This document discusses somatic hybridization techniques for crop improvement using potato and its wild relatives as an example. Somatic hybridization involves fusing plant protoplasts from two different species to create a hybrid plant. The author has used this technique to introduce disease resistances from wild potato species into cultivated potato. Specifically, late blight resistance was captured from Solanum bulbocastanum and introgressed into potato breeding lines. Somatic hybridization also allows for the combination of traits from different species and the creation of novel germplasm. The author discusses applications to citrus improvement as well, including creating seedless varieties and improving rootstocks.
2. John P. Helgeson
USDA/ARS, Dept of Plant Pathology
University of Wisconsin - Madison
Protoplast Preparation and Somatic Hybrids
3. • In many cases the somatic hybrids can be
crossed back to one or the other of the two
parents.
• In that way the DNA of a wild species can
be incorporated into a breeding line of a
crop species.
• Our work has been done primarily with
potato and its wild relatives.
Somatic Hybridization
18. • The shoots can be excised from
the calli and rooted on nutrient
media in a test tube. This will
result in a whole new potato
plant.
• The new potato plant can be
grown in a test tube
Somatic Hybridization
19. A form of DNA
fingerprinting
called RFLP
analysis can be
used to
demonstrate
that one really
does have a
somatic hybrid.
Note that the somatic hybrids have the DNA
bands from S. bulbocastanum and potato
PI203900, the parents of the hybrids.
20. • Characteristics of both of the
combined species can be expressed
in the somatic hybrids.
• Note the purple stems vs the green
stems and compound vs simple
leaves stems in the following slide
Somatic Hybridization
23. We have been using this procedure
to capture disease resistances
from wild potato species that can’t
be crossed with the domestic
potato
Somatic Hybridization
24. Somatic hybrids obtained in the Helgeson Lab
S. brevidens PL 218228 (2x) + S. tuberosum
(4x)
S. brevidens PL 218228 (2x) + Russet Burbank
(4x)
S. brevidens PL 218228 (2x) + S. tuberosum
(2x)
S. bulbocastanumPL 245763 (2x) + S. tuberosum (4x)
S. bulbocastanumPL 245310 (2x) + S. tuberosum (4x)
S. cardiophyllum PL 279272 (2x) + S. brevidens
(2x)
S. commersonii PL 320266 (2x) + S. tuberosum
(4x)
S. commersonii PL 320266 (2x) + S. tuberosum
(2x)
S. etuberosum PL 245939 (2x) + S. tuberosum
25. Some new disease resistances from somatic hybrids
Disease/pathogen Somatic
hybrid
Late blight
Early blight
M. Chitwoodii
Erwinia Soft
rot
Bacterial wilt
PVY
PLRV
S. bulbocastanum + S. tuberosum
S. bulbocastanum + S. tuberosum
S. bulbocastanum + S. tuberosum
S. brevidens + S. tuberosum
S. commersonii + S. tuberosum
S. etuberosum + S. tuberosum
S. brevidens + S. tuberosum
26. We have been able to capture late
blight resistance from the wild
potato species, Solanum
bulbocastanum and, using sexual
crosses of the somatic hybrid we
have developed a number of
highly resistant potato breeding
lines.
Somatic Hybridization
29. View of test field - Hancock Research
Station. The green plants are those
with the new resistance gene
30. Backcross 1
line (J101- K27)
in field at
Hancock, WI
The field
received no
fungicide and
the dead plants
are from the
cultivar Russet
Burbank, killed
by late blight.
31. BC1 line J138A12 in Toluca, Mexico.
The arrow points to what is left of a
plant of the susceptible cultivar
“Alpha.”
The resistance to late
blight is effective in
Toluca Mexico, where
the variation in the late
blight organisms is the
greatest in the world.
32. By somatic hybridizations such as
those of potato and S.
bulbocastanum we hope to
introduce new genes into potato so
that pesticide use can be
decreased substantially.
For more information contact John Helgeson,
JPH@plantpath.wisc.edu
Protoplast Preparation and Somatic Hybrids
33. SOMATIC HYBRIDIZATION
Involves fusion of two distantly related,
to closely related plant protoplasts at
intraspecific, interspecific, intergeneric,
and interfamily levels, with subsequent
regeneration of hybrid cells into hybrid
plants.
