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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.

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 holds great potential in citrus improvement by circumventing several biological barriers such as nucellar polyembryony, long juvenility and pollen/ovule sterility. To date more than 300 intergeneric and interspecific citrus somatic hybrids have been produced by standard symmetric fusion; among which, more than forty parental combinations produced leaf-parent-type cybrids or putative cybrids. Herein somatic cybrid production by protoplast fusion aiming to produce citrus cybrids with male sterile cytoplasm being transferred simply via symmetric fusion is detailed including methods to produce cybrids, possible mechanism and factors involved in cybrid production via symmetric fusion, strategy of targeted cybrid production for potential seedlessness, and agronomic performance and omics-based studies of some selected cybrids. This review provides the most comprehensive and updated information concerning citrus cybrid production over the past 20 years.

Pummelo (Citrus grandis Osbeck) is one of the ancestors of sweet orange and economically important cultivated species of the family Rutaceae. The availability of the EST SSR markers for Pummelo represents a promising source to increase the number of markers available for the citrus species. In this study, we evaluated 212 Pummelo EST derived SSR markers (CgEMS) for their transferability across the genera, polymorphism, mapping ability and utility for genetic diversity analysis. Among these markers, 136 were amplified bands, 99 were transferable across the genera. Transferability of CgEMS to C. sinensis, C. reticulata, C. lemon, Fortunella sp and Poncirus sp was 76%, 76%, 75%, 74% and 73%, respectively. In contrast 52 (53%) markers were found to be polymorphic and segregating in a mapping population. Segregating markers can be categorized into four groups: full informative (8%), male informative (15%), female informative (19%) and partly informative (59%). The phylogenetic relationship between the citrus and its relatives obtained with CgEMS was in a good agreement with the established citrus taxonomy and phylogeny. CgEMS could potentially serve as perfect markers for determining variation in phenotype, fingerprinting, mapping and genetic diversity study in C. grandis. Their high level of cross genera transferability of the CgEMS markers has allowed phylogenetic inference within the Rutaceae.

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.

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 precocious trifoliate orange (Poncirus trifoliata [L.] Raf.), an early flowering mutant of P. trifoliata, has a short juvenile phase of about 14 months, significantly shorter than other citrus. In this report, using the stems of precocious trifoliate orange seedlings as explants, an improved protocol mediated by Agrobacterium tumefaciens was developed to evaluate regeneration and transformation frequency. The transformed plants continued to reveal the precocious trait of non-transgenic mature precocious trifoliate orange, and as such could prove to be an efficient system for the verification of gene function in the fruits/flower via genetic transformation in woody plants.

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.