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Highlights: [•] More than forty leaf-parent-type cybrids or putative cybrids were produced in citrus. [•] Somatic cybridization aiming to produce male sterile and seedless citrus cybrids was detailed. [•] Agronomic performance and omics-based studies of some selected cybrids were reviewed. [ABSTRACT FROM AUTHOR]

Abstract: More than 150 plants were regenerated from our previous somatic hybridization between embryogenic callus line of Page tangelo (Citrus reticulata Blanco× C. paradisi Macf) and mesophyll protoplasts of rough lemon (C. jambhiri Lush) mediated by electrofusion. Preliminary screening showed that 78% of these plants were tetraploids while the rest were diploids morphologically resembling the leaf parent (rough lemon). Herein, eight plants (six tetraploids and two diploids) were selected and further analyzed by flow cytometry, simple sequence repeat (SSR), mitochondria (mt) RFLP and chloroplast (cp) SSR techniques. The results showed that four of these six tetraploids were somatic hybrids, one tetraploid was autotetraploid of Page tangelo, and the remaining one tetraploid was cybrid with nuclear and cpDNA from rough lemon and mtDNA from Page tangelo; the two diploids were verified being cybrids with nuclear DNA from rough lemon and mtDNA from Page tangelo, cpDNA was randomly inherited. The regeneration mechanism of these novel cybrids was discussed. [Copyright &y& Elsevier]

Abstract: Previous experience with citrus somatic fusion suggests that hybrid cells are generally more vigorous and have higher capacity for embryogenesis, compared to unfused cells from the embryogenic callus parent. However, this observation has never been validated due to the unavailability of an appropriate experimental design. Citrus mesophyll protoplasts never divide and regenerate into plants. Herein, mesophyll protoplasts of transgenic Valencia orange (Citrus sinensis) which expressed the green fluorescent protein (GFP) gene were fused with protoplasts isolated from an embryogenic callus line of Shekwasha mandarin (C. reticulata) to visually screen somatic hybrids. At the multi-cell cluster stage, the regenerated callus lines expressing GFP were morphologically identical to the callus parent regenerate. All hybrid callus lines expressing the GFP developed into embryoids, while most callus parent regenerates still remained in a callus stage. Embryoids with strong or weak GFP expression were verified to be diploid cybrid and tetraploid somatic hybrid embryoids, respectively, by flow cytometry, simple sequence repeat (SSR) and chloroplast SSR analyses. Diploid cybrid globular embryoids appeared light green in color and had a tendency to enter subsequent developmental stages earlier than the dark green tetraploids; however, GFP plants were regenerated from both sources. Thus, expression of the GFP marker gene was effectively used to visually screen somatic hybrids, and confirmed our previous observation that somatic hybrid tissue has a regeneration advantage. The intensity of GFP fluorescence and the color difference of regenerated embryoids was also an early indicator of ploidy level (based on visual GFP observation and confirmed by flow cytometry analysis), and could serve as a tool to study the regeneration mechanism of diploid cybrids via symmetric fusion in citrus and other higher plants. [Copyright &y& Elsevier]

Highlights • Four 2x × 4x crosses were conducted using three diploid seedy cultivars as seed parents. • High germination rate and shoot regeneration was achieved via an optimized embryo rescue approach. • Numerous triploid and tetraploid progenies were obtained as determined by flow cytometry analysis. • Hybrid nature of the selected triploids and two different origins of the selected tetraploids were confirmed. Abstract Seedlessness is a primary breeding objective for citrus fresh fruit market, and triploids have been proven to have great value to produce seedless fruits. In this study, aiming to produce triploid plants for developing some seedless cultivars, four 2x × 4x interploid crosses were conducted using three elite but seedy cultivars as seed parents and one newly flowered doubled diploid and two allotetraploid somatic hybrids as pollen parents. As a result, a total of 1454 developed and 3409 undeveloped seeds from 341 fruits were obtained. Using an optimized embryo rescue approach, 669 developed and 1301 undeveloped seeds germinated in vitro, with an average germination rate as 52.5% for the crosses using 'Nadorcott' tangor and 'Bendizao' tangerine (polyembryonic) as seed parents and 31.0% for the crosses using 'Orah' mandarin (monoembryonic) as seed parent. Then by shoot and root induction, totally 1354 plantlets were regenerated, among which 401 and 54 plants were proved to be triploids and tetraploids by flow cytometry (FCM) analysis and chromosome counting. Hybrid nature of the selected triploid progenies, as well as two different origins (doubled diploid and hybrid origins) for the selected tetraploid progenies was further confirmed by single nucleotide polymorphism (SNP) markers. [ABSTRACT FROM AUTHOR]

