified, among which there were 487 differentially expressed proteins (DEPs) in one or more developmental stages of anthers between MT and WT. The main functional subcategories associated with the main category biological process into which the DEPs were classified were sporopollenin biosynthesis process and pollen exine formation. The enriched pathways were phenylpropanoid biosynthesis, flavonoid biosynthesis, and phenylalanine metabolism. Moreover, there were eight pathways linked in terms of being related to phenylpropanoid metabolism. Eighteen important genes related to phenylpropanoid metabolism were also analysized by qRT-PCR (quantitative real time PCR). An integrative analysis of the fold change at the transcript (log2 FPKM ratios) and protein (log1.2 iTRAQ ratios) levels was performed to reveal the consistency of gene expression at transcriptional and proteomic level. In general, the expression of genes and proteins tended to be positively correlated, in which the correlation coefficients were 0.3414 (all genes and all proteins) and 0.5686 (DEPs and according genes).ConclusionThis study is the first to offer a comprehensive understanding of the gene regulation in ‘Wuzi Ougan’ and its wild type, especially during the microsporocyte to meiosis stage. Specifically, the involved genes include those in phenylpropanoid biosynthesis, flavonoid biosynthesis, and phenylalanine metabolism, as determined by integrative transcriptome and proteome analysis.Electronic supplementary materialThe online version of this article (10.1186/s12863-018-0693-9) contains supplementary material, which is available to authorized users.

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BackgroundChimeras synthesized artificially by grafting are crucial to the breeding of perennial woody plants. ‘Hongrou Huyou’ (Citrus changshan-huyou + Citrus unshiu) is a new graft chimera originating from the junction where a Citrus changshan-huyou (“C”) scion was top-grafted onto a stock Satsuma mandarin ‘Owari’ (C. unshiu, “O”). The chimera was named OCC because the cell layer constitutions were O for Layer 1(L1) and C for L2 and L3. In this study, profiles of primary metabolites, volatiles and carotenoids derived from different tissues in OCC and the two donors were investigated, with the aim of determining the relationship between the layer donors and metabolites.ResultsThe comparison of the metabolite profiles showed that the amount and composition of metabolites were different between the peels and the juice sacs, as well as between OCC and each of the two donors. The absence or presence of specific metabolites (such as the carotenoids violaxanthin and β-cryptoxanthin, the volatile hydrocarbon germacrene D, and the primary metabolites citric acid and sorbose) in each tissue was identified in the three phenotypes. According to principal component analysis (PCA), overall, the metabolites in the peel of the chimera were derived from donor C, whereas those in the juice sac of the chimera came from donor O.ConclusionThe profiles of primary metabolites, volatiles and carotenoids derived from the peels and juice sacs of OCC and the two donors were systematically compared. The content and composition of metabolites were different between the tissues and between OCC and the each of the two donors. A clear donor dominant pattern of metabolite inheritance was observed in the different tissues of OCC and was basically consistent with the layer origin; the peel of the chimera was derived from C, and the juice sacs of the chimera came from O. These profiles provide potential chemical markers for genotype differentiation, citrus breeding assessment, and donor selection during artificial chimera synthesis.

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Citrus is widely grown all over the world, and citrus fruits have long been recognized for their nutritional and medical value for human health. However, some local citrus varieties with potentially important value are still elusive. In the current study, we elucidated the biological characteristics, phylogenetic and phytochemical profiling, antioxidants and antioxidant activities of the two local citrus varieties, namely Zangju and Tuju. The physiological and phylogenetic analysis showed that Zangju fruit has the characteristics of wrinkled skin, higher acidity, and phylogenetically closest to sour mandarin Citrus sunki, whereas, Tuju is a kind of red orange with vermilion peel, small fruit and high sugar content, and closely clustered with Citrus erythrosa. The phytochemical analysis showed that many nutrition and antioxidant related differentially accumulated metabolites (DAMs) were detected in the peel and pulp of Zangju and Tuju fruits. Furthermore, it was found that the relative abundance of some key flavonoids and phenolic acids, such as tangeritin, sinensetin, diosmetin, nobiletin, and sinapic acid in the peel and pulp of Zangju and Tuju were higher than that in sour range Daidai and satsuma mandarin. Additionally, Zangju pulp and Tuju peel showed the strongest ferric reducing/antioxidant power (FRAP) activity, whereas, Tuju peel and pulp showed the strongest DPPH and ABTS free radical scavenging activities, respectively. Moreover, both the antioxidant activities of peel and pulp were significantly correlated with the contents of total phenols, total flavonoids or ascorbic acid. These results indicate that the two local citrus varieties have certain nutritional and medicinal value and potential beneficial effects on human health. Our findings will also provide an important theoretical basis for further conservation, development and medicinal utilization of Zangju and Tuju.

