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  • Author or Editor: Frederick G. Gmitter Jr x
  • Journal of the American Society for Horticultural Science x
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A method was developed to produce nonchimeric, autotetraploid Citrus plants via in vitro somatic embryogenesis in the presence of colchicine. Undeveloped ovules from immature fruit of `Valencia' sweet orange (Citrus sinensis [L.] Osb.) and `Orlando' and `Minneola' tangelos (Citrus reticulata Blanco × Citrus × paradisi Macf.) were held on Murashige and Tucker medium with 500 mg malt extract/liter and 0.0090, 0.01%, or 0.10% colchicine for 21 days. Embryogenesis from tangelo ovules was suppressed by 0.10% colchicine, but no such effect was observed among sweet orange ovules. Colchicine treatments had no subsequent effect on embryo germination. The numbers of chromosomes in root tip cells showed that both tetraploid and diploid `Valencia' and `Orlando' plants were recovered from colchicine treatments. `Minneola' cultures produced only diploid plants. Tetraploid plant morphology was typical for Citrus tetraploids. Examination of chromosome numbers in root tip, shoot, and leaf meristems indicated that the regenerants were nonchimeric. Such nonchimeric tetraploids will be useful parents for interploid hybridization directed toward development of seedless triploid Citrus scion cultivars.

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The inheritance of resistance to a host-specific isolate (Shinn) of Alternaria alternata (Fr.:Fr.) Keissl. from `Minneola' tangelo (a cross between Citrus paradisi Macf. `Duncan' and C. reticulata Blanco `Dancy') was shown to be controlled by a single recessive allele, aaM1, within the citrus genome. A backcross between resistant `Clementine' mandarin (C. reticulata) and susceptible LB#8-10 (a hybrid of `Clementine' mandarin and `Minneola' tangelo) resulted in 61 resistant (R) and 58 susceptible (S) plants (χ2 = 0.0756, P ≥ 0.05), but the reciprocal cross deviated from the expected 1R:1S ratio (87 R and 36 S plants (χ2 = 21.1463, P ≥ 0.05). A dominant allele, AaM1, of this resistance gene was found in a loose coupling phase linkage with two RAPD markers, P12850 (15.3 cM) and AL31250 (36.7 cM), after JOINMAP computer analysis.

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Protoplasm culture following polyethylene glycol (PEG) -induced fusion resulted in the regeneration of somatic hybrid plants from the following six parental combinations: Citrus sinermis (L.) Osbeck cv. Hamlin + Severinia buxifolia (Poir.) Tenore (Chinese box-orange); C. reticulate Blanco cv. Cleopatra + Poncirus trifoliata (L.) Raf. cv. Flying Dragon; C. reticulate cv . Cleopatra + Swingle citrumelo (C. paradisi Macf. × P. trifoliata); C. sinensis cv . Hamlin + C. jambhiri cv . Rough lemon; C. sinensis cv . Valencia + C. jambhiri cv . Rough lemon; and C. paradisi cv . Thompson + `Murcott' tangor (purported hybrid of C. reticulate × C. sinensis). Diploid plants were regenerated from nonfused embryogenic culture-derived protoplasts of `Cleopatra' mandarin and `Hamlin' and `Valencia' sweet orange, and from nonfused leaf-derived protoplasts of Rough lemon and `Mnrcott'. Regenerated plants were classified according to leaf morphology, chromosome number, and isozyme analyses. All of the somatic hybrids reported herein are tetraploid (2n = 4x = 36), with the exception of the `Hamlin' + S. buxifolia hybrid, which was unexpectedly found to have a chromosome number of 2n = 27. These six new somatic hybrids have potential in citrus scion and rootstock improvement for commercial use.

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Citrus fruit with sector chimeras were collected in commercial packinghouses and from the field. Chimeric fruit from eight cultivars of sweet oranges [Citrus sinensis (L.) Osbeck], grapefruit (C. paradisi Macf.), tangelo (C. paradisi × C. reticulate Blanco), and tangors (C. reticulate × c. sinensis) were found at a frequency of 0.009% to 0.271%. Tetraploid plants obtained from one type of sector mutant (termed gigas) and albino plants obtained from another type of sector mutant confirmed that some genetic mutations observed in fruit rind can be recovered in nucellar seedlings. The gigas chimeras were identified as a source of citrus tetraploids. Several types of potentially useful sector mutants with altered rind color were observed, and plants were produced from some mutant sectors by developed seed or culture of aborted ovules. HPLC analysis of rind tissues from sectors of one chimeric fruit revealed substantial quantitative and qualitative differences in pigment composition. Propagation of plants from mutant sectors may yield cultivars with improved fruit color, altered maturation date, and reduced disease or mite susceptibility and may eventually lead to breeding of seedless triploid hybrids.

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Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.

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Citrus (Citrus sp.) germplasm collections are a valuable resource for citrus genetic breeding studies, and further utilization of the resource requires knowledge of their genotypic and phylogenetic relationships. Diverse citrus accessions, including citron (Citrus medica), mandarin (Citrus reticulata), pummelo (Citrus maxima), papeda (Papeda sp.), trifoliate orange (Poncirus trifoliata), kumquat (Fortunella sp.), and related species, have been housed at the Florida Citrus Arboretum, Winter Haven, FL, but the accessions in the collection have not been genotyped. In this study, a collection of 80 citrus accessions were genotyped using 1536 sweet orange–derived single nucleotide polymorphism (SNP) markers, to determine their SNP fingerprints and to assess genetic diversity, population structure, and phylogenetic relationships, and thereby to test the efficiency of using the single genotype-derived SNP chip with relatively low cost for these analyses. Phylogenetic relationships among the 80 accessions were determined by multivariate analysis. A model-based clustering program detected five basic groups and revealed that C. maxima introgressions varied among mandarin cultivars and segregated in mandarin F1 progeny. In addition, reciprocal differences in C. maxima contributions were observed among citranges (Citrus sinensis × P. trifoliata vs. P. trifoliata × C. sinensis) and may be caused by the influence of cytoplasmic DNA and its effect on selection of cultivars. Inferred admixture structures of many secondary citrus species and important cultivars were confirmed or revealed, including ‘Bergamot’ sour orange (Citrus aurantium), ‘Kinkoji’ (C. reticulata × Citrus paradisi), ‘Hyuganatsu’ orange (Citrus tamurana), and palestine sweet lime (Citrus aurantifolia). The relatively inexpensive SNP array used in this study generated informative genotyping data and led to good consensus and correlations with previously published observations based on whole genome sequencing (WGS) data. The genotyping data and the phylogenetic results may facilitate further exploitation of interesting genotypes in the collection and additional understanding of phylogenetic relationships in citrus.

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