34. SOMATIC HYBRIDIZATION BASICS
1. In vitro culture
2. Protoplast isolation and purification
3. Protoplast fusion
4. Growth and selection of somatic
hybrid tissues
5. Confirmation of hybridity
6. Exploitation of S.H.
35. 2. PROTOPLAST ISOLATION
Protoplast:
Bacterial, fungal or plant cell deprived of its rigid
wall but with its plasma membrane intact
Procedure for protoplast isolation
1. Mechanical production
(Klercker, 1892)
2. Enzymatic digestion
(EC Cocking, 1960)
39. ELECTROFUSION
A two-stage process:
1. A non-uniform AC field stimulates close
protoplast plasmamembrane contact
1. Short pulse of DC stimulates breakdown
of closely aligned plasma membranes
producing pores through which cytoplasm
of adjacent ppts. flows and causes fusion
42. 4. SELECTION OF SOMATIC HYBRIDS
1. Manual selection
Physical isolation of the hybrid (using physical
differentiation like pigmentation orregenerability)
2. Dual fluorescent labeling system
(FDA labelled green florescing protoplasts fused
with RIT labelled protoplasts emitting a red
fluorescence
3. Complementation
Procedures inhibiting growth of homokaryons
4. FACS
physical sorting of heterokaryons
44. BENEFITS OF SOMATIC HYBRIDIZATION
SHs are not GMOs
Elimination of crossing barriers
Specific addition of the genomes of two plants
Unique combinations of nuclear and organellar
genomes generate novel germplasm
No strict maternal inheritance of organelles
45. LIMITATIONS OF SOMATIC HYBRIDIZATION
a) Somatic hybridization does not always produce
plants that give fertile and viable seeds.
b) Tedious selection procedures
c) After confirmation, hybrid material is field
evaluated to check stable integration of
desired character
d) Protoclonal variations, chromosomal
elimination, organelle segregation etc. all lead
to variable somatic hybrids
46. Applications of Somatic Hybridization and
Cybridization in Scion and Rootstock
Improvement with Focus on Citrus
47. CITRUS CULTIVAR IMPROVEMENT
Ineffective results of conventional breeding
owing to:
High heterozygosity
Pollen and ovule sterility
Sexual incompatibility
Nucellar polyembryony
Juvenility
48.
49. Citrus Scion improvement objectives:
Seedlessness
Fruit quality traits
Root Stocks:
Better adaptability to biotic/
abiotic stresses
Tree size control
50. Scion Improvement
Triploid production:
1.By crossing 4X SH with 2X parent
creation of allotetraploid SH (2X + 2X= 4X)
4X SH are then used in interploidal
crosses as breeding parents (4X x 2X) = 3X
2.By fusion of diploid and haploid parents
creation of triploid SH (2X + 1X)
3. Cybridization
creation of cybrids to transfer Satsuma CMS
cytoplasm to seedy cultivars
51. Rootstock Improvement
- somatic hybridization of complementary parents
building a better sour orange
-Production of Tetrazygs
– fusion of selected mandarin + pummelo parents
68. Interploid hybridization using tetraploid somatic
hybrids as pollen parents to produce seedless
triploids for mandarin improvement:
- more than 12000 triploids using pollen from
somatic hybrids
(under direction of FG Gmitter,CREC)
- oldest hybrids are now fruiting and most are
seedless!!!!!
69. NOVA + SUCCARI SOMATIC HYBRID FRUIT
(father of several hundred triploid progeny)
70. VALENCIA + (ROBINSON X TEMPLE)
HARVESTED JANUARY 22,2004
BRIX=11.4, ACID=0.57, RATIO=20
3 SEEDS/FRUIT
71. ROHDE RED VALENCIA + DANCY
HARVESTED JANUARY 28,2004
BRIX=11.4, ACID=1.57, RATIO= 7.26
3 SEEDS/FRUIT
78. 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)
83. WHAT DO WE NEED?
- Objectives
- 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
86. 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.