Highlights • The expression of CsFUS 3 is positively related with the capacity of citrus callus for somatic embryogenesis (SE). • Overexpression of CsFUS 3 promotes SE and restores normal late embryogenesis in fus 3. • Overexpression of CsFUS 3 has effects on the expression of SE-related transcription factors and hormone pathways, especially the ABA and GA pathways. Abstract In citrus, genetic improvement via biotechnology is challenging due to insufficient understanding of molecular barriers that prevent regeneration by somatic embryogenesis (SE). Our previous study indicated that LEC genes were involved in SE in citrus, but their regulatory roles remain to be elucidated. Here, we cloned one of the LEC genes, CsFUS3 , and show that it is preferentially expressed during SE and in the embryogenic callus (EC) derived from citrus varieties with strong embryogenic competence. The overexpression of CsFUS3 in recalcitrant citrus callus restored embryogenic competence. Complementation of the loss-of-function Arabidopsis fus3 mutant with the CsFUS3 gene restored normal late embryogenesis, which is consistent with the CsFUS3 and AtFUS3 proteins contributing to the same regulatory network in Arabidopsis. Transcriptome profiling revealed that the expression of particular TFs that promote SE was up-regulated in the citrus overexpression (OE) line. The 104 differentially expressed genes associated with hormone biosynthesis, catabolism, and signaling are particularly noteworthy. The dynamic change in the ratio of ABA to GA during SE in wild-type callus mirrored the expression pattern of CsFUS3. In contrast, in the OE line, the ratio of ABA to GA was higher and the capacity for SE was greater when the OE line was separately treated with ABA and GA biosynthesis inhibitors. Taken together, our results demonstrate that the overexpression of CsFUS3 appears to establish a cellular environment favorable to SE, at least in part by promoting a high ABA to GA ratio and by regulating the expression of TFs that promote SE. [ABSTRACT FROM AUTHOR]

• Autotetraploid of citrus junos shows higher salt tolerance than the corresponding diploid. • Transcriptomes of diploid and autotetraploid differ greatly under salt stress. • Plant chitin signaling is the key pathway responding to salt stress in autotetraploid citrus junos. Polyploidization is a widespread phenomenon in flowering plants, and it often enhances abiotic stress tolerance. However, the related molecular mechanism remains largely unknown, especially for perennial woody plants. Here, we found that autotetraploid citrus (Citrus junos Sieb. ex Tanaka) had greater salt tolerance than their diploid progenitors. Transcriptome analysis identified a large number of differentially expressed genes (DEGs) between autotetraploid and diploid roots under salt stress. GO analysis showed that chitin response-related genes were enriched in roots of autotetraploid. Consistently, the autotetraploids exhibited higher chitinases activity than the diploids under salt stress. The top five transcription factor (TF) families with the largest number of TFs were bHLH, ERF, MYB, NAC, and WRKY in autotetraploids and diploids, and 25 TFs were higher expressed in autotetraploids than in diploids under salt stress. Our results revealed that salt stress induced the activation of chitin pathway, thus reducing in ROS accumulation, eventually enhancing salt tolerance of autotetraploid citrus. Our research provides the theoretical basis for citrus breeding aiming at abiotic stress tolerance enhancement. [ABSTRACT FROM AUTHOR]

