Applications of Protoplasts in Citrus Improvement

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1 Applications of Protoplasts in Citrus Improvement Jude Grosser and Ahmad Omar University of Florida, Citrus Research and Education Center, Lake Alfred, FL USA Basic Elements of UF/CREC Citrus Plant Improvement Protocol Genomics Transgenic Plant Production (Core Lab) Sources of Genetic Variation 1. Seed/budwood introductions 2. Natural mutants 3. Somaclones 4. Sexual hybrids 5. Somatic hybrids/cybrids 6. Irradiated budlines 7. Molecular genetics Replicated field trials Horticultural evaluation Pests and diseases Regional sites Appropriate rootstocks Planting density Selection and Screening RELEASE (with germplasm protection as appropriate) 1

2 -Somatic Hybridization Scion Improvement - building high quality tetraploid breeding parents - triploid recovery from interploid crosses -Somatic Hybridization Rootstock Improvement - somatic hybridization of complementary parents - building a better sour orange fusion of selected mandarin + pummelo parents -Genetic Transformation - No need for Agrobacterium, - focus on disease resistance - potential for all-plant transformation system SWEET ORANGE EMBRYOGENIC CALLUS 2

3 SWEET ORANGE ORGANOGENESIS ADVENTITIOUS SHOOT BUD INDUCTION 3

4 SUCROSE-MANNITOL GRADIENT FOR PROTOPLAST PURIFICATION SWEET ORANGE SUSPENSION CULTURE PROTOPLASTS 4

5 LEAF-DERIVED CITRUS PROTOPLASTS TYPICAL SUSPENSION PROTOPLAST + LEAF PROTOPLAST PEG-INDUCED FUSION 5

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7 NEW SOMATIC HYBRID PLANT Ploidy Analysis Showing Diploid, Triploid and Tetraploid Peaks, Using a Partec Tabletop Flow Cytometer 7

8 Somaclonal Variation: variability in plants regenerated from tissue culture that is either induced or uncovered by a tissue culture process. Most somaclonal variation is negative, but if enough plants are examined, positive changes can usually be recovered. Sources of somaclones in citrus: organogenesis, somatic embryogenesis, protoplasts 8

9 Valenfresh Tm sweet orange, a seedless selection of Valencia for fresh market or processing, late maturing and holds quality into June! Valquarius Tm processing sweet orange, an early-maturing selection of Valencia that can be harvested mid-january through February, with typical Valencia quality. 9

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11 NOVA + SUCCARI SOMATIC HYBRID TREE Interploid hybridization using tetraploid somatic hybrids as pollen parents to produce seedless triploids for mandarin improvement: - more than 20,000 triploids produced to date, many fathered by somatic hybrids (under direction of FG Gmitter,CREC) - oldest hybrids are now fruiting and most are seedless!!!!! 11

12 LB8-9, Sugar Belle Tm : a New Diploid Interspecific Mandarin Hybrid Rohde Red Valencia + Dancy harvested January 28,2004 brix=11.4, acid=1.57, ratio= seeds/fruit 12

13 Nova + Osceola harvested December 6, 2005 brix= 14.8, acid=1.15, ratio= 12.9 Page + (Clementine x Satsuma) First zipperskin tetraploid with large fruit size and rich flavor! First triploids in the ground! 13

14 Page + Ortanique Tetraploid breeding parent for late maturity goal: seedless mandarins after Murcott. First triploids in the ground! 2011 Interploid Crosses Progenitors Progenies Seed Pollen 2x 3x 4x 6x #fd Ellendale x [Page + Ortanique] x [RhodeRed + Dancy] Fortune x [Page + Ortanique] x [Hamlin + Dancy] x [Succari + Murcott] x [Succari + Changsha] x [Page + (ClemxSats)] x [Murcott + LB8-8] x [RhodeRed + Dancy] Marisol Clementine x [Succari + Murcott] Nules Clementine x [Page + Ortanique] x [Succari + Murcott] x [Murcott + LB8-8] Monreal Clementine x [Succari + Murcott] x [Murcott + LB8-8] Lee x [Page + Ortanique] x [Hamlin + Dancy] Fallglo x [Page + Ortanique] x [Hamlin + Dancy] x [Succari + Murcott] x [Succari + Changsha] x [Page + (ClemxSats)] x [Murcott + LB8-8] SugarBelle x [RhodeRed + Dancy] x [Page + (ClemxSats)] Ambersweet x [Valencia + Page] x [Succari + Page] x [Valencia + Minneola]

