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 - 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

SWEET ORANGE EMBRYOGENIC CALLUS

SWEET ORANGE ORGANOGENESISADVENTITIOUS SHOOT BUD INDUCTION

SUCROSE-MANNITOL GRADIENT FOR

PROTOPLAST PURIFICATION

SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS

LEAF-DERIVED CITRUS PROTOPLASTS

TYPICAL SUSPENSION PROTOPLAST + LEAF

PROTOPLAST PEG-INDUCED FUSION

RMAN

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

NEW SOMATIC HYBRID PLANT

NOVA + SUCCARI SOMATIC HYBRID TREE

TETRAPLOID SOMATIC HYBRID COMBINATIONS – PLANTS REGENERATED FROM EMBRYOGENIC CULTURE + LEAF FUSIONS AT THE CREC:

Intergeneric, sexually compatible 41Intergeneric, sexually incompatible 16Interspecific, sexually compatible >200

Total: >250

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

Somatic Hybrid Pollen Parents Used For Mandarin Citrus Fruit Improvement To Date Include:

1. Nova mandarin + Succari sweet orange2. Valencia sweet orange + Murcott tangor3. Succari sweet orange + Murcott tangor4. Rohde Red Valencia + Dancy mandarin5. Succari + Page tangelo6. Valencia + Page tangelo7. Succari + Minneola tangelo8. Hamlin sweet orange + Ponkan mandarin9. Hamlin + LB8-4 (Clementine x Minneola)10. Valencia + (Robinson x Temple)11. Pink Marsh grapefruit + Murcott12. Succari + Dancy13. Murcott + LB8-9 tangelo14. Nova + Osceola mandarin

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!!!!!

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

Valencia + (Robinson x Temple)harvested January 22,2004

brix=11.4, acid=0.57, ratio=203 seeds/fruit

Rohde Red Valencia + Dancyharvested January 28,2004

brix=11.4, acid=1.57, ratio= 7.263 seeds/fruit

Nova + Osceolaharvested December 6, 2005

brix= 14.8, acid=1.15, ratio= 12.9

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)

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

- > 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

Somatic Hybridization Results - Recent Scion Improvement Fusions

  Embryogenic Parent Leaf Parent Microcalli Embryos Plantlets Ploidy  

Changsha W-Murcott x x Changsha Fremont x xChangsha Dancy x xW-Murcott Kinnow x x x 2x, 4xPage Kinnow x x x 4xPage Dancy x x x 4xPage Fremont x xMurcott Dancy x x x 4xMurcott Kinnow x xMurcott FG#307 x x x 2xW. Murcott SF Lee Hybrid x x x 2x, 4xW. Murcott FG#306 x xValencia SF14W-62 W-Murcott x x x 2x, 4xValencia SF14W-62 Changsha x x x 2x, 4xValencia SF14W-62 Kinnow x x x 2x, 4xValencia SF14W-62 Fremont x x x 2x, 4xValencia SF14W-62 Osceola x x x 2x, 4xValencia SF14W-62 Dancy x x x 2x, 4x

 New Murcott + Dancy somatic hybrid

Seedless fresh fruit varieties at the Tetraploid level?

Seedless somatic hybrids with excellentfruit quality:

1. Succari sweet orange + Page tangelo - peelable, early maturing, excellent flavor

2. Valencia sweet orange + Murcott tangor - peelable, excellent flavor

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

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

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.

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 pummelo2. Guoqing Satsuma + Sunburst mandarin3. Guoqing Satsuma + (Clementine x Murcott) Lee hybrid4. Guoqing Satsuma + LB8-9 (Clementine x Minneola)

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

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. 

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.

‘Sunburst’

James Saunt, 1990. Citrus Varieties of the World

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

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

‘Murcott’ tangor The Honey Tangerine

James Saunt, 1990. Citrus Varieties of the World

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

Somatic Cybridization Results – Recent Scion Improvement Fusions

 Embryogenic Parent Leaf Parent Microcalli Embryos Plantlets PloidyG1 Satsuma Kinnow x x x 2xG1 Satsuma W-Murcott x x x 4xG1 Satsuma Dancy x x x 2xG1 Satsuma Changsha x G1 Satsuma Fremont x x x 4xG1 Satsuma FG#307 x x x 2xG1 Satsuma Murcott x x x 2xG1 Satsuma ScabFree Lee H. x x x 2x, 4xG1 Satsuma FG#303 x x x 2xG1 Satsuma FG#304 x x x 2x, 4x  

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

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

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

ROOTSTOCK IMPROVEMENT

An Exercise in “Creative

Packaging”

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

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.

