Polyploidy is a widespread phenomenon in nature and is thought to play a major role in the evolution of flowering plants. Additionally, polyploidization produces novel phenotypes that through plant breeding have enhanced the production of biomass and improved the stress tolerance of major economic crops. However, the effect of polyploidization on plant metabolism is still unclear.In order to test whether there are common metabolic responses following genome doubling, we performed a comparative metabolomic analysis of mature leaves from doubled diploids and the corresponding diploids of red tangerine (Citrus reticulata Blanco), trifoliate orange (Poncirus trifoliata L. Raf.) and precocious trifoliate orange (P. trifoliata).Non-targeted and targeted metabolic profiling of mature leaves from three doubled diploids and their diploid controls were performed by using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS).About 11–34% of the detected metabolic features differentially accumulated in the doubled diploids, mostly by less than fivefold. The levels of primary metabolites tended to increase in the doubled diploids. Concentrations of tricarboxylic acid cycle intermediates—citric acid, malic acid, fumaric acid and succinic acid, enhanced in all of the doubled diploids. The levels of secondary metabolites, including phenylpropanoids and terpenoids, tended to decrease in the doubled diploids. This is consistent with the lower C/N ratios in the doubled diploids.Polyploidization had a significant but relatively limited influence on the accumulation of metabolites in these citrus species. We conclude that primary metabolism takes priority over secondary metabolism in doubled diploid plants to relieve the “genomic stress” encountered during the early stages of genome doubling, probably to promote vitality and growth.

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate, symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyll-parent-type cybrids. It suggested that mitochondrial DNA (mtDNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis (L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein (GFP) gene, in an effort to elucidate whether mtDNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids’ with mtDNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mtDNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

Homozygous genotypes are valuable for breeding and genomic studies in higher plants. The production of haploids and DHs through gametic embryogenesis allows a single-step development of complete homozygous lines from heterozygous parents, requiring much less time than the conventional selfing method. Here, we report the regeneration of haploid and double haploid lines of citrus species through anther culture. The anthers of seven citrus cultivars at the uninucleate stage were cultured and induced using four previously reported mediums. Ten haploid lines (2n = x = 9), six DH lines (2n = 2x = 18), two tetraploid lines (2n = 4x = 36) of ‘Early Gold’ sweet orange, and one haploid line of ‘Rohde Red’ Valencia sweet orange were obtained, as identified by ploidy, karyotype and simple sequence repeats (SSRs) analysis. All of them were confirmed to be fully homozygous by SSR analysis using 31 primer pairs that are distributed evenly on each of the chromosomes. Among them, plants regenerated from two DH lines of ‘Early Gold’ sweet orange grew vigorously in the greenhouse. To our knowledge, this is the first report on sweet orange anther culture with successful DH plant regeneration. The haploid, DH and tetraploid lines reported here hold great potential for future citrus genome resequencing in genetic studies and seedless breeding via somatic fusion.

Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1. Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation. Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP) with the aim to clarify their potential roles in anther development and male sterility. On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed differentially expressed profiles in one or more stages. Proteins up- or down-regulated in G1+HBP were mainly involved in carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase), nucleotide binding (RNA-binding proteins), protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits). Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.

Seedlessness, an important economic trait for fresh fruit, is among the prior goal for all citrus breeding programs. Symmetric somatic hybridization provides a new strategy for citrus seedless breeding by creating cybrids transferring mitochondrial DNA (mtDNA) controlled cytoplasmic male sterility (CMS) from the callus parent Satsuma mandarin (C. unshiu Marc.) to seedy cultivars. In this study, protoplast fusion was adopted to transfer CMS from C.unshiu Marc. cv. Guoqing No. 1 (G1) to three seedy sweet oranges (C. sinensis L. Osb.), i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’. Flow cytometry analysis showed that 12 of 13 regenerated plants from G1 + ‘Early gold’, 9 of 12 from G1 + ‘Taoye’ and both two plants from G1 + ‘Hongjiang’ were diploids, while the remaining regenerated plants were tetraploids. Molecular analysis using 23 simple sequence repeat (SSR) markers previously proven to map to the citrus genome showed that the nuclear DNA from all recovered diploid and tetraploid plants derived from their corresponding leaf parent, while cleaved amplified polymorphic sequence analysis showed that the mtDNA of all regenerated plants derived from the callus parent, indicating that the regenerated 2X and 4X plants from all these three combinations are authentic cybrids. Furthermore, the Chloroplast SSR analysis revealed that somatic cybrid plants from the three combinations possessed either of their parental chloroplast type in most cases. These results demonstrated that mtDNA of G1 Satsuma mandarin was successfully introduced into the three seedy sweet orange cultivars for potential seedlessness via symmetric fusion.

