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

6. SUCROSE-MANNITOL GRADIENT FOR PROTOPLAST PURIFICATION

7. SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS

8. LEAF-DERIVED CITRUS PROTOPLASTS

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

10. RMAN

13. Ploidy Analysis Showing Diploid and Tetraploid Peaks Using a Partec Tabletop Flow Cytometer

14. NEW SOMATIC HYBRID PLANT

16. NOVA + SUCCARI SOMATIC HYBRID TREE

17. 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

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

20. Somatic Hybrid Pollen Parents Used For Mandarin Citrus Fruit Improvement To Date Include: 1.Nova mandarin + Succari sweet orange 2.Valencia sweet orange + Murcott tangor 3.Succari sweet orange + Murcott tangor 4.Rohde Red Valencia + Dancy mandarin 5.Succari + Page tangelo 6.Valencia + Page tangelo 7.Succari + Minneola tangelo 8.Hamlin sweet orange + Ponkan mandarin 9.Hamlin + LB8-4 (Clementine x Minneola) 10.Valencia + (Robinson x Temple) 11.Pink Marsh grapefruit + Murcott 12.Succari + Dancy 13. Murcott + LB8-9 tangelo 14. Nova + Osceola mandarin

21. 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!!!!!

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

23. Valencia + (Robinson x Temple) harvested January 22,2004 brix=11.4, acid=0.57, ratio=20 3 seeds/fruit

24. Rohde Red Valencia + Dancy harvested January 28,2004 brix=11.4, acid=1.57, ratio= 7.26 3 seeds/fruit

25. Nova + Osceola harvested December 6, 2005 brix= 14.8, acid=1.15, ratio= 12.9

26. 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)

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

29. - > 12000 triploid mandarin hybrids (FG Gmitter, Jr.) - Focus: seedless fresh market - CREC 9505 – proof of concept – 0 seed! - many beginning to fruit! MID-TERM SCION CANDIDATES (5-10 years) Crec-9505 triploid mandarin hybrid

30. 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

31. New Murcott + Dancy somatic hybrid

32. Seedless fresh fruit varieties at the Tetraploid level? Seedless somatic hybrids with excellent fruit quality: 1.Succari sweet orange + Page tangelo - peelable, early maturing, excellent flavor 2. Valencia sweet orange + Murcott tangor - peelable, excellent flavor

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

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

35. 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.

36. 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): 1.Guoqing Satsuma + Hirado Buntan pink pummelo 2.Guoqing Satsuma + Sunburst mandarin 3.Guoqing Satsuma + (Clementine x Murcott) Lee hybrid 4.Guoqing Satsuma + LB8-9 (Clementine x Minneola)

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

38. 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.

39. 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.

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

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

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

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

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

45. 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

47. Somatic Hybrid Parents Used For Grapefruit Improvement To Date Include: 1.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

48. X Breeding Canker Tolerant Triploid Seedless Grapefruit-like Hybrids 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

49. 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

50. ROOTSTOCK IMPROVEMENT An Exercise in “Creative Packaging”

52. WHAT DO WE NEED? - Wide adaptability, tolerance of high pH, calcareous and heavy soils - Resistance to CTV-induced quick decline - Resistance to Citrus Blight - High yields of good fruit quality - Optimum tree size for harvesting - New rootstocks must be able to tolerate mechanical damage inflicted by Diaprepes larvae, and resist secondary infections of Phytophthora nicotianae and P. palmivora, and other invading fungi - Must be capable of vigorous root growth following Diaprepes damage. - tolerance of salinity in some areas

53. Our primary strategy for rootstock improvement has been to produce allotetraploid somatic hybrids by combining complementary diploid rootstocks. Tetraploid rootstocks usually (but not always) have a built in tree-size control mechanism due to some unknown physiological reaction with the diploid scion.

