6. 5/28/2012
HISTORY
Klercker isolated protoplast in 1892 by cutting plasmolaised
cells of tobacco leaf.
1960 Cocking used cellulase to isolate protoplas from fungi
1960 Power and Cocking isolated protoplast by single step
method.
1968 Takebe et al. used pectinase and cellulase to isolate
protoplast from tobacco by two step method.
1972 Power and Cocking fused the protoplast of 2 Nicotiana
sp and regenerated somatic hybrid. 6
7. 5/28/2012
PROPERTIES OF PROTOPLAST
Lack cell wall and have plasma membrane
Semi permeable and selectively permeable.
Spherical in shape
Sensitive to osmotic presser
Phagocytises and phenocytises
Fusion property
Regenerate the cell wall (totipotent) 7
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SOMATIC HYBRIDIZATION TECHNIQUE
1. Isolation & purification of protoplast
2. Fusion of the protoplasts of desired species
3. Identification and Selection of hybrid protoplasts
4. Culture of the hybrid protoplasts
5. Regeneration & characterization of hybrid plants
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11. 5/28/2012
1. MECHANICAL METHOD
Plant Tissue Cells Plasmolysis
Microscope Observation of cells
Cutting cell with knife Release of protoplasm
Collection of protoplasm
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CONTED.,
Used for vacuolated cells like onion bulb
scale, radish and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability
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2. ENZYMATIC METHOD
Leaf sterlization, removal of
epidermis
Plasmolysed Plasmolysed
cells cells
Pectinase
Pectinase +cellulase
Release of Protoplasm
isolated cells released
Protoplasm released
cellaulase
Isolated
Protoplasm 13
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CONTED.,
Used for variety of tissues and organs
Mesophyll tissue - most suitable source
High yield of protoplast
Easy to perform
More protoplast viability
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SPONTANEOUS FUSION
Protoplast fuse spontaneously during isolation process
mainly due to physical contact
• Intraspecific produce homokaryones
• Interspecific and Intergeneric have no importance
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Concentration of Replications A Replications B Replications C Mean
PEG Fusion Fusion Fusion Fusion
and Time of frequency (%) frequency (%) frequency (%) frequency
incubation (%)
PEG20% & 15 min 2.0 0.8 4.0 2.26
PEG 20% & 20 min 1.4 1.2 3.9 2.5
PEG 25% & 15 min 2.9 1.5 5.04 3.14
PEG 25% & 20 min 8.06 6.56 9.1 7.1
PEG 30% & 15 min 6.9 6.76 9.6 7.75
PEG 30% & 20 min 8.13 5.86 13.2 9.06
Kilima et al., 2009
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CULTURE OF THE HYBRID CELLS
Hybrid cells are cultured on suitable medium provided with
the appropriate culture conditions.
culture in liquid medium
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Production of unique nuclear cytoplasmic combinations
Production of heterozygous lines in vegetatively propagated
crops Eg:- Sugar cane, potato, other tubers root crops
Protoplast of sexually sterile plants
(haploid, triploid, anueploid) can be fused to produce fertile
diploids and polyploidy
Photosynthetic efficiency of plants can be enhanced through
the transfer of efficient foreign chloroplast into the plants
having less photosynthetic systems.
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CONTI….
For cpDNA For nuclear DNA
Suthern bloting Peroxidase isozyme pattern44
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Production of novel intergeneric hybrids
Fig. 1 (a) M. incana; (b) A somatic hybrid plant (pot cultivation); (c) B. oleracea; (d) Leaves
of (from left to right) M. incana (M), hybrids (H1 and H3), nonfusion plant
(R1), and B. oleracea(K)
Xiaoguang et al., 2008
45
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Characterisation of somatic hybrids with molecular
markers
Brassica oleracea=K and Matthiola incana=M
RAPD
RAPD
Xiaoguang et al., 2008
46
SRAP
47. 5/28/2012
1. T1 +T2 +S1
2. T1 +T2 +S2
Fusion combinations
3. T1 +T2 +S3
4. T1 +T2 +S4
Hybrid lines
cell line No. 6 in combination 2.
cell line No. 5 in combination 2.
cell line No. 3 in combination 1.
cell line No. 1 in combination 1. Xiang et.al., 2004
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TRANSFER OF DESIRABLE GENES
Transfer of PLRV resistance gene from
Solanum verrucosum to potato
(S. tuberosum L.)
Carrasco et al.,2000
49
50. clone A405nm Response
S. tuberosum (ver R3064) 0.5675 0.03 S
S. verrucosum(Ver 1340) 0.154 0.005 R
VR1 0.142 0.001 R
5/28/2012
VR2 0.149 0.002 R
VR3 0.133 0.002 R
VR4 0.146 0.005 R
VR5 0.136 0.001 R
VR6 0.153 0.001 R
VR7 0.287 0.003 S
VR8 0.309 0.002 S
VR9 0.146 0.001 R
VR10 0.130 0.001 R
VR11 0.143 0.002 R
VR12 0.138 0.001 R
VR14 0.376 0.005 S
VR16 0.140 0.001 R
VR17 0.136 0.001 R
Kennebec 0.689 0.011 S
50
Uninoculated Cntrol 0.115 0.001 ………
Carrasco et al.,2000
55. 5/28/2012
Atrazin resistance from S. nigrum to
tomato(VF36)
Fusion combinations
1. S. nigrum + tomato(VF36)
2. UV irradiated S. nigrum + tomato
3. UV irradiated S. nigrum + iodoacetate
treated tomato
4. Back fusion of tomato with somatic
hybrid
Jain et al.(1988)
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Hybrids can be produced in vegetatively
propagated crops
In completely sterile plants
Ex : Monoploids
Breeding duration can be reduced by 6
years in transfer of cytoplasm
Hybrids can be produced in juvenile
phase of perennial crops
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Poor regeneration(somatic incompatibility)
Non-viability of fused products
Production of unfavorable hybrids
Lack of an efficient method for selection of
hybrids
No confirmation of expression of
particular trait in somatic hybrids
Sexual reproduction of somatic hybrids. 62
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Species Useful traits transferred Reference
Brassica
Qian et al.