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.

Male sterility, which could result from abnormal anther development, is a main contributor to seedless fruit production in higher plants, including citrus. MicroRNAs (miRNAs) play important roles in plant development by regulating various biological processes. In this study, by high-throughput sequencing of small RNA libraries constructed from two Ponkan mandarin (Citrus reticulata Blanco) lines, i.e., male fertile and seedy “Egan No. 1” and its male sterile and seedless mutant “Qianyang seedless,” a total of 156 known miRNAs from 32 families and 24 novel miRNAs were identified. Analyses of the expression profiles in sequencing data showed that 71 known miRNAs and 11 novel miRNAs were differentially expressed between the seedy and seedless types. By degradome sequencing, a total of 138 targets were identified for 44 miRNA families, among which 5 targets were experimentally validated by RLM-5′RACE. Consistent with the results in previous studies, target annotation demonstrated that most targets of the known miRNAs were transcription factors. Forty-one targets were also detected to be cleaved by 16 novel miRNAs. Expression profile analyses by qRT-PCR revealed a negative regulation between eight known miRNA and their targets during anther development. Our research provides new information about the miRNA regulatory network during anther development and further advances the study about the mechanism of citrus male sterility.

2nmegagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement.Tetraploid plants are frequently observed in the seedlings of diploid polyembryonic citrus genotypes. However, the mechanisms underlying the formation of tetraploids are still indistinct when apomictic citrus genotypes are used as female parent to cross with tetraploids. Herein, 54 tetraploid progenies, which were unexpectedly obtained previously from four 2x × 4x crosses using polyembryonic ‘Nadorcott’ tangor as seed parent, were analyzed by 22 simple sequence repeat (SSR) markers, aiming to reveal their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent; while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confirmed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2n megagametophytes. Based on the genotypes of 2n megagametophytes, the analysis of maternal heterozygosity restitution (HR) for each marker showed that it varied from 0.00 to 87.80 % with a mean value of 40.89 %. In addition, it was observed that 13 markers displayed a lower rate than 50 %. On the basis of the above results, it can be speculated that the second division restitution (SDR) is the mechanism underlying the 2n megagametophyte formation in ‘Nadorcott’ tangor. The elucidation of the mechanism of 2n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus.

An efficient Agrobacterium rhizogenes mediated transformation system of Poncirus trifoliata was developed. The combination of A. rhizogenes MSU440 with epicotyl segments was confirmed to be a suitable system for genetic transformation in P. trifoliata. About 3 weeks after inoculation with bacteria, hairy roots emerged at the cut surfaces of epicotyl segments on Murashige and Tucker selective medium free of plant growth regulators. The presence of rol B, β-glucuronidase (GUS) and yellow fluorescent protein (YFP) genes in co-transformed hairy roots was confirmed by fluorescence observation, GUS staining and polymerase chain reaction (PCR) analysis. The co-transformation rate based on GUS staining was as high as 68.3 %. A regeneration procedure of transgenic plantlets from hairy roots was also established. Out of 45 hairy root segments from different co-transformed lines, 23 were successfully regenerated into rooted plantlets. The presence and expression of YFP gene in the regenerated co-transformed plants were further confirmed by Southern blot hybridization and quantitative reverse transcription PCR. The DR5∷GUS–YFP reporter system was used to study the auxin response maxima at the apices of primary hairy roots and their lateral roots. Auxin distribution and response maxima during lateral root primordium development were also studied. The hairy roots obtained by A. rhizogenes mediated transformation were confirmed to be a good system for investigating root development in P. trifoliata. Furthermore, the genetic transformation and plant regeneration system via A. rhizogenes provides an alternative method for genetic engineering in Citrus.