55. SOMATIC HYBRID ROOTSTOCKS – TREE SIZE CONTROL McTeer Sandridge Trial – Planted 1995 - Data from 2003 harvest Rootstock boxes/tree S.S./box Avg. Diam. Yield Eff. Kg/m3 SO + FD 2.64 6.73 3.2 0.46 FD 2.0 5.74 3.1 0.36 Succ + APT 3.17 6.52 3.5 0.45 Cleo + APT 3.67 6.21 3.7 0.40 SO + rangpur 4.11 6.10 3.8 0.40 Swingle 4.89 5.70 4.0 0.37 SO + CZO 4.89 6.10 4.1 0.36 Cleo 5.71 6.16 4.4 0.36 Palestine SL 7.0 5.32 4.8 0.29

56. Effect of Polyploidy on Tree Size (based on % of Carrizo average canopy volume) – Sweet orange scion, McTeer Trial, Dundee, Somatic hybrids indicated by plus sign. Trees 4-7 years old. Rough lemon 8166 141 CARRIZO 100 Cleo 100 Kinkoji 95 C-35 90 Sour orange + Carrizo 84 Swingle 78 Sour orange + rangpur 65 WGFT+50-7 63* Cleo + Carrizo 61 Cleo + Argentine trifoliate orange 58 Nova + Hirado Buntan pummelo zyg. 56 Cleo + rangpur 54 SO+50-7 51* Nova + Palestine sweet lime 49 Milam + Kinkoji 42 Changsha + Benton 38 Cleo + Swingle 35 Cleo + Flying Dragon 35 Hamlin + Flying Dragon 34 Flying Dragon 33 Sour orange + Benton 29

57. Overview of somatic hybrid seedlings at Reed Brothers Nursery - thanks to Chuck Reed and Mike Baker

58. OTHER NEW SOMATIC HYBRID ROOTSTOCKS PRODUCED RECENTLY: Milam + Kinjoji Changsha + 50-7 trifoliate orange White grapefruit + 50-7 trifoliate orange Sour orange + 50-7 trifoliate orange Sour orange + Benton citrange Amblycarpa + Carrizo citrange Amblycarpa + C-35 citrange Amblycarpa + Benton citrange Amblycarpa + Volk Amblycarpa + Flying Dragon Amblycarpa + Rubidoux trifoliate orange Murcott + Rubidoux trifoliate orange Amblycarpa + 50-7 trifoliate orange

59. SOUR ORANGE ROOTSTOCK - formerly the most important rootstock worldwide - widely adapted to most soil types, tolerant of blight - produces fruit of high quality, holds fruit well -- very nursery friendly - no longer being used due to susceptibility to quick decline disease caused by citrus tristeza virus - introduction of the brown citrus aphid (an efficient tristeza vector) to many areas threatens existing trees - replacement rootstock badly needed!

60. WHAT IS SOUR ORANGE? RAPD Markers Identified in Various Citrus Genotypes For Analysis of Origin (from Nicolosi et al. 2000). Genotype Markers Markers shared with Extra Pumm, Mand, Citron, Sweet, Sour sweet or. 71 35 36 sour or. 84 42 36 6 grapefr. 72 45 27 lemon 78 45 31 2 Volk 56 6 27 22 1 rough lem 79 32 46 5 Palestine 73 48 21 4 Rangpur 85 32 46 7

61. Screening Zygotic Pummelo Seedlings from a superior pummelo seed parent

62. Screening of Open-Pollinated Pummelo Seedlings for Tolerance of Phytophthora and High pH, Calcareous Soil Tree I.D. Pummelo Variety % Seedling Survival DPI 8-1 Liang Ping Yau sdlg. (China) 69% DPI 7-2 Large Pink Pummelo (SE Asia) 67% J.L.C. Hirado Buntan sdlg. (Japan) 56% DPI 5-4 Red Shaddock Pummelo (SE Asia) 47% DPI 4-3 Sha Ten Yau sdlg. (China) 30% DPI 7-3 Siamese Sweet (Thailand) 27% DPI 4-4 Siamese Pummelo (Thailand) 18% DPI 5-1 Hirado Buntan sdlg. (Japan) 17% DP”I 8-2 Pummelo NW (Florida) 12% DPI 7-1 Chinese Pummelo (China) 4% DPI 6-1 Kao Phuang (Thailand) 0% >200 seedlings selected for fusion experiments