15 Embryo Rescue to Recover Triploids from Interploid Crosses Mandarin C : a new and distinct early maturing seedless triploid tangerine for the fresh market. Proposed Name: Kid s Favorite 15

16 Interspecific Somatic Hybrid Parents Used For Grapefruit/Pummelo 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 2500 triploid plants produced to date Red pummelo B Murcott+Chandler #81 somatic hybrid (4x) Breeding for canker resistance: canker epidemic causing a natural screen of CREC germplasm leading to the identification of superior canker tolerant diploid and tetraploid breeding parents for use in interploid crosses to generate seedless triploids crosses underway! 16

17 Triploid hybrid fruit 2/3 pummelo, 1/3 sweet orange 15 ratio October 1 st! 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 17

18 C Ornamental potential? Now being grafted to dwarfing rootstocks to investigate this possibility. Todo el ano lemon x [Hamlin sweet orange+femminello lemon somatic hybrid] triploid lime - LATE 18

19 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 new production systems - 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 --NOW Tolerance to HLB (huanglongbing)! 19

20 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. 20

21 Projected Valencia fruit yield with trees planted at optimum spacing in rows with 20 foot middles (based on 2011 actual yield data). Somatic Hybrid In-row spacing Trees/Acre Boxes/Acre SO (ft) WGFT Changsha SO+Carrizo Changsha + Benton NEW STRATEGY: BREEDING SOMATIC HYBRID ROOTSTOCKS AT THE TETRAPLOID LEVEL CREATION OF TETRAZYGS -Use of allotetraploid somatic hybrid breeding parents allows the mixing of genes from 3-4 diploid rootstocks at once. - Progeny can be screened at the seed/seedling level for wide soil adaptability and Phytophthora resistance. - Products can have direct rootstock potential including adequate polylembryony, ability to control tree size due to polyploidy, and improved disease resistance. 21

22 Vigorous trifoliate hybrid growing in high ph-calcareous soil infested with P. nicotianae and P. palmivora SALINITY TOLERANCE Selection of progeny from crosses designed for salinity tolerance: healthy seedlings from Nova + HBpummelo with Cleo + rangpur 22

23 NEW PROBLEM: -New rootstocks must be able to tolerate mechanical damage inflicted by Diaprepes larvae (the sugar cane root weevil), and resistant to secondary infections of Phytophthora nicotianae and P. palmivora, and other invading fungi - Must be capable of vigorous root growth following Diaprepes damage. Amenability of Tetrazyg Rootstock Candidates to Traditional Seed Propagation Hybrid Seeds per Fruit Seed Type (based on SSR**) Orange 1 (Nova+HBPxCleo+APT) 15 Nucellar Orange 2 10 Nucellar Orange 3 4 Nucellar* Orange 4 23 Nucellar Orange 6 3 Zygotic Orange 8 18 Zygotic Orange Zygotic Orange Nucellar Orange Nucellar Orange 15 6 Zygotic Orange Nucellar* White 4 (Nova+HBP x Succ+APT) 20 Nucellar* Green 2 (Nova+HBP x SO + CZO) 23 Zygotic Green 6 20 Zygotic Green 7 21 Nucellar Purple 2 (Nova+HBP x Cleo+SO 20 Zygotic Purple 4 15 Zygotic Blue 1 (Nova+HBP x SO+PSL) 15 Nucellar Blue 2 12 Zygotic Blue 3 7 Nucellar Blue 4 26 Zygotic Blue 9 7 Zygotic 6058x S1 (salt-tolerant) 8 Nucellar* hybrid performed well in Diaprepes screen, **SSR = Simple sequence repeats (microsatellite) analyses performed by J. Gmitter and C. Chen.HBP = Hirado Buntan pummelo seedling; APT = Argentine trifoliate orange; SO = sour orange; Succ = Succari sweet orange; PSL = Palestine sweet lime 23

24 Tetrazyg nucellar liners prior to budding at commercial nursery 5 year old Valencia on Orange#14, between two rough lemon trees planted at the same time blight resets in Alligator grove (Mr. Lee) 24

25 St. Helena Project c/o Mr. Orie Lee 8/9/12 photo, trees 4.5 years old Rootstock effects on HLB emerging! Candidate for ACPS Valquarius on Orange #15 tetrazyg rootstock just < 5 years at St. Helena, Dundee FL 25