87. Selection of progeny from crosses designed for salinity tolerance:
healthy seedlings from Nova + HBpummelo with Cleo + rangpur
88. Selection of progeny from crosses designed for salinity tolerance: healthy
seedlings from Nova + HBpummelo with Cleo + rangpur
Salinity Tolerance
92. 5 year old Valencia on Orange#14, between two rough lemon trees
planted at the same time – blight resets in Alligator grove (Mr. Lee)
93. HLB tolerance from new rootstocks?
Vernia/Orange #4 Vernia/Orange #19 New
photos
New photos of trees PCR+ since last September – St.
Helena
94. Rootstock improvements regarding HLB are like likely
to come in stages:
First stage: Rootstocks that reduce the frequency of
HLB infection, and reduce the severity of the disease
once infected – these will still require efficient psyllid
control and optimized production systems.
Second stage: Potential rootstock mitigation of the
disease – research is underway to possibly identify
rootstocks that can protect the entire tree –
regardless of the scion. Psyllid control may not be
necessary. No horticultural performance data would
be available on such selections initially, but the
hybrids would have good rootstock pedigree.
95. Concluding Remarks:
1.Tolerance/resistance to canker and HLB is being discovered in commercial quality
CREC scion and rootstock germplasm – making these epidemics a mixed blessing for
our breeding program. 2. Successful rootstocks should be tolerant/resistant to all
known vascular diseases of citrus including CTV-quick decline and blight (is there a
blight/HLB synergy, the double whammy??). Commercial rootstocks are lacking in
this regard – especially Swingle and Carrizo!
3.Management programs should minimize any stress on the trees, including
nutritional or water stresses; and provide constant nutrition. Don’t forget about
ground fertilizer applications!
4.New rootstock candidates are showing a lower frequency of HLB infection, and
reduced severity of disease symptoms once trees become infected. Tolerant
rootstock types being identified include complex tetrazygs, citranges, citrandarins,
and sour orange types.
5.Complex new rootstocks in combination with the right tree care and good psyllid
control have potential to prevent and/or mitigate HLB. Imagine the potential of
ACPS when combined with the use of HLB-tolerant, tree-size controlling precocious
rootstocks!
6.Differences observed in HLB responses among unselected new rootstock
candidates suggest great potential for the selection of rootstock genotypes that
can completely protect trees – a solution for all commercial scions. Stay tuned for
the second wave of new rootstocks! THERE IS HOPE!
96. CITRUS BLIGHT
-still a major killer of trees in Florida
-if you solve the sour orange problem, you
solve blight!
-trifoliate orange hybrids susceptible –
strategy is to dilute trifoliate contribution
while retaining disease resistance and fruit
quality attributes
-field screening of new hybrids by resetting
in blight holes– Primary cooperator is Mr.
Orie Lee, Secondary cooperator David and
Sharon Garrett.
-build in tree size control along with the
CTV resistance
NEW STRATEGY FOR BUILDING A
BETTER SOUR ORANGE
-Somatic Hybridization of Superior
Pummelo + Mandarin parents. - All
pummelos are not created equal. We have
developed an effective greenhouse screen
for selecting zygotic pummelo seedlings
adapted to high pH, calcareous soils, and
that are resistant to both Phytophthora
nicotianae and P. palmivora. A
trememdous amount of genetic
diversity can be quickly screened.
Superior individuals will serve as fusion
partners with selected mandarins
including widely adapted Shekwasha
and C. amblycarpa. Resulting somatic
hybrids should have tremendous
rootstock potential.
99. 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
100. Overview of somatic hybrid seedlings at Reed Brothers
Nursery - thanks to Chuck Reed and Mike Baker
101. 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.
102. 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
108. 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
109. 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.
115. 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!
116. 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
120. 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.
