New Somatic Hybrid Mandarin Tetraploids Generated by Optimized Protoplast Fusion and Confirmed by Molecular Marker Analysis and Flow Cytometry

in Journal of the American Society for Horticultural Science

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.

Contributor Notes

Corresponding author. E-mail: jwg@ufl.edu.

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    Division of fused protoplasts and creation of colonies following polyethylene-glycol-mediated protoplast fusion (400×): (A) protoplasts obtained from fusion of protoplast isolated from nucellar callus (from ‘W. Murcott’ tangor) and leaf-derived protoplast (from ‘Ponkan’ mandarin); (B) the first cell division of fused protoplast; (C) the second cell division of fused protoplast; (D) immature colony; and (E) mature colony.

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    The effect of three induction media on callusogenesis (calculated as % of ovules that responded to the media after 9 months of in vitro culture). The four citrus cultivars are Ponkan mandarin (Po), Page tangelo (Pg), Murcott tangor (Mt), and W. Murcott tangor (WM). Different letters above columns indicate that mean values are significantly different at P < 0.05 according to Duncan’s multiple range test. Capital letters (A, B, C) refer to differences in callogenesis for each individual cultivar grown on three different induction media while small letters (a, b, c) refer to differences in callogenesis among four different cultivars for each tested induction medium.

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    The effect of three embryo induction media on embryogenesis (calculated as the percentage of ovules that responded to the media after 9 months of in vitro culture). The four citrus cultivars are Ponkan mandarin (Po), Page tangelo (Pg), Murcott (Mt), and W. Murcott (WM) tangor. Different letters above columns indicate that mean values are significantly different at P < 0.05 according to Duncan’s multiple range test. Capital letters (A, B, C) refer to differences in embryogenesis for each individual cultivar grown on three different induction media while small letters (a, b, c) refer to differences in embryogenesis among four different cultivars for each tested induction medium.

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    Phases between culture of mature cell colonies and regenerated plants. Parental material came from ‘W. Murcott’ tangor and ‘Ponkan’ mandarin. (A) Mature colonies in the liquid media; (B) generation of somatic embryos upon transfer of colonies to 0.15 m EME-malt solid embryo induction medium (Grosser et al., 2010b); (C, D) growth of somatic embryos on the surface of aceto-cellulose filter papers placed on top of solid medium; (E) maturation of somatic embryos on EME 1500 embryo enlargement medium (Grosser et al., 2010b); (F) elongation growth of embryos on B+ embryo germination medium (Grosser and Gmitter, 1990); (G, H) induction of organogenesis in undeveloped embryos on DBA3 shoot induction/multiplication medium (Grosser and Gmitter, 1990); and (I) growth and rooting of plants on RMAN rooting medium (Grosser and Gmitter, 1990) in Magenta vessels (GA-7; Magenta, Lockport, IL).

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    Leaf morphology of parents (top row) and six somatic hybrids (bottom row). Top: (A) ‘Ponkan’ mandarin, (B) ‘W. Murcott’ tangor, (C) ‘Willowleaf’ mandarin, (D) ‘Murcott’ tangor. Bottom: (E) ‘Ponkan’ + ‘W. Murcott’ allotetraploid, (F) ‘W. Murcott’ autotetraploid that was produced in ‘W. Murcott’ + ‘Ponkan’ fusion, (G) ‘Willowleaf’ autotetraploid that was produced in ‘W. Murcott’ + ‘Willowleaf’ fusion, (H) ‘W. Murcott’ autotetraploid that was produced in ‘W. Murcott’ + ‘Willowleaf’ fusion, and (I) ‘Murcott’ autotetraploid that was produced in ‘Murcott’ + ‘Willowleaf’ fusion.

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    Identification of mandarin hybrid plants with the use of flow cytometry and expressed sequence tag–simple sequence repeat analysis (EST-SSR). (A) Flow cytometry–histogram presentation of fluorescence intensities from nuclei extracted from regenerant plants. Fluorescence intensity is proportional to the amount of DNA in the nuclei. Each peak in the histogram represents the group of cells with the same ploidy level: 2x, 3x, or 4x. (B) Representative chromatograms of polymerase chain reaction products (primer CX2007) produced in EST-SSR analyses. Uppermost graph shows the homozygous allele [171 base pairs (bp)] in ‘Ponkan’ mandarin; middle graph shows the homozygous allele (177 bp) in ‘W. Murcott’ tangor; and the lowest graph shows the combined two alleles (171 and 177 bp) in their somatic allotetraploid.

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    New tetraploid mandarins grafted to UFR-15 rootstock (Citrus maxima × Citrus reshni) and planted in the field at University of Florida, Citrus Research and Education Center, Lake Alfred. Left: Somatic hybrid of ‘Ponkan’ mandarin + ‘W. Murcott’ tangor; middle: somatic hybrid of ‘W. Murcott’ + ‘Snack’ tangor; right: autotetraploid ‘W. Murcott’. Trees are just older than 1 year.

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