26 Initially Susceptible (2 dead) Initially Tolerant HLB screening of complex new rootstock candidates by grafting hot PCR+ HLB infected Valencia budsticks into each hybrid (after propagation of seed trees). Valencia trees growing out from the infected tissue with no symptoms will be checked by PCR, and if negative are passed through a hot psyllid house prior to field trial. Gauntlet survivor after 10 months in the field Valencia/tetrazyg [Nova+HBP] x [(sour orange+rangpur) x Cleo+Arg. trifoliate orange)] 26

27 Protoplast/GFP transformation Jellyfish (Aqueorea victoria) green fluorescent protein (GFP) is excited by UV or blue light and emits green fluorescence Common reporter gene to study gene function Detection of EGFP expression is noninvasive Gene expression can be directly observed using a microscope equipped for epifluorescent illumination 27

28 Plasmids for Co-Transformation and Direct transformation (Omar, et al. JHSB 2007) 28

29 Cellulase Macerozyme Protoplast ring 29

30 protoplasts 24 hours after transformation 4-6 weeks 6-8 weeks 8-9 months 3-4 months 5-6 months Transgenic One year 8-9 months 3-4 months (Omar, et al. JHSB 2007) 5-6 months non-transgenic RMAN 30

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32 Embryo-derived shoots (transformed) on DBA3 medium 4 months after transformation 32

33 GFP expression in the root (Omar, et al. JHSB 2007) 33

34 Newhall navel orange Early Gold sweet orange In vitro micro-grafting of transgenic shoots on Carrizo citrange rootstock. Left: Newhall navel orange Middle: Early Gold sweet orange Right: Hamlin sweet orange 34

35 Molecular characterization of the transgenic plants DNA RNA Proteins Replication Transcription Translation PCR analysis to detect the presence of the GFP (0.7 kb) and the cdna of the Xa21 (1.3 kb) genes in transgenic W. Murcott plants 35

36 Multiple PCR analysis to detect the presence of the GFP (0.8 kb) and the cdna of the Xa21 (1.4 kb) genes in transgenic citrus plants Southern blot analysis Lanes 1-8 transgenic plants and lane 9 non-transgenic plant. 36

37 Western blot analysis Soluble extracts of the transgenic rice expressing the wild-type Xa21 gene (lane 20), non-transgenic rice (lane 21), transgenic citrus expressing the GFP gene only (lane 1), non-transgenic citrus (lane 2), transgenic citrus positive for GFP and Xa21 genes (lanes 4-19) were subjected to Western blotting with reference to molecular mass markers (indicated in lane 1) Transformation vs. Co-transformation Transformation Cotransformation GFP % PCR positive for GFP % PCR positive for Xa % Western positive

38 Comparison of endoplasmic reticulum targeted and nontargeted cytoplasmic GFP as a selectable marker in Citrus protoplast transformation (Omar and Grosser, Plant Science 2007) 38

39 p524egfp.1 pars108 ER-targeted GFP Non-ER-targeted GFP (Omar and Grosser, Plant Science 2007) 39

40 Hey, I have an ER-targeted GFP, Would you like to see it? Expose me to blue light! You can compare us, too! Hey, I have a non-er-targeted GFP, Would you like to see it? Expose me to blue light! 40

41 pars108 p524egfp.1 (Omar and Grosser, Plant Science 2007) 41

42 pars108 p524egfp.1 (Omar and Grosser, Plant Science 2007) A B C D pars108 42

43 A B C D p524egfp.1 pars108 p524egfp.1 (Omar and Grosser, Plant Science 2007) 43

44 SPECIAL THANKS! To HALL OF FAME CITRUS GROWER-RESEARCHERS And Outstanding Collaborators: Mr. Orie Lee and the late Mr. Harold McTeer Funding: Mr. Orie Lee, Citrus Variety Improvement Grants from the Citrus Research and Development Foundation (CRDF) and the New Varieties Development and Management Corporation (NVDMC); the Citrus Research and Education Foundation (CREF), and USDA-CSREES. Thanks also to: Gary Barthe, J.L. Chandler, Zenaida Viloria, US Sugar/Southern Gardens Citrus (Skip McGuire and Mike Irey), Chuck Dunning, Mauricio Rubio, Ralph Chandler Story, Julie Gmitter, Chunxian Chen, Xiuli Shen, many others, and especially Troy Gainey and the CREC Grove Crew and many others! 44