122. 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
131. A B
A: Leaf and stem explants of S. chacoense
B: Shoot regeneration from S.
chacoense leaves
C D
C: Regeneration from leaf and stem
explants of Desiree
D: Regeneration from leaf
Explant of Desiree
133. Culture of Protoplasts
IPE FPE
Plating density
Culture methods
Liquid culture Agarose droplets
5x104, 1x105, 1.5x105 ,2x105
Plating efficiency
134. G H
I J
G: Dividing protoplast of Desiree H: Agarose droplet culture of
Desiree protoplasts
I: Protoplast calli on proliferation
medium
J: Regenerating protoplast calli of
Desiree
135. Somatic Hybridisation
1:1 mix. of Desiree and S. chacoense protoplasts
Electrofusion
Incubation (30 min.) and centrifugation of fused protoplasts
Droplet culture in MS (modified) medium (4-6 weeks)
Transfer microcolonies to CG/LSR1 medium (6-8 weeks)
Transfer heterotic calli to LSR2 medium (6-8 weeks)
Rooting of shoots and transfer to glasshouse
136. K
L M
K: Fusion of Desiree and S. chacoense protoplasts
L: Somatic hybrid callus M: Regeneration from hybrid callus
137. N
N: Plants of S. chacoense (L), Desiree (R), and somatic hybrids (centre)
138. P
O
O: Flower of S. chacoense
P: Flower of Somatic hybrid
Q
Q: Flower of Desiree
139. S
T
U
U: Tubers of Desiree
T: Tubers of Somatic hybrid
S: Tubers of S. chacoense
140. R
R: Leaves of S. chacoense (L), Desiree (R) and Somatic hybrid (Centre)
R1: 2n=6x=72 chromosomes of a somatic hybrid
R1
141. V
V: RAPD profiles generated by primer OPA-2. Lane 1: potato cv. Desiree, Lane 2: S.
chacoense, Lane 3: 1:1 mix. of parental DNA, Lanes 4-21: somatic hybrids.
Molecular marker = 100 bp.
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
142. SOMATIC HYBRIDIZATION
Somatic hybridization involves fusion of two
distantly related, to closely related plant
protoplasts at intraspecific, interspecific,
intergeneric, and interfamily levels, with sub
sequent regeneration of hybrid cells into
hybrid plants.
143. SOMATIC HYBRIDIZATION
BASICS
1. In vitro culture
2. Protoplast isolation and purification
3. Protoplast fusion
4. Growth and selection of somatic hybrid
tissues
5. Confirmation of hybridity
6. Exploitation of S.H.
144. 1. PROTOPLAST ISOLATION
Source material
Procedure for protoplast isolation
1. Mechanical production
2. Enzymatic digestion
145. PROTOPLAST FUSION AND REGENERATION OF
SOMATIC HYBRIDS
Protoplast fusion must occur before new walls of
plant ppts
New walls begin to regenerate within 2-5 minutes of
isolation
Detect with UV microscopy and use of Calcafluor
stain
Mitosis occurs about 2-7 days after isolation
Colonies seen in 1-3 weeks
146.
147. METHODS OF PPT. FUSION
Spontaneous
Fuse during isolation (callus ppt)
May contain 2-40 nuclei
Thought to be due to coalescence of plasmosdesmatal
connections between cells
Prevented by strong plasmolysis
Mechanical
protoplasts are brought into intimate physical contact mechanically under
microscope using micromanipulator and perfusion micropipette. This
micropipette is partially blocked within 1 mm of the tip by a sealed
glass rod. In this way the protoplasts are retained and compressed by
the flow of liquid. By this technique occasional fusion of protoplast has
been
Induced
Sodium nitrate (NaNo3)
Artificial sea water
Lysozyme
High pH/Ca++
Polyethylene glycol (PEG)
148. INDUCED FUSION
Sodium nitrate (Power et al., 1970)
Transfer 1:1 mix of protoplasts to 0.25M NaNO3, incubate for 35 min. and
then centrifuge (200 xg), ppt pellet kept at 300 C for 30 min for fusion,fusion
mix replaced with 0.1% NaNO3 for 15 min. and washed.
Low rate of heterokaryons (leaf ppt.)
