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Somatic Hybridization – Scion Improvement - building high quality tetraploid breeding parents

Applications of Somatic Hybridization and Cybridization in Scion and Rootstock Improvement with Focus on Citrus Jude Grosser and Milica Ćalović University of Florida, Citrus Research and Education Center, Lake Alfred, FL USA. Somatic Hybridization – Scion Improvement

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Somatic Hybridization – Scion Improvement - building high quality tetraploid breeding parents

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  1. Applications of Somatic Hybridization and Cybridization in Scion and Rootstock Improvement with Focus on Citrus Jude Grosser and Milica Ćalović University of Florida, Citrus Research and Education Center, Lake Alfred, FL USA

  2. Somatic Hybridization – Scion Improvement • - building high quality tetraploid breeding parents • - triploid recovery from interploid crosses • Somatic Cybridization • -transfer of Satsuma CMS (Cytoplasmic Male Sterility) • cytoplasm to seedy cultivars for potential seedlessness • -Somatic Hybridization – Rootstock Improvement • -somatic hybridization of complementary parents • - building a better sour orange – fusion of selected • mandarin + pummelo parents

  3. SWEET ORANGE EMBRYOGENIC CALLUS

  4. SWEET ORANGE ORGANOGENESIS ADVENTITIOUS SHOOT BUD INDUCTION

  5. SUCROSE-MANNITOL GRADIENT FOR PROTOPLAST PURIFICATION

  6. SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS

  7. LEAF-DERIVED CITRUS PROTOPLASTS

  8. TYPICAL SUSPENSION PROTOPLAST + LEAF PROTOPLAST PEG-INDUCED FUSION

  9. RMAN

  10. Ploidy Analysis Showing Diploid and TetraploidPeaks Using a Partec Tabletop Flow Cytometer

  11. NEW SOMATIC HYBRID PLANT

  12. NOVA + SUCCARI SOMATIC HYBRID TREE

  13. TETRAPLOID SOMATIC HYBRID COMBINATIONS – PLANTS REGENERATED FROM EMBRYOGENIC CULTURE + LEAF FUSIONS AT THE CREC: Intergeneric, sexually compatible 41 Intergeneric, sexually incompatible 16 Interspecific, sexually compatible >200 Total: >250

  14. ‘Tacle’ – a seedless triploid mandarin hybrid produced by A. Starrantino.

  15. Somatic Hybrid Pollen Parents Used For Mandarin • Citrus Fruit Improvement To Date Include: • Nova mandarin + Succari sweet orange • Valencia sweet orange + Murcott tangor • Succari sweet orange + Murcott tangor • Rohde Red Valencia + Dancy mandarin • Succari + Page tangelo • Valencia + Page tangelo • Succari + Minneola tangelo • Hamlin sweet orange + Ponkan mandarin • Hamlin + LB8-4 (Clementine x Minneola) • Valencia + (Robinson x Temple) • Pink Marsh grapefruit + Murcott • Succari + Dancy • Murcott + LB8-9 tangelo • Nova + Osceola mandarin

  16. Interploid hybridization using tetraploid somatic • hybrids as pollen parents to produce seedless • triploids for mandarin improvement: • more than 12000 triploids produced to date,many fathered by somatic hybrids • (under direction of FG Gmitter,CREC) • oldest hybrids are now fruiting and most are seedless!!!!!

  17. NOVA + SUCCARI SOMATIC HYBRID FRUIT (father of several hundred triploid progeny)

  18. Valencia + (Robinson x Temple)harvested January 22,2004brix=11.4, acid=0.57, ratio=203 seeds/fruit

  19. Rohde Red Valencia + Dancyharvested January 28,2004brix=11.4, acid=1.57, ratio= 7.263 seeds/fruit

  20. Nova + Osceolaharvested December 6, 2005brix= 14.8, acid=1.15, ratio= 12.9

  21. EMBRYO RESCUE • Facilitates triploid plant recovery from interploid crosses (circumvents embryo abortion due to endosperm failure) • Also useful for triploid recovery exploiting • 2n gametes (unreduced) from cultivars such as • Clementine and Temple (several thousand • triploids recovered at IVIA, Spain)

  22. New triploid mandarin hybrids following embryo rescue and micro-grafting (F.G. Gmitter, Jr.)

  23. MID-TERM SCION CANDIDATES (5-10 years) Crec-9505 triploid mandarin hybrid • > 12000 triploid mandarin hybrids (FG Gmitter, Jr.) • Focus: seedless fresh market • CREC 9505 – proof of concept – 0 seed! • many beginning to fruit!