Accuracy of the quantitative real-time reverse transcription-PCR (qRT-PCR) depends on the stability of the reference gene(s), i.e. housekeeping gene(s) used for data normalization. Recent studies have shown that the expression of common reference genes can vary considerably in certain experimental conditions. However, reference genes of qRT-PCR in fruit trees have not been well identified. In this study, stability of expression of ten potential reference genes in citrus, including CitACT7, CiteIF-1A, CiteIF4α, CitHistone H3, CitHistone H4, CitTUA3, CitTUB8, CitUBL5, CitUBQ1 and CitUBQ14 was assessed. Furthermore, this was validated by qRT-PCR in diverse sets of biological samples, including embryonic callus at seven stages, embryos maintained at three different temperatures, five distinct plant organs, four floral tissues and four stages of flower development. Three distinct statistical algorithms, geNorm, NormFinder and Bestkeeper, were used to evaluate the expression stability of the candidate reference genes. The three outputs were merged by means of a non-weighted unsupervised rank aggregation method. GeNorm was also used to determine both the optimal number and the best combination of reference genes for each experimental set. The expression of CitUBQ1 was the most stable one across the set of all samples, flower developmental stages and somatic embryogenesis process under two conditions i.e. different temperature treatment and normal temperature treatment. CitUBQ14 presented more stable expression in different plant organs and floral tissues. CitHistone H3 was the most unsuitable reference gene in many citrus sample sets. In addition, the relative gene expression profile of citrus receptor-like kinase gene CitSERK1-like was conducted to confirm the validity of the reference genes in this study. Taken together, this study identified the reference genes that are most suitable for normalizing the gene expression data in citrus. These results provide guidelines for the selection of reference gene(s) under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of samples in citrus.

Somatic embryogenesis (SE) is a most promising technology that is used for in vitro germplasm conservation and genetic improvement via biotechnological approaches in citrus. Herein, three suppression subtractive hybridization (SSH) libraries were constructed using calluses of Citrus sinensis cv. ‘Valencia’ to explore the molecular mechanisms that underlie the SE in citrus. A total of 880 unisequences were identified by microarray screening based on these three SSH libraries. Gene ontology analysis of the differentially expressed genes indicated that nucleolus associated regulation and biogenesis processes, hormone signal transduction, and stress factors might be involved in SE. Transcription factors might also play an important role. LEC1/B3 domain regulatory network genes (LEC1, L1L, FUS3, ABI3, and ABI5) were isolated in citrus SE. Some new transcription factors associated with citrus SE, like a B3 domain containing gene and HB4, were identified. To understand the influence of these isolated genes on SE competence, their expression profiles were compared among callus lines of seven citrus cultivars with different SE competence. The expression dynamics suggested that these genes could be necessary for the SE initiation and might play a role in embryogenic competence maintenance in different cultivars. On the basis of gene expression profiles, an overview of major physiological and biosynthesis processes at different developmental stages during citrus SE is presented. For the first time, these data provide a global resource for transcriptional events important for SE in citrus, and the specific genes offer new information for further investigation on citrus SE maintenance and development.

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants, so transgene copy number analysis is identified as one most important task after obtaining transgenic plants. In this paper, TaqMan real-time PCR was used to estimate the copy number of exogenous MAC12.2 and NPTII genes in transgenic precocious trifoliate orange (Poncirus trifoliata [L.] Raf) in order to overcome the limitations of Southern blot analysis, which is labor-intensive, time-consuming, in considerable needs of DNA, etc. We developed a real-time PCR assay which permitted the determination of the copy number of transgene (MAC12.2 and NPTII), relative to a conserved endogenous gene (PtLTP) in transgenic lines. R value is 0.92 by comparing the results to that of Southern blot analysis, indicating a strong correlation coefficient between TaqMan real-time PCR assay and Southern blot method.

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants.Key message Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified.