Not in use
High pH/Ca
++
(Kelier and Melchers, 1973)
A calcium solution buffered at high pH induces aggregation of the
protoplasts and their fusion. (Solution of 0.4M mannitol containing 0.05 M CaCl2 with
pH at 10.5, 370 C)
Ca
++
reduces the potential of the surface negative charge on protoplasts,
facilitating protoplast adhesion. (-ve charge due to intramembranous phosphate
groups)
The high alkalinity (pH 9.5 - 10.4) induces the formation of
intramembranous lysophospholipids such as lysolecithin and
lysophosphatidyl -ethanolamine that increase membrane fluidity and fusion
149.
150. INDUCED FUSION(CONT.)
PEG (polyethylene glycol) – MWt. 200-20,000
Effective: 6000-8000 MW
Reproducible high production of heterokaryons
Add 0.6 ml of PEG solution to protoplast pellet
Incubate at room temperatuer for 40 minutes with
occasional rocking
After fusion, add 0.5 - 1.0 ml culture medium and
centrifuge
Resuspend protoplasts in culture medium
Disturbs the charge on the membrane
151. PEG-INDUCED FUSION
A highly water-soluble, non-ionic weak surfactant
A strong affinity of PEG for water causes local membrane dehydration and increased
fluidity
PEG itself induces aggregation, but α-tocopherol present as an impurity in
commercial grade PEG, actually promotes membrane fusion
When the chain of PEG polymer molecule is long enough (m.wt ≥ 1000) it may act as
a mol. bridge between the surfaces of adjacent protoplasts , allow agglutination to
occur
PEG of molecular wt 2000-6000 is active in fusion and PEG 200 and 20,000 inactive
Other compounds structurally-related to PEG namely PVP (Polyvinyl pyrrolidone),
PVA (Polyvinyl alcohol) and Polyglycerol are also known to induce fusion
PEG can be used with other fusogens to enhance fusion frequency like
Pre-incubation of protoplasts with lysozymes
Combination of PEG with high pH and Ca++
Addition of DMSO
Factors influencing the efficiency of PEG-induced fusion are:
Concentration, MWT. and purity of PEG
pH, Osmolarity
Method of application of solutions in fusion and their elution
Physiological status of protoplasts
Density of protoplasts in fusion solution
Limitations
Impurities in the commercial preparations of PEG cause cyto-toxicity or reduced
viability of fusion products
Cause deformation of mitochondria
152.
153. ELECTROFUSION
A two-stage process:
1.A non-uniform AC field stimulates close
protoplast plasmamembrane contact
2.Short pulse of DC stimulates breakdown of
closely aligned plasma membranes
producing pores through which cytoplasm of
adjacent ppts. flows and causes fusion
154.
155.
156. FACTORS AFFECTING ELECTROFUSION
voltage, frequency and duration of AC and DC
Shorter DC pulses (10-50 µs) at higher voltage (1000-2000 V/cm)
are preferable leading to higher viability and PE of ppts.
Higher voltage of DC pulse facilitates overall fusion but t the cost of
binary fusions and viability of fusion products
Ppt density, size and cell memebrane properties
Mesophyll ppts tend to fuse readily than suspensioo-derived ones
Large ppts tend to fuse more readily than smaller ones
Calcium ions (micromolar concen.) are required for
better alignment of protoplasts
Biochemical means have been tested to improve fusion
frequency
(Avidin-biotin high affinity for Higher heterokaryon formation
Exogenous protease (Dispase, Pronase E) to improve fusion freq. of
mammalian cells
160. MAINTENANCE OF PROTOPLAST CULTURES
Maintain the protoplast cultures initially for 10-15 days
under dark at 25-30°C
Keep at low continuous illumination first at 1,000 lux and
gradually increase to 3,000 lux.
Maintain relative humidity in containers during this period
to avoid desiccation.