  24. Somatic Hybridization Results - Recent Scion Improvement Fusions Embryogenic Parent Leaf Parent Microcalli Embryos Plantlets Ploidy Changsha W-Murcott x x Changsha Fremont x x Changsha Dancy x x W-Murcott Kinnow x x x 2x, 4x Page Kinnow x x x 4x Page Dancy x x x 4x Page Fremont x x Murcott Dancy x x x 4x Murcott Kinnow x x Murcott FG#307 x x x 2x W. Murcott SF Lee Hybrid x x x 2x, 4x W. Murcott FG#306 x x Valencia SF14W-62 W-Murcott x x x 2x, 4x Valencia SF14W-62 Changsha x x x 2x, 4x Valencia SF14W-62 Kinnow x x x 2x, 4x Valencia SF14W-62 Fremont x x x 2x, 4x Valencia SF14W-62 Osceola x x x 2x, 4x Valencia SF14W-62 Dancy x x x 2x, 4x

  25. New Murcott + Dancy somatic hybrid

  26. Seedless fresh fruit varieties at the • Tetraploid level? • Seedless somatic hybrids with excellent • fruit quality: • Succari sweet orange + Page tangelo • - peelable, early maturing, excellent flavor • 2. Valencia sweet orange + Murcott tangor • - peelable, excellent flavor

  27. Succari + Page fruits taken on Sep 27, 2002 (seedless)

  28. Valencia + Murcott, fruits taken on Jan 15, 2003 (nearly seedless)

  29. SOMATIC CYBRIDIZATION • Unfused leaf protoplasts not capable of plant • regeneration • Diploid plants often regenerated from symmetric • fusions of embryogenic callus + leaf that are • morphologically identical to the leaf parent (from • more than 50 parental combinations). • RFLP analyses indicates that these plants are always • cybrids containing the mitochondrial genome of the • callus parent! The chloroplast genome inheritance is • random.

  30. PRACTICAL APPLICATION OF CYBRIDIZATION: • Transfer of cytoplasmic male sterility (CMS) from the • seedless Satsuma mandarin to other superior seedy diploid • commercial cultivars via symmetric fusion. • Objective: remove seed from successful cultivars without • otherwise altering cultivar integrity. • Cybrid combinations produced to date (Grosser, Guo, • Prasad and Deng): • Guoqing Satsuma + Hirado Buntan pink pummelo • Guoqing Satsuma + Sunburst mandarin • Guoqing Satsuma + (Clementine x Murcott) Lee hybrid • Guoqing Satsuma + LB8-9 (Clementine x Minneola)

  31. Somatic cybrid plant of Hirado Buntan pink pummelo containing Guoqing Satsuma cytoplasm (mitochondrial genome).

  32. SSR analysis (nuclear) of G1 + HBP diploid cybrid plant. 1: 100 bp ladder; 2, 5: G1; 3, 6: G1 + HBP diploid cybrid plant; 4, 7: HBP. Primer pairs: 2-4, TAA15; 5-7, TAA41.

  33. Mt- and cp-DNA analysis of G1 + HBP diploid cybrid plant by CAPS markers. 1, 4, 8, 11: G1; 2, 5, 9, 12: G1 + HBP diploid cybrid plant; 3, 6, 10, 13: HBP; 7, 14:100 bp ladder. Primer pair/enzyme combinations, 1-3: nad4Exon1-nad4Exon2/HaeIII; 4-6: nad4exon1-nad4exon2/HinfI; 8-10: rbcL-psaI/TaqI; 11-13: trnD-trnT/TaqI.

  34. ‘Sunburst’ James Saunt, 1990. Citrus Varieties of the World

  35. Somatic cybrid plant of Sunburst tangerine containing Guoqing Satsuma cytoplasm (mitochondrial genome).

  36. Somatic cybrid plant of Lee hybrid (Murcott x Clementine) containing Guoqing Satsuma cytoplasm (mitochondrial genome).

  37. ‘Murcott’ tangor The Honey Tangerine James Saunt, 1990. Citrus Varieties of the World

  38. ‘Kinnow’ James Saunt, 1990. Citrus Varieties of the World

  39. Somatic Cybridization Results – Recent Scion Improvement Fusions Embryogenic Parent Leaf Parent Microcalli Embryos Plantlets Ploidy G1 Satsuma Kinnow x x x 2x G1 Satsuma W-Murcott x x x 4x G1 Satsuma Dancy x x x 2x G1 Satsuma Changsha x G1 Satsuma Fremont x x x 4x G1 Satsuma FG#307 x x x 2x G1 Satsuma Murcott x x x 2x G1 Satsuma ScabFree Lee H. x x x 2x, 4x G1 Satsuma FG#303 x x x 2x G1 Satsuma FG#304 x x x 2x, 4x

  40. Somatic Hybrid Parents Used For Grapefruit • Improvement To Date Include: • Nova mandarin + Hirado Buntan sdl. Pummelo • (male and female) • 2. Succari sweet orange + Hirado Buntan sdl. Pummelo • (male and female) • 3. Succari sweet orange + Murcott tangor • pollen parent • - more than 1100 triploid plants produced to date

  41. Breeding Canker Tolerant Triploid Seedless Grapefruit-like Hybrids X Tetraploid Somatic Hybrid Canker Tolerant Pummelo • Several good breeding parents now flowering • Hundreds of triploid hybrids already produced • Embryo rescue not required when tetraploid parent is used • as the female

  42. Interploid hybridization using tetraploid somatic • hybrids as pollen parents to produce seedless • triploids for acid-fruit (lemon/lime) improvement: • - other objectives: improved cold-hardiness and disease resistance (canker, witches broom); new industrial oils. • - more than 600 triploids produced to date • (Grosser, Viloria and Chandler,CREC)- • Somatic hybrid breeding parents include: • Mexican lime + Valencia • Mexican lime + Lakeland limequat • Valencia + Femminello lemon • Hamlin + Femminello lemon • Milam lemon + Femminello lemon • Early Gold sweet orange + C. micrantha

  43. ROOTSTOCK IMPROVEMENT An Exercise in “Creative Packaging”

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