Background: Chimeras synthesized artificially by grafting are crucial to the breeding of perennial woody plants. ‘Hongrou Huyou’ (Citrus changshan-huyou + Citrus unshiu) is a new graft chimera originating from the junction where a Citrus changshan-huyou (“C”) scion was top-grafted onto a stock Satsuma mandarin ‘Owari’ (C. unshiu, “O”). The chimera was named OCC because the cell layer constitutions were O for Layer 1(L1) and C for L2 and L3. In this study, profiles of primary metabolites, volatiles and carotenoids derived from different tissues in OCC and the two donors were investigated, with the aim of determining the relationship between the layer donors and metabolites. Results: The comparison of the metabolite profiles showed that the amount and composition of metabolites were different between the peels and the juice sacs, as well as between OCC and each of the two donors. The absence or presence of specific metabolites (such as the carotenoids violaxanthin and β-cryptoxanthin, the volatile hydrocarbon germacrene D, and the primary metabolites citric acid and sorbose) in each tissue was identified in the three phenotypes. According to principal component analysis (PCA), overall, the metabolites in the peel of the chimera were derived from donor C, whereas those in the juice sac of the chimera came from donor O. Conclusion: The profiles of primary metabolites, volatiles and carotenoids derived from the peels and juice sacs of OCC and the two donors were systematically compared. The content and composition of metabolites were different between the tissues and between OCC and the each of the two donors. A clear donor dominant pattern of metabolite inheritance was observed in the different tissues of OCC and was basically consistent with the layer origin; the peel of the chimera was derived from C, and the juice sacs of the chimera came from O. These profiles provide potential chemical markers for genotype differentiation, citrus breeding assessment, and donor selection during artificial chimera synthesis.

Background: Chimeras synthesized artificially by grafting are crucial to breeding of perennial woody plants. 'Hongrou Huyou' ( Citrus changshan-huyou + C . unshiu Marc.) was a new grafting chimera originated from the junction where a scion Citrus changshan-huyou ("C") was top-grafted onto a stock Satsuma mandarin 'Owari' ( C . unshiu Marc., "O"). The chimera was simplified as OCC because the cell layer constitutions were O for L1 and C for L2 & L3, respectively. In this study, profiles of primary metabolites, volatiles and carotenoids derived from different tissues were investigated between OCC and two donors, aiming to figure out the relationship between layer donor and metabolites. Results:Comparison of the metabolite profiles showed that the amount and composition of metabolites were different between the peels and the juice sacs, as well as between OCC and two donors. Metabolites (such as violaxanthin and β -cryptoxanthin in carotenoids, germacrene D in volatiles, citric acid and sorbose in primary metabolites) specifically present or absent in the certain tissue were identified in three phenotypes. According to the principal component analysis (PCA), the total metabolites of chimeric peel properties were derived from the donor C, whereas those of chimeric juice sac properties came from the donor O. Conclusion: Profiles of primary metabolites, volatiles and carotenoids derived from the peels and the juice sacs were systematically compared between OCC and two donors. The content and composition of metabolites differed in tissues as well as between OCC and two donors. Donor dominant metabolite inheritance was dramatic in the different tissues of OCC and basically consistent with the layer origin that the chimeric peels were derived from C and the chimeric juice sacs came from O. These profiles provide potentially chemical markers for genotype differentiation and citrus breeding assessment, more than that, for donor selection during chimera synthesis artificially. BackgroundPlant chimeras refer to those plants composed of more than two genotype cells. According to the theory of 'Tunica-Corpus', the shoot apical meristem (SAM) of dicotyledonous plants are composed of three cell layers, that is L1, L2 and L3, from the outermost layer [1]. For citrus fruits, the juice sacs and epidermal pericarps are derived from cell layer L1; the color and aroma of the fruit rind, seeds 4 and segment walls are developed from L2; L3 produces vascular bundles, and fruit shape was determined by L2 and/or L3 [2]. Up to now, there have been some reports on the discovery and identification of citrus chimeras. Zhou et al. found that the interaction between cells derived from different genotypes caused the mutation of DNA levels in periclinally chimeric fruits NFF (L1-L2-L3=N-F-F) and FNN [2]; Wu et al. found that the fruit characteristics of chimera Ekuliku were inconsistent with source donor and cross-sectional structure of the blade was quite different from two donors [3] Zhang and his colleagues investigated two citrus chimeras named...