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miR156, as one of the first discovered and most conserved microRNAs (miRNAs) in plants, was known to play versatile roles in plants, through regulation of the target SBP-box genes, a plant-specific transcription factor family. In this study, four MIR156 and 15 SQUAMOSA promoter binding protein-like (SPL) genes were retrieved from Citrus sinensis genome. According to the phylogenetic grouping with plant SPLs, synteny analysis with Arabidopsis SPLs, characterization of exon–intron structure and motif organization, the CsSPL genes were clustered into two major groups, and one major group was further divided into seven subgroups. The possible function of CsSPLs was inferred based on their likely orthologous relationship to SPLs in Arabidopsis. The precursor of csi-miR156a was isolated and overexpressed in citrus callus. The transformed callus cells were found to over-accumulate amyloids, resulting in increased starch content. Among the nine CsSPLs targeted by csi-miR156a/b, only CsSPL2 and CsSPL14 showed downregulated transcript level in miR156 overexpressed lines, suggesting the involvement of ‘miR156-CsSPL’ module in starch accumulation. In this study, genomic analysis of miR156 and SPL gene family of C. sinensis was conducted, and preliminary function of miR156-SPL was validated in citrus callus lines.

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.

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.

A member of the LEAFY COTYLEDON gene family encoding a HAP3 (heme activated protein 3) subunit of the CCAAT box-binding factor was isolated and termed as Citrus sinensis LEAFY COTYLEDON 1-LIKE (CsL1L). The deduced amino acid sequence shared a high similarity with LEAFY COTYLEDON 1-LIKE (L1L) in Arabidopsis thaliana, Phaseolus coccineus, Theobroma cacao, and Helianthus annuus. Quantitative RT-PCR results indicated that CsLIL was highly expressed in embryogenic callus, somatic embryos and immature seeds, but was rarely detected in non-embryogenic callus, vegetative and floral tissues. Ectopic expression of CsL1L in vegetative tissues could induce embryo-like structures, suggesting that CsL1L has the capability to transit cells from vegetative to embryogenic phase. Comparison of CsL1L expression in the newly formed and long-term subcultured embryogenic calli of W. Murcott tangor (C. sinensis × C. reticulata) and Hongkong kumquat (Fortunella hindsii Swingle) revealed that the potency of embryogenesis was related to the level of CsL1L expression. Sub-cellular localization analysis indicated that CsL1L was a nuclear protein in plant. A microsatellite in CsL1L was verified with polymorphism among the citrus species.

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.

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.

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.

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.

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.

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.

‘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.