161. 3. SELECTION OF SOMATIC HYBRIDS
1. Manual selection
Physical isolation of the hybrid (using physical
differentiation like pigmentation or regenerability)
2. Dual fluorescent labeling system
(FDA labelled green florescing protoplasts fused with RIT
labelled protoplasts emitting a red fluorescence
3. Complementation
Procedures inhibiting growth of homokaryons
4, FACS
physical sorting of heterokaryons
164. PLOIDY ESTIMATION BY STOMATA OBSERVATION
1. COLLECT A MATURE LEAF
2. PEEL OFF THE SURFACE CELL LAYER FROM UNDERSURFACE OF THE LEAF USING A
PAIR OF TWEEZERS
3. PLACE THE CELL LAYER ON A SLIDE GLASS, AND STAIN WITH A DROP OF 2%
SILVER NITRATE SOLUTION
4. AFTER STAINING FOR 1 MINUTE, COVER WITH A COVER SLIP
5. COUNT THE NUMBER OF CHLOROPLAST GRAINS IN A GUARD CELL OF A STOMA
UNDER A MICROSCOPE
NOTE: APPROXIMATE NUMBER OF CHLOROPLAST GRAINS IN ONE GUARD CELL IS,
2X ··· 2-4
3X ··· 5-6
4X ··· 8-12
(SLIGHTLY VARIED DEPENDING ON SPECIES
AND CULTIVARS)
165. CHROMOSOME NUMBER IN SOMATIC HYBRIDS
The chromosome number in the somatic hybrids is
generally varied compared to both of the parental
protoplasts.
If the chromosome number in the hybrid is the sum of
the chromosomes of the two parental protoplasts, the
hybrid is said to be symmetric hybrid.
Asymmetric hybrids have abnormal or wide variations
in the chromosome number than the exact total of two
species.
In 1972, Carlson and his associates produced the first
inter-specific somatic hybrid between Nicotiana glauca
and N. langsdorffii.
In 1978, Melchers and his co-workers developed the
first inter-generic somatic hybrids between Solanum
tuberosum (potato) and Lycopersicon esculentum
(tomato). The hybrids are known as ‘Pomatoes or
Topatoes’.
166. BENEFITS OF
SOMATIC HYBRIDIZATION
Elimination of crossing barriers
No loss of genetic information during the
formation of gametes
Specific addition of the genomes of two plants
Combination of complex traits without loosing
any gene
Unique combinations of nuclear and organellar
genomes generate novel germplasm
No strict maternal inheritance of organelles
167. Somatic hybrids developed in crop plants belonging to
following major families
Brassicaceae, Fabaceae, Poaceae, Solanaceae, Rutaceae
· Interspecific hybrids have been obtained in Nicotiana,
Brinjal, Potato, Tomato,Brassica Medicago, Soyabean,
Sugarcane, Datura, Petunia, Sorghum etc.
· Intergeneric hybrids are obtained between Tomato x Potato,
Tomato x Tobacco, Festuca x Lolium, sugarcane x Bajra,
Wheat x Bajra, Brasica x Eruca etc.
· Inter tribal hybrids have been developed between Brassica
x Arabdopsis and Brassica x lesquerella Fendleri
168. LIMITATIONS OF SOMATIC HYBRIDIZATION
a) Somatic hybridization does not always produce plants that
give fertile and viable seeds.
b) There is genetic instability associated with protoplast
culture.
c) Tidious selection procedures
e) It is not certain that a specific character will get expressed
in somatic hybridization.
f) Regenerated plants obtained from somatic hybridization
are often variable due to protoclonal variations, chromosomal
elimination, organelle segregation etc.
g) Protoplast fusion between different species/genus is easy,
but the production of viable somatic hybrids is not always
possible.
169. CYBRIDS
The cytoplasmic hybrids where the nucleus is derived from
only one parent and the cytoplasm is derived from both the
parents are referred to as cybrids.
The process of formation of cybrids is called cybridization.
During the process of cybridization and heterokaryon
formation, the nuclei are stimulated to segregate so that one
protoplast contributes to the cytoplasm while the other
contributes nucleus alone.
The irradiation with gamma rays and X-rays and use of
metabolic inhibitors makes the protoplasts inactive and non-
dividing.
Some of the genetic traits in certain plants are cytoplasmically
controlled. This includes certain types of male sterility,
resistance to certain antibiotics and herbicides. Therefore
cybrids are important for the transfer of cytoplasmic male
sterility (CMS), antibiotic and herbicide resistance in
agriculturally useful plants.
