Applications of Somatic Hybridization and Cybridization in Scion and Rootstock Improvement with Focu

1
Applications of Somatic Hybridization and
Cybridization in Scion and Rootstock Improvement
with Focus on Citrus Jude Grosser and Milica
Calovic 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


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
11
(No Transcript)
12
(No Transcript)
13
Ploidy Analysis Showing Diploid and Tetraploid
Peaks Using a Partec Tabletop Flow
Cytometer
14

NEW SOMATIC HYBRID PLANT

15


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
gt200 Total
gt250
18
(No Transcript)
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
• 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

21

• Interploid hybridization using tetraploid somatic
• hybrids as pollen parents to produce seedless
• triploids for mandarin improvement
• more than 12000 triploids produced to datemany
  fathered by somatic hybrids
• (under direction of FG GmitterCREC)
• 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
222004brix11.4 acid0.57 ratio203
seeds/fruit
24
Rohde Red Valencia Dancyharvested January
282004brix11.4 acid1.57 ratio 7.263
seeds/fruit
25
Nova Osceolaharvested December 6 2005brix
14.8 acid1.15 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.)
28
(No Transcript)
29
MID-TERM SCION CANDIDATES (5-10 years)
Crec-9505 triploid mandarin hybrid

• gt 12000 triploid mandarin hybrids (FG Gmitter
  Jr.)
• Focus seedless fresh market
• CREC 9505 proof of concept 0 seed!
• many beginning to fruit!

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
FG307 x
x x 2x W.
Murcott SF Lee Hybrid x
x x 2x
4x W. Murcott FG306
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
• 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)
• Guoqing Satsuma Hirado Buntan pink pummelo
• Guoqing Satsuma Sunburst mandarin
• Guoqing Satsuma (Clementine x Murcott) Lee
  hybrid
• 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 14100 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 FG307
x x
x 2x G1 Satsuma
Murcott x x
x 2x G1 Satsuma
ScabFree Lee H. x x
x 2x 4x G1 Satsuma
FG303 x
x x 2x G1 Satsuma
FG304 x
x x 2x 4x
 
46
(No Transcript)
47

• 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

48
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

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 ChandlerCREC)-
• 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
51
(No Transcript)
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.
54
(No Transcript)
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 WGFT50-7

63 Cleo Carrizo
61 Cleo
Argentine trifoliate orange
58 Nova Hirado Buntan pummelo zyg.
56 Cleo rangpur

54 SO50-7
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 DPI 8-2 Pummelo NW (Florida)
12 DPI 7-1 Chinese Pummelo
(China) 4 DPI 6-1 Kao
Phuang (Thailand) 0
gt200 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 Shekwa
sha 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
Gel -2
1 2 3 4 5 6 7
8 9 10 11
1 2 3 4 5 6 7 8
9 10 11 12
Primer c-11
Primer c-11
Primer c-64
Gel -3
Primer c-64
Gel -4
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 SOCarrizo 13
Nucellar

Data Julie Gmitter and Chunxian Chen
72
SPECIAL THANKS!

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
VarietiesCSREES/USDA Special Research Grants
Program Citrus Tristeza Virus USDA/IFAS
Cooperative Agreements Research on Citrus
tristeza virus and other exotic diseases/
Diaprepes.

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!