       

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

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 141CARRIZO 100Cleo 100Kinkoji 95C-35 90Sour orange + Carrizo 84Swingle 78 Sour orange + rangpur 65WGFT+50-7 63*Cleo + Carrizo 61Cleo + Argentine trifoliate orange 58Nova + Hirado Buntan pummelo zyg. 56Cleo + rangpur 54SO+50-7 51*Nova + Palestine sweet lime 49Milam + Kinkoji 42Changsha + Benton 38Cleo + Swingle 35Cleo + Flying Dragon 35Hamlin + Flying Dragon 34Flying Dragon 33Sour orange + Benton 29

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

OTHER NEW SOMATIC HYBRID ROOTSTOCKS PRODUCED RECENTLY:

Milam + KinjojiChangsha + 50-7 trifoliate orangeWhite grapefruit + 50-7 trifoliate orangeSour orange + 50-7 trifoliate orangeSour orange + Benton citrangeAmblycarpa + Carrizo citrangeAmblycarpa + C-35 citrangeAmblycarpa + Benton citrangeAmblycarpa + VolkAmblycarpa + Flying DragonAmblycarpa + Rubidoux trifoliate orangeMurcott + Rubidoux trifoliate orangeAmblycarpa + 50-7 trifoliate orange

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!

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 6grapefr. 72 45 27lemon 78 45 31 2Volk 56 6 27 22 1rough lem 79 32 46 5Palestine 73 48 21 4Rangpur 85 32 46 7

Screening Zygotic Pummelo Seedlings from a superior pummelo seed parent

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

NEW MANDARIN + PUMMELO SOMATIC HYBRIDS PRODUCED DURING 2000-2003.

Murcott + Hirado Buntan pummelo (HBP)Murcott + HBP sdl-JL1Murcott + Chandler sdl-80Murcott + Siamese Pummelo sdl 4-4-99-6Murcott + Chandler sdl-11-A1 (best in Diaprepes test)Amblycarpa + HBPAmblycarpa + Chandler pummeloAmblycarpa + Chandler sdl-69Amblycarpa + HBP sdl-5-1-99-1BAmblycarpa + HBP sdl-JL2BAmblycarpa + LingPingYau-sdl-8-1-99-4AAmblycarpa + Large Pink Pummelo sdl-7-2-99-5Amblycarpa + HBP sdl-JL1Amblycarpa + HBP sdl-JL4Amblycarpa + HBP sdl-5-1-99-3Shekwasha + HBPShekwasha + Chandler Shekwasha + HBP sdl-JL2B

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.

NEW MANDARIN + PUMMELO SOMATIC HYBRIDS PRODUCED DURING 2004-2006.

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

NEW MANDARIN + PUMMELO SOMATIC HYBRIDS PRODUCED DURING 2006.

W. Murcott + HBP sdl. JL-3W. Murcott + HBP sdl. JL-7W. Murcott + HBP sdl JL-12W. Murcott + Large Pink sdl 7-2-99-2W. Murcott + Sha Tian You sdl 4-3-99-2W. Murcott + HBP sdl 5-1-99-2Nova + Large Pink sdl 7-2-99-2Nova + Pummelo 8-2-99-1Nova + Siamese sweet sdl 7-3-99-1Nova + Ling Ping Yau sdl 8-1-99-4BMurcott + HBP sdl. JL-5

red = pummelo parent tolerant of CTV-induced quick-decline after 2 yearsin field

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

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.

Gel -1

Primer c-11

Gel -2

Primer c-11

Gel -4 Primer c-64

Gel -3 Primer c-64

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11 12

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).

Field Assay for TristezaVirus Resistance – Sour orange not growing, Mandarin+ Pummelo somatic hybrid not affected by virus!

Sour orange

Amenability to Traditional Seed Propagation – 2006

Tree Size Controlling Somatic Hybrids

Hybrid Seeds per Fruit Seed Type

WGFT + 50-7 20 NucellarChangsha + 50-7 17 NucellarSO + 50-7 14 NucellarSO+Carrizo 13 Nucellar

Data: Julie Gmitter and Chunxian Chen

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!

Some of the Key Contributors with the ‘Protoplast Gene’

J.L. Chandler Dr. XiuXin DengDr. Fred Gmitter, Jr Dr. Wenwu GuoDr. Bill Castle Karla Lopez-MaderaDr. Nicasio Tusa Dr. G. AnanthakrishnanDr. Eliezer Louzada Dr. Ahmad OmarDr. Francisco Mourao Dr. Cecile CabassonDr. Victor Medina-Urrutia Dr. Hesham El-SmamyDr. Oscar Olivares-Fuster Dr. Iqrar KahnDr. Zenaida Viloria Dr. Geraldine FlemingMilica Calovic Patricia BrickmanJulie Gmitter Dr. Manjul DuttDr. Jim Graham Dr. Larry Duncan

AND MANY MORE!