Homozygous genotypes have great potential in breeding and genomic researches in higher plants. Induction of parthenogenesis via pollination with gamma-irradiated pollens is an efficient method to obtain haploid plants that can overcome the biological limitations of fruit trees. In this study, we investigated the induction of haploid plants in ‘Hirado Buntan’ pummlo ( Citrus grandis ) through in-situ gynogenesis by pollination with irradiated pollens of trifoliate orange (255, 325 and 500 Gy) and ‘Tongshui 72-1 Jincheng’ sweet orange (255, 395 and 500 Gy). The pollen stainability, pollen tube behavior after pollination were evaluated. The seeds extracted from the fruits were rescued by in vitro embryo culture, and the ploidy level of the obtained plantlets was determined by flow cytometry. It showed that two haploid plants and one triploid plant, were obtained from the cross-pollination of ‘Hirado Buntan’ pummelo with irradiated pollens of trifoliate orange; two haploid plants were obtained from the cross-pollination of ‘Hirado Buntan’ pummlo with irradiated pollens of ‘Tongshui 72-1 Jincheng’ sweet orange. The parthenogenic origin of the haploid plants was further confirmed by chromosomal cytogenetic and SSR analysis. The ‘Hirado Buntan’ haploid plants obtained here hold great potential in citrus genetic and genomic studies in the future. [ABSTRACT FROM AUTHOR]

The homeodomain–leucine zipper (HD-Zip) transcription factors, which belong to a class of Homeobox proteins, has been reported to be involved in different biological processes of plants, including growth and development, photomorphogenesis, flowering, fruit ripening and adaptation responses to environmental stresses. In this study, 27 HD-Zip genes ( CsHBs ) were identified in Citrus . Based on the phylogenetic analysis and characteristics of individual gene or protein, the HD-Zip gene family in Citrus can be classified into 4 subfamilies, i.e. HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV containing 16, 2, 4, and 5 members respectively. The digital expression patterns of 27 HD-Zip genes were analyzed in the callus, flower, leaf and fruit of Citrus sinensis . The qRT-PCR and RT-PCR analyses of six selected HD-Zip genes were performed in six citrus cultivars with different embryogenic competence and in the embryo induction stages, which revealed that these genes were differentially expressed and might be involved in citrus somatic embryogenesis (SE). The results exhibited that the expression of CsHB1 was up-regulated in somatic embryo induction process, and its expression was higher in citrus cultivars with high embryogenic capacity than in cultivars recalcitrant to form somatic embryos. Moreover, a microsatellite site of three nucleotide repeats was found in CsHB1 gene among eighteen citrus genotypes, indicating the possible association of CsHB1 gene to the capacity of callus induction. [ABSTRACT FROM AUTHOR]

Abstract: Most of what we know about the molecular genetics of flowering time regulation comes from studies in the model plants. However, little is known about the regulation of flowering transition in perennial species or in species with particular growth habits compared with model plants. Here comparative transcriptome and proteome profiling of spring shoots was performed on an early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) and its wild-type. A total of 19,215 read-mapped genes were observed in two genotypes by RNA-Seq. Of these, 1450 and 1159 genes were specifically observed in the mutant and wild-type libraries, respectively. There were 355 genes that were expressed differently in the two genotypes. A total of 1664 proteins were identified by the iTRAQ technique, and transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. In addition, a global survey of messenger RNA splicing events identified 16,343 splice junctions among 12,688 genes and showed that alternative 3′ splice is the most prevalent form of alternative splicing. We further identify 5698 novel transcribed regions that are not overlapping with annotated citrus transcriptome in two genotypes. Understanding of the regulation of flowering transition in citrus can help in the development of new genetic or management strategies to improve fruit production. [Copyright &y& Elsevier]

Abstract: The present study was to assess informativeness and efficiency of three different molecular markers for genetic diversity among 24 Citrus and its relative species. Sixty one SSR, 33 SRAP and 24 CAPS-SNP markers were used to evaluate the level of polymorphism and discriminating capacity. A total of 596, 656 and 135 polymorphic amplicons were observed in SSR, SRAP and CAPS-SNP markers with average polymorphism information content (PIC) of 0.97, 0.98 and 0.89, respectively. High levels of polymorphism were recorded for SSR and SRAP compared with CAPS-SNP markers. The highest correlations (r =0.930) were obtained between SSR and SRAP markers, whereas SSR and CAPS-SNP were poorly correlated (r =0.833). Cluster analysis was performed to construct dendrograms using UPGMA. And the dendrogram from SSR data was most congruent with the general dendrogram. These findings provide basis for future efficient use of these molecular markers in the genetic analysis of Citrus and its relatives. [Copyright &y& Elsevier]