63. NEW MANDARIN + PUMMELO SOMATIC HYBRIDS PRODUCED DURING 2000-2003. Murcott + Hirado Buntan pummelo (HBP) Murcott + HBP sdl-JL1 Murcott + Chandler sdl-80 Murcott + Siamese Pummelo sdl 4-4-99-6 Murcott + Chandler sdl-11-A1 (best in Diaprepes test) Amblycarpa + HBP Amblycarpa + Chandler pummelo Amblycarpa + Chandler sdl-69 Amblycarpa + HBP sdl-5-1-99-1B Amblycarpa + HBP sdl-JL2B Amblycarpa + LingPingYau-sdl-8-1-99-4A Amblycarpa + Large Pink Pummelo sdl-7-2-99-5 Amblycarpa + HBP sdl-JL1 Amblycarpa + HBP sdl-JL4 Amblycarpa + HBP sdl-5-1-99-3 Shekwasha + HBP Shekwasha + Chandler Shekwasha + HBP sdl-JL2B

64. Test for CTV-induced quick-decline: selected pummelo seedlings are budded with MCA-13-CTV positive sweet orange. After 2+ years in the field, 20 best identified.

65. NEW MANDARIN + PUMMELO SOMATIC HYBRIDS PRODUCED DURING 2004-2006. Amblycarpa + Siamese Pummelo sdl 4-4-99-6 Amblycarpa + Siamese Sweet sdl. 7-3-99-1 Amblycarpa + Shao Tian You sdl. 4-3-99-2 Amblycarpa + Hirado Buntan Pink (HBP) sdl. JL-3 Amblycarpa + HBP sdl. JL-5 Amblycarpa + HBP sdl. JL-7 Amblycarpa + HBP sdl JL-12 Amblycarpa + HBP sdl 5-1-99-2 Amblycarpa + HBP sdl. MG-10 Amblycarpa + Chandler sdl-11-A1 (best in Diaprepes test) Changsha + HBP sdl. JL-3 Changsha + HBP sdl. JL-5 Changsha + HBP sdl. JL-7 Page + HBP sdl. JL-3 Page + HBP sdl. JL-4 Page + HBP sdl. JL-5 red = pummelo parent tolerant of CTV- Page + HBP sdl. JL-7 induced quick-decline after 2 years Page + HBP sdl. JL-12 in field

66. NEW MANDARIN + PUMMELO SOMATIC HYBRIDS PRODUCED DURING 2006. W. Murcott + HBP sdl. JL-3 W. Murcott + HBP sdl. JL-7 W. Murcott + HBP sdl JL-12 W. Murcott + Large Pink sdl 7-2-99-2 W. Murcott + Sha Tian You sdl 4-3-99-2 W. Murcott + HBP sdl 5-1-99-2 Nova + Large Pink sdl 7-2-99-2 Nova + Pummelo 8-2-99-1 Nova + Siamese sweet sdl 7-3-99-1 Nova + Ling Ping Yau sdl 8-1-99-4B Murcott + HBP sdl. JL-5 red = pummelo parent tolerant of CTV- induced quick-decline after 2 years in field

67. Vigorous new mandarin + pummelo somatic hybrid: Amblycarpa + Hirado buntan sdl. 5-1-99-1B

68. Figure.1. Leaf morphology of sour orange and some new somatic hybrids of mandarin + pummelo (‘mandelos’). 1- Sour orange; 2 - Amblycarpa + C. grandis ‘Chandler’; 3 - Amblycarpa + HBP (Hirado Buntan Pummelo); 4 - Amblycarpa + HBP sdl-JL-2B; 5 - Amblycarpa + HBP sdl-5-1-99-1B; 6 - Amblycarpa + ‘LingPing Yau’-sdl-8-1-99-4A.