Cybrids of Brassica raphanus that contain nucleus of B.
napus, chloroplasts of atrazinc resistant B. compestris and
male sterility from Raphanus sativas have been developed.
170. Status of mt. and ch genomes in somatic hybrids
The somatic hybrids have novel traits due to recombination in mitochondrial
genomes. While, Chloroplast genome of either one of the parents stays and
the other gets eliminated with the mitotic division in fusion products.
The chloroplasts and mitochondrial genomes of parental protoplasts behave
differently in the fusion products, that may be due to the structural
organization of theses organelles and their genomes. Mitochondrial
genomes are able to disperse and fuse in fusion products. While fusion of
chloroplast genomes is rare in higher plants.
Ch Genome possess two inverted repeats. Intramolecular homologous
recombination between these regions leads to the inversion of the unique
sequences inserted between them.
In contrast, the mitochondrial genome possesses at least two direct repeats,
which normally can recombine to generate two or more separate smaller
molecules containing intact the sequences of the parent molecule. This
model explains the apparent heterogeneity of the mitochondrial genome.
Recombination is the rule governing plant mitochondrial DNA organization.
171. Damage occurs when the free radical
encounters another molecule and seeks to
find another electron to pair its unpaired
electron. The free radical often pulls an
electron off a neighboring molecule,
causing the affected molecule to become a
free radical itself. The new free radical can
then pull an electron off the next molecule,
and a chemical chain reaction of radical
production occurs.
An antioxidant is a molecule that inhibits the oxidation of other
molecules. Oxidation is a chemical reaction that transfers electrons
or hydrogen from a substance to an oxidizing agent. Oxidation
reactions can produce free radicals. In turn, these radicals can start
chain reactions. When the chain reaction occurs in a cell, it can
cause damage or death to the cell. Antioxidants terminate these
chain reactions by removing free radical intermediates, and inhibit
other oxidation reactions. They do this by being oxidized
themselves, so antioxidants are often reducing agents such as
thiols, ascorbic acid, or polyphenols
172. When two or more protoplasts fuse to form a new cell, the parent nuclei may remain
separate or fuse to form a somatic hybrid. If one of the nuclei is lost after fusion the
cytoplasms of the two parent protoplasts will still coalesce and form what is known as
a cytoplasmic hybrid or cybrid. These contain nucleus of one parent ppt but a mixture
of the cytoplasic organelles of both parents. The production of cybrids is an important
tech. in breeding programmes where CMS is desirable, because the CMS trait is
inheritted through mitochondria. Whereas, the heterokaryons is what we need in s.h
experiments, which contains a mixture of cytoplasm and nuclei of both the parent
ppts.
PPTs are aligned in an AC field which is inhomogeneous and of low voltage and
high frequency. The AC causes dielectrophoresis of ppts by inducing a dipole in each
ppt. and masking the net surface charge distribution on membranes.The ppts
undergoing dielectrophoresis are mutually attracted and consequently bind together to
form chains parallel to the field direction.
The electrofusion is composed of a non-electrolyte (usually mannitol) to ensure
appropriate osmolarity and at the same time enable the dielectrophoretic alignment to
occur. Calcium ions at greater than micromolar concentration are required for better
alignment of protoplasts.
The aligned ppts. Are fused by applying a direct current pulse. DC pulse is generally
of high voltage and short duration, which causes a reversable breakdown at the
contact zone of two membranes. Such membrane breakdown occurs at several points
in the contact zone, simultaneously leading to fusion of cell membranes and
subsequent fusion of cell contentsOptimum electrofusion should produce a large
number of viable and binary heterokaryons. Parameters such as voltage, frequency
and duration of AC and DC, ppt density and cell memebrane properties can all affect
the alignment and fusion of protoplasts as well as the viability of fusion products.