Somatic embryogenesis (SE) is a remarkable process of plant somatic cells developing into an embryo capable of forming a complete plant. MiRNAs play important roles in plant development by regulating expression of their target genes, but its function in SE has rarely been studied. Herein, ten conserved miRNAs with critical functions in plant development are detected by stem-loop qRT-PCR in the SE system of Valencia sweet orange. Sixteen unigenes from citrus are predicted to be targeted by six of the miRNAs. Cleavage sites on 15 of these target mRNAs are mapped by 5′RACE, of which ten are reported in this study. Transcript abundances of the 16 target unigenes are detected by qRT-PCR during SE process. Stage and tissue-specific expressions of miRNAs and their targets suggest their possible modulation on SE of citrus callus: miR156, 168 and 171 exert regulatory function during somatic embryo induction process; miR159, 164, 390 and 397 are related to globular-shaped embryo formation; miR166, 167 and 398 are required for cotyledon-shaped embryo morphogenesis; in addition, target genes of miR164, 166 and 397 are associated with SE disability of nonembryogenic callus. Exploration of miRNA-mediated modulation on SE is expected to provide new insights into plant cell totipotency, as well as how to maintain and enhance the embryogenic capacity of somatic cells.

To explore and isolate genes related to flowering and fruit development, we constructed a cDNA library from floral organs and fruitlets of Ponkan mandarin (Citrus reticulata Blanco). A total of 661 high-quality expressed sequence tags (ESTs) were generated and submitted to GenBank with the accession numbers from GO343532 to GO344192. All these ESTs were assembled into 43 contigs and 296 singletons (totally 339 unigenes). The BLAST2GO software was employed to annotate the unigenes, among which 77 ones had no significant homology with the sequences in NCBI non-redundant proteins database by BLASTX analysis. Additionally, gene ontology (GO) analysis revealed an overview of sequences distribution, which implied some specially expressed genes related to flower and fruit development. Furthermore, some abundantly expressed unigenes involved in several crucial metabolic pathways related to fruit quality were highlighted and three types of homologues of miraculin-like protein2 were analyzed by both semiquantitative RT-PCR and real-time PCR. The results showed different expression profiles of these genes, which meant that they contribute distinctly to fruit development.

MicroRNAs (miRNAs) are a group of single-stranded noncoding RNAs with general size of 21–24 nucleotides, which negatively regulate gene expression posttranscriptionally by repressing gene translation or degrading targeted mRNAs. Despite the important functions of miRNAs in plants, little is known about miRNAs in citrus. Here we present a study of bioinformatics identification of microRNA precursors in citrus by comparing known plant miRNAs against 560,271 citrus ESTs. Seventy-eight ESTs from ten citrus species and three hybrids were identified as putative miRNA precursors, encoding 51 unique miRNA sequences, representing 28 miRNA families. Blastn search of the putative miRNAs against citrus unigenes and ESTs was performed to identify the target genes. As a result, 36 unigenes and 77 ESTs were identified as putative targets of 25 citrus miRNA families. The putative targets are mainly transcription factors and play important roles in development, metabolism, and stress resistance. To validate the predicted miRNAs, qRT-PCR was applied to detect the tissue-specific expression of nine putative miRNAs in Citrus sinensis cv. Valencia. The cleavage sites on five mRNAs targeted by three miRNA families were mapped by 5′RACE. This study provided information on citrus miRNA precursors, mature miRNAs, and miRNA targets and is helpful for future research of miRNA function in citrus.

In transgenic plants, multiple T-DNA copies and cytosine methylation usually correlate with a loss of transgene expression while methylation of promoter is associated with transgene silencing. Here, six independent GFP transgenic ‘Bingtang’ sweet orange (Citrus sinensis (L.) Osb.) plants were analyzed with copy number, T-DNA repeat, cytosine methylation and transgene expression. The fluorescence of transgenic plants was normal and bright except that the transgenic B4 plant showed mottled fluorescence. Five of the six transgenic plants had multiple transgene copies and three transgenic plants had direct repeat sequences. The degrees of cytosine methylation within GFP and CaMV 35S promoter region were higher in multiple copies than in single copy transgenic plant B6. Cytosine methylation of nptII in multiple copies transgenic plants except B4 was slightly greater than single copy transgenic plant B6. Furthermore, transgenic B4 plant with mottled GFP expression had the highest degree of methylation of CG cytosine pattern. The transcript level of GFP and nptII was lower in three transgenic plants (B1, B2 and B3) with direct repeat, as revealed by real-time RT–PCR analysis. These results suggest that multiple T-DNA copies and methylation of CaMV 35S promoter affect transgene expression levels but do not cause gene silencing in transgenic sweet orange.