69. Gel -1 Primer c-11 Gel -2 Primer c-11 Gel -4 Primer c- 64 Gel -3Primer c-64 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11 12 RAPD patterns of new mandarin + pummelo interspecific somatic hybrids and their parental genotypes. Gels-1 and 3 are products amplified by operon primers C-11 and C-64 respectively. Lane 1- 1kb marker; 2- Amblycarpa mandarin (C. amblycarpa); 3- Amblycarpa + ‘Siamese’ sdl-4-4-99-6; 4- ‘Siamese’ sdl-4-4-99-6; 5- Amblycarpa + ‘Chandler’ sdl. A1-11; 6- ‘Chandler’ sdl. A1-11; 7- Amblycarpa + ‘Large Pink’ sdl-7-2-99-5; 8- ‘Large Pink’ sdl-7-2-99-5; 9- Amblycarpa + HBP sdl-JL-4; 10- HBP sdl-JL-4; 11- Amblycarpa. Gels-2 and 4 are products amplified by operon primers C-11 and C-64 respectively. Lane 1- 1kb marker; 2- Amblycarpa mandarin (C. amblycarpa); 3- Amblycarpa + HBP sdl-JL-1; 4- HBP sdl-JL-1; 5-‘Succari’; 6- ‘Succari’ + C.grandis ‘Hirado Buntan Pink’ (HBP); 7- C.grandis ‘Hirado Buntan Pink’ (HBP); 8- ‘Murcott’ tangor; 9-‘Murcott’ + ‘Chandler’ sdl. A1-11; 10- ‘Chandler’ sdl.A1-11; 11- ‘Murcott’ + ‘Siamese’ sdl-4-4-99-6; 12- ‘Siamese’ sdl-4-4-99-6. (White arrows indicate complementary bands from the embryogenic parents and red diamond arrows from the pummelo leaf parents).

70. Field Assay for Tristeza Virus Resistance – Sour orange not growing, Mandarin + Pummelo somatic hybrid not affected by virus! Sour orange

71. Amenability to Traditional Seed Propagation – 2006 Tree Size Controlling Somatic Hybrids Hybrid Seeds per Fruit Seed Type WGFT + 50-7 20 Nucellar Changsha + 50-7 17 Nucellar SO + 50-7 14 Nucellar SO+Carrizo 13 Nucellar Data: Julie Gmitter and Chunxian Chen

72. To a host of CREC scientists and Excellent Florida Citrus Industry Cooperators! Funding: Citrus Production Research Advisory Council for Financial Support via Block Grant # 0110-031 “A Comprehensive Program for the Genetic Improvement of Florida Citrus Scion and Rootstock Varieties”; CSREES/USDA Special Research Grants Program, Citrus Tristeza Virus; USDA/IFAS Cooperative Agreements – Research on Citrus tristeza virus and other exotic diseases/ Diaprepes. SPECIAL THANKS!

73. Some of the Key Contributors with the ‘Protoplast Gene’ J.L. Chandler Dr. XiuXin Deng Dr. Fred Gmitter, Jr Dr. Wenwu Guo Dr. Bill Castle Karla Lopez-Madera Dr. Nicasio Tusa Dr. G. Ananthakrishnan Dr. Eliezer Louzada Dr. Ahmad Omar Dr. Francisco Mourao Dr. Cecile Cabasson Dr. Victor Medina-Urrutia Dr. Hesham El-Smamy Dr. Oscar Olivares-Fuster Dr. Iqrar Kahn Dr. Zenaida Viloria Dr. Geraldine Fleming Milica Calovic Patricia Brickman Julie Gmitter Dr. Manjul Dutt Dr. Jim Graham Dr. Larry Duncan AND MANY MORE!