173. For practical breeding purpose, it is necessary to either select hybrid cells immediately
after fusion or inhibit non-hybrid cells with pre- and/ or post-fusion treatments. There
are a no. of factors to be considered:1. There are more no.of hybrid cells among
fusion products, the non-hybrid cells include homokaryons as well as non-fused
parental protoplasts. Unless culture conditions made favorable for the fusant cells,
they will generally be over-grown by the more numerous non-hybrid cells, though
there are some exceptions of hybrid vigor. 2. Cell division from protoplasts requires
high culture density (104 to 106/ml). But the density of hybrid cells in a fusion popn. Is
generally v. low and they are often surrounded by the toxic dead cell debris.3.
Selection at the cell level is far more economical than at plant level.
1. Physical isolation of heterokaryon selection is feasible only if the two ppt popn differe
cosiderably in physical traits. Such as fusion of green leaf ppts with colorless cell
suspension ppts.such heterokaryons can be isolated using a micromanipulator or
hand-helled micropippeter, but being too tedious, such application is not popular
2. Dual fluorescent labeling system
(FDA labelled green florescing protoplasts fused with RIT labelled protoplasts
emitting a red fluorescence
2. Physical separation is automated with Fluorescence-activated cell sorte)Once the
labelled ppts are labelled with different fluorochromes (Carboxyfluorescein and
scopoletin) the FACS is capable of selecting fusant cells that display dual
fluorescence. Major drawback with this tech. is high cost.
2. Genetic or phyiological complementations are highly effective, but the mutants
required are not always available. Metabolic complementation relies on irreversible
inhibitors rather than mutants and is more versatile. The parental ppts are treated
with 2 diff. metabolic inhibitors(one with iodoacetate; the other with Rhodamine 6-
G)Following fusion, metabolic complementation allows hybrids to survive, while non-
fused ppts are died of the irreversible inhibition.
174. EVER SINCE THE FIRST REPORT ON PROTOPLAST FUSION DERIVED SOMATIC HYBRID
PLANTS OF NICOTIANA GUAUCA +- N. LANGSDORFFII BY CARLSON ET AL. (1972),
SOMATIC HYBRIDIZATION HAS OPENED UP SEVERAL POSSIBILITIES FOR THE
PARASEXUAL MANIPULATION OF PLANTS.
175. intraspecific ( n tr -sp -s f k) also intraspecies ( n tr -sp sh z, -s z)
Arising or occurring within a species or between members of the same species.
Interspecific competition, in ecology, is a form of competition in which individuals of
different species vie for the same resource in an ecosystem (e.g. food or living
space). The other form of competition is intraspecific competition, which involves
organisms of the same species. electrolyte /elec·tro·lyte/ (e-lek´tro-lit) a substance
that dissociates into ions when fused or in solution, thus becoming capable of
conducting electricity electrolyte
a chemical substance which, when dissolved in water or melted, dissociates into
electrically charged particles (ions), and thus is capable of conducting an electric
current. The principal positively charged ions in the body fluids (cations) are sodium
(Na+), potassium (K+), calcium (Ca2+), and magnesium (Mg2+). The most important
negatively charged ions (anions) are chloride (Cl-), bicarbonate (HCO3-), and
phosphate (PO43-). These electrolytes are involved in metabolic activities and are
essential to the normal function of all cells. Concentration gradients of sodium and
potassium across the cell membrane produce the membrane potential and provide
the means by which electrochemical impulses are transmitted in nerve and muscle
fibers.
The concentration of the various electrolytes in body fluids is maintained within a
narrow range. However, the optimal concentrations differ in the extracellular fluid and
intracellular fluid. An electrolyte imbalance exists when the serum concentration of an
electrolyte is either too high or too low.
Stability of the electrolyte balance depends on adequate intake of water and the
electrolytes, and on homeostatic mechanisms within the body that regulate the
absorption, distribution and excretion of water and its dissolved particles.
The effects of an electrolyte imbalance are not isolated to a particular organ or
system. In general, however, imbalances in calcium concentrations affect the bones,
kidney and gastrointestinal tract. Calcium also influences the permeability of cell
membranes and thereby regulates neuromuscular activity. sodium affects the
osmolality of blood and therefore influences blood volume and pressure and the
retention or loss of interstitial fluid. potassium affects muscular activities, notably
those of the heart, intestines and respiratory tract, and also affects neural stimulation
of the skeletal muscles.