The green fluorescent protein (GFP) has become an ideal visual marker to monitor and quantify the expression of the transgene. It can be targeted to specific subcellular locations, including the endoplasmic reticulum, mitochondria, actin cytoskeleton and nuclei through the addition of signal peptides. Our previous work has resulted in transgenic citrus plants expressing cytoplasmic targeted GFP (Cy-GFP) or endoplasmic reticulum targeted GFP (Er-GFP) gene. To evaluate the localization of three different subcellular targeted GFP, i.e., Cy-GFP, Er-GFP and mitochondria targeted GFP (Mt-GFP) in citrus tissues and to utilize cell lines containing Mt-GFP for basic research in cell fusion, the plasmid pBI-mgfp4-coxIV encoding the Mt-GFP gene was successfully transferred into embryogenic callus of Valencia sweet orange (Citrus sinensis (L.) Osbeck) via Agrobacterium tumefaciens-mediated transformation. Furthermore, we compared the specific expression of these three different subcellular localized GFP constructs in cells of different mature leaf tissues (upper epidermis, palisade parenchyma, spongy parenchyma and lower epidermis) by a confocal laser scanning microscope (CLSM). Cytoplasmic-localized GFP expression was observed throughout the cytoplasm but appeared to accumulate within the nucleoplasm. The Er-GFP occurred within a layer very close to the cell wall. In addition, a stable fluorescence on the ER network throughout the guard cells was detected. Interestingly, the Mt-GFP specifically expressed in the guard cells to particles of about 1–2 μm within the cytoplasm in this case. To verify that the fluorescent particles observable in the guard cells are indeed mitochondria, we co-localize the Mt-GFP fusion protein with a mitochondrial-specific dye in citrus protoplasts. These results demonstrate that the subcellular distribution of the three subcellular targeted GFP is very distinct in citrus leaf cells and the cell lines containing Mt-GFP gene can be further used in citrus basic cell fusion research.

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.

In order to predict the performance of growth, development and resistance of a citrus diploid cybrid plant between Citrus unshiu Marc. cv. Guoqing No. 1 (G1) and Hirado Buntan pummelo (Citrus grandis (L.) Osbeck) (HBP), the proteomes of leaves from this cybrid and its parents were investigated. First, the diploid cybrid plant (G1 + HBP) created with its nuclear and chloroplast genomes from HBP and mitochondria genome from G1 was further verified by ploidy and molecular analysis (SSR, CAPS and cp-SSR). Then, 2-DE combined with MALDI-TOF/TOF MS were employed to analyze the variation of proteomes in the cybrid and its parental plants. Significant analysis allowed 90 (P < 0.05) differentially expressed protein spots between G1 and the cybrid, 20 between HBP and cybrid, and 116 between G1 and HBP. The comparative proteome patterns well validated the genetic background of the cybrid and its parental plants, suggesting that there is a good correlation between genome constitution and proteome expression in this cybrid. Seventy differentially expressed spots were selected for MALDI-TOF/TOF analysis and 25 proteins were identified. The identified proteins were mainly involved in photosynthesis, stress response, anti-oxidative stress and metabolism. Five proteins involved in photosynthesis such as Rubisco and Rubisco activase were significantly up-regulated in the cybrid, indicating the photosynthesis is enhanced in the cybrid. Expression analysis of proteins involved in stress response and anti-oxidative stress suggested that the resistance might be improved while anti-oxidative system exhibited a complex effect in the cybrid. The nitrogen, sulfur, carbon and energy metabolism might also be affected in the cybrid plant.

Somatic hybridization by protoplast fusion from cell suspension cultures and leaf parent has been a well-established technique holding great potential for citrus variety improvement. In this study, somatic hybrid plants were regenerated from the following two fusion combinations: ‘Murcott’ tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) and ‘Bingtang’ orange (C. sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge). Somatic hybrids were selected at an early stage based on their higher capacity for embryogenesis comparing to non-hybrid cells. Flow cytometry analysis showed that all plants from pre-selected lines of the two combinations were tetraploid. SSR analysis confirmed their hybrid nature, with nuclear DNA from both fusion parents, and absence of parental specific bands was also detected. Cytoplasmic compositions of the recovered plants were further revealed by CAPS and cpSSR analysis. The allotetraploid somatic hybrids from the ‘Murcott’ tangor + HBP combination will be applied to develop triploid seedless cultivars by interploid crossing with diploid seedy citrus cultivars, and those from ‘Bingtang’ orange + Calamondin could be valuable for Asiatic citrus canker-tolerant and ornamental citrus breeding.