B. napus (+) B. rapa Increased biomass and yield
(2003)
B. napus (+) Crambe Increased erucic acid content in Wang et al.
abyssinica seeds* (2003)
B. napus (+) Orychophragmus Improved fatty acid composition Hu et
violaceus in seeds* al(2002b)
Enhanced resistance to
Hu et al.
B. napus (+) Sinapsis arvensis Blackleg (Leptosphaeria
(2002)
maculans)*
B. oleracea (+) Moricandia Introduction of the C3–C4 Ishikawa et al.
arvensis intermediate trait† (2003)
Raphanus sativus (+) Diplotaxis Introduction of the C3–C4 Bang et al. 63
†
64. Solanum 5/28/2012
S. melongena (+) S. Resistance to bacterial wilt Collonnier et al.
aethiopicum (Ralstonia solanacearum)* (2001)
S. melongena (+) S. Resistance to bacterial and fungal Collonnier et al.
sisymbrifolium wilts* (2003)
S. tuberosum (+) S. Gavrilenko et al.
Resistance to potato virus Y*
etuberosum (2003)
Resistance to potato blight Szczerbakowa
S. tuberosum (+) S. nigrum
(Phytophthora infestans)† et al. (2003)
S. tuberosum (+) S. Resistance to bacterial wilt (R. Fock et al.
stenotomum solanacearum)* (2001)
Citrus
C. amblycarpa (+) Citroncirus Improved rootstock for Mexican Medina-Urrutia
webberri C35 lime† et al. (2004)
C. limonia (+) C. sunki cv. Tolerance to citrus blight, tristeza Costa et al.
Tanaka virus, and Phytophthora† (2003)
Production of mixoploid plants
C. reticulata cv. Blanco (+) C. Liu and Deng
tolerant to citrus exocortis virus
paradisi (2002)
(CEV)†
C. reticulata cv. Blanco (+) C. Tolerance to citrus blight, tristeza Costa et al. 64
65. 5/28/2012
C. reticulata cv. Blanco (+) Resistance to CEV†
Guo et al. (2002)
Poncirus trifoliata
C. sinensis cv. Rohde Red (+) Tolerance to citrus blight, tristeza Costa et al.
C. volkameriana virus, and Phytophthora† (2003)
C. sinensis cv. Ruby Blood (+) Tolerance to citrus blight, tristeza Costa et al.
C. volkameriana virus, and Phytophthora† (2003)
C. sinensis (+) Fortunella Cheng et al.
Increased plant vigour*
crassifolia (2003)
Tolerance to citrus blight, tristeza Costa et al.
C. sinensis (+) F. obovata
virus, and Phytophthora† (2003)
C. sinensis (+) Clausena
Production of triploid plants* Fu et al. (2003)
lansium
C. unshiu cv. Guoqing No. 1 (+)
Generation of seedless cybrids† Guo et al. (2004)
C. grandis cv. Buntan Pink
C. unshiu cv. Guoqing No. 1 (+)
Generation of seedless cybrids† Guo et al. (2004)
C. reticulata cv. Blanco
C. unshiu cv. Guoqing No. 1 (+)
Generation of seedless cybrids† Guo et al. (2004)
C. reticulata×C. sinensis
Michael et al., 200565
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Reference
Species Application
Gene recognition mechanisms Leister and Katagiri
Arabidopsis thaliana
involved in plant pathogenicity (2000)
Elucidation of plant signal
A. thaliana/Zea mays Sheen (2001)
transduction mechanisms
Electrophysiological studies of
Brassica chinensis Fan et al. (2003)
outward K+ channels
Yasukawa et al.
Bryopsis plumosa Electrochemical assays of metabolic
(2002), Zhou et al.
(marine green alga) flux; enzyme (peroxidase) activity
(2003)
Cucurbita pepo Viral pathogenicity Choi et al. (2003)
Synthetic peptide import through the
Helianthus annuus Cormeau et al. (2002)
plasma membrane
Hibiscus cannabinus Viral replication processes Liang et al. (2002)
Comparison of stress mechanisms in 66
Hordeum vulgare Zhou et al. (2000)
67. Nicotiana Shanka
5/28/2012 et al.
Viral recombination and replication
benthamiana (2004)
Nicotiana Genetic basis of developmental regulation and Chesnokov et al.
plumbaginifolia specificity (2002)
Nicotiana Regulation of osmotic water transport across cell
Ding et al. (2004)
tabacum membranes
Oryza
sativa/Pisum
sativum/Sorghu Ishikawa et al.
Membrane permeability and tolerance to Al3+
m (2001)
bicolor/Triticum
vulgare/Z. mays
Phaseolus Electrophysiological studies of inward-rectifying Etherton et al.
vulgaris K+ channels (2004)
Raphanus Immunocytochemical evaluation of aquaporin
Suga et al. (2003)
sativus accumulation
Fluorometric analysis of photosynthetic electron
Vicia faba Goh et al. (2002)
transport
Intracellular responses to drought and salinity
Vigna radiata Kim et al. (2004)
stress
Vermeer et al.
V. unguiculata Studies on plasma membrane organisation
Shapka et al., 200467
(2004)