Somatic embryogenesis receptor-like kinase (SERK) belonging to the receptor-like kinases (RLKs) has been shown to be implicated in somatic embryogenesis (SE). In this study, a somatic embryogenesis receptor-like gene CitSERK1-like was cloned and characterized from Citrus sinensis cv. ‘Valencia’, a genotype with high somatic embryogenesis capacity for over 26 years. Fifteen consecutive amino acids in putative leucine zipper domain of CitSERK1-like gene were different from the reported CitSERK1 gene. Homology search and sequence analysis demonstrated that the deduced CitSERK1-like protein shared a high degree of identity with SERKs from other species in sequence and structure. Real-time PCR analysis revealed that the transcript of CitSERK1-like was enhanced during the induction of SE. At subsequent embryo-transition phase, a moderate level of expression was detected in heart-torpedo and cotyledon embryos, while low expression was detected in globular embryo. Among the different tissues, the expression of CitSERK1-like was highest in young leaves. Further analysis of its spatial expression by in situ hybridization revealed that CitSERK1-like was mainly located in the embryogenic callus and vascular cells of different embryos or tissues. The results of temporal and spatial expression of CitSERK1-like showed that it played critical roles throughout the process of SE and had a broader role in plant development. In addition, CitSERK1-like expression was up-regulated by 2, 4-D and NAA at the early stage, but down-regulated afterward. Taken together, it suggested that CitSERK1-like activated complex developmental pathways associated with somatic embryogenesis and plant growth.

‘Meiwa’ kumquat (Fortunella crassifolia Swingle.) is famous for its relatively short juvenility, delicious flavor, human health benefits and high resistance to citrus canker. To establish kumquat transformation system and to further shorten its juvenility, Agrobacterium-mediated epicotyledon segment transformation of APETALA1 (AP1 from Arabidopsis) gene was conducted. Transformation efficiency ranged from 1.00 to 4.08% depending on seedling age, and 20 day age seedlings proved to be the best explants for transformation. Five stable transgenic plants were obtained as revealed by GUS assay, and further confirmed by specific PCR and Southern blot analyses. After transfer to the greenhouse, one transgenic line (J3) flowered at the 11th month and continued to flower in the next years, till the third year when all non-transformed and transgenic plants but J66 flowered. Gene expression analysis of AP1 and four endogenous flowering genes CiAP1, CiFT, CiLFY, and CiTFL1 by real-time RT-PCR suggested that CiFT and CiTFL1 played important roles in the regulation of flowering in transgenic AP1 kumquat. Factors influencing kumquat transformation efficiency and the relationship between flowering time in transgenic AP1 kumquat and expression levels of endogenous FT and TFL1 genes were discussed.

Precocious trifoliate orange (Poncirus trifoliata [L.] Raf), an extremely early flowering mutant of P. trifoliata, is an attractive model for functional genomics research in Citrus. A procedure for efficient regeneration and transformation of this genotype was developed by using green fluorescent protein (GFP) gene as visual marker and etiolated stem segments as explants. In vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic shoots and escapes. Transformation efficiency was 20.7% and the transformants were identified by polymerase chain reaction (PCR) and Southern blot analysis. Moreover, the transgenic lines expressed variable amounts of the GFP gene as revealed by real-time PCR analysis. Fifteen transgenic plants flowered 18 months after transfer to the greenhouse and six of them set fruits. GFP expression was also observed in the transgenic flowers and fruits. To test the utility of this system for functional genomics studies, an Arabidopsis thaliana MAC12.2 gene with the potential to produce seedless fruits was introduced into this genotype, and the traits of the transgenic fruits were characterized. The successful transformation of this perennial woody genotype with extremely short juvenility will allow us to test the function of cloned genes in citrus, the improvement of which is hindered by a long juvenility period.

Transformation and high efficient regeneration of transgenic plants from the trimmed etiolated shoot/root region (TESRR) of Anliucheng sweet orange [Citrus sinensis (L.) Osb.] seedling was reported. A visual green fluorescent protein (GFP) marker gene was introduced to evaluate transformation efficiency by using the explants from TESRR and epicotyls. The transformation protocol was: infection 20 min, co-culture 3 d, selection culture 30 d, and rooting 15 d. Out of a total of 288 sprouted shoots obtained from TESRR, 34 shoots (11.8 %) yielded GFP expression. In contrast, only 2 (3.0 %) of the 67 sprouted shoots from epicotyl transformation yielded GFP expression. In all plants showing the green fluorescence an expected 500 bp GFP fragment was proved by PCR analysis. Southern blot analysis further confirmed the integration of GFP gene into citrus genome. Transgenic plantlets were obtained within 80 d using the TESRR, compared within 150 d by using epicotyls.

Transformation and high efficient regeneration of transgenic plants from embryogenic calluses of Bingtang sweet orange [Citrus sinensis (L.) Osbeck] was reported. Embryogenic calluses were inoculated with Agrobacterium tumefaciens strain EHA105, harboring the binary Ti plasmid pROK II and carrying a neomycin phosphotransferase II (NPTII) gene, an intron β-glucuronidase (GUS) gene and the Arabidopsis APETALA1 (AP1) gene. Transformation treatment was with inoculation time of 30 min, co-culture of 3 d at 23 °C and supplementation of the co-culture medium with 2 mg dm−3 acetosyringone (AS). Kanamycin (50 mg dm−3) was effective to inhibit the growth of non-transformed calluses while it did not affect the transformed ones. The total number of transformed callus lines was 7 with 100 % embryo induction. High efficient regeneration of the transgenic embryos (88 % with 4–5 shoots per embryoid) was realized within 3 months. Integration of the transgene into the citrus genome was confirmed by histochemical GUS staining, polymerase chain reaction (PCR) analysis with AP1-specific primer and Southern blot hybridization with a 712 bp PCR fragment of AP1 as the probe.

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR (pattern recognition receptor) gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene (HPT) was introduced into Anliucheng sweet orange (Citrus sinensis Osbeck) via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin (HYG) and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identified by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was briefly discussed.

•Four haploid plants of ‘Hirado Buntan’ pummelo generated by pollination with gamma irradiated pollen.•The homozygosity and origin of the obtained haploid plants were validated by molecular characterization and cytogenetic analysis.•The pollen tube behaviors were evaluated to explored the mechanism of parthenogenesis induction by gamma irradiated pollen.•The haploid plants obtained in this study would play an important role in citrus genetic and breeding studies.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.

Somatic hybridization provides an alternative for transferring mitochondria-encoded cytoplasmic male sterility (CMS). Herein, symmetric protoplast electrofusion was conducted between embryogenic callus protoplasts of Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a CMS cultivar, and mesophyll-derived protoplasts of seedy ‘Page’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)] or ‘Nova’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)], to transfer CMS trait. Flow cytometry analysis showed that 14 plants recovered from G1 + ‘Page’ tangelo that displayed typical morphological character of ‘Page’ were diploid, and 6 plants regenerated from G1 + ‘Nova’ tangelo were tetraploid. Genetic compositions of regenerated plants from the two fusions were determined by SSR, CAPS and chloroplast-SSR analysis. Cybrid nature of diploids from G1 + ‘Page’ tangelo with nuclear DNA from ‘Page’, mitochondrial DNA (mtDNA) from the G1 and chloroplast DNA (cpDNA) derived from either parent was confirmed. Tetraploid plants from G1 + ‘Nova’ tangelo were identified as somatic hybrids with random cpDNA inheritance. The regenerated cybrid and hybrid plants hold great potential for Citrus seedless breeding at diploid or triploid levels.

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.Graphical abstractAn unweighted pair-group method with arithmetic averages (UPGMA) dendrogram of genetic relationships among 24 genotypes of Citrus and its relative species based on the Dice similarity coefficients obtained using SSR, SRAP and CAPS-SNP combined data.Download full-size imageResearch highlights► A comparative investigation on the performance of SSR, SRAP and CAPS-SNP in the genus Citrus was conducted. ► PIC values were high in both SSR and SRAP compared with that in CAPS-SNP. ► The dendrogram from SSR data was more congruent with the general dendrogram. ► Combining different marker systems was better for diversity study of Citrus and its relatives.