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SEMINAR-I
PRESENTER:-
NAME: SHILPA . V. MALAGHAN.
Jr.MSc(AGRI), GPB.
ID NO :PG11AGR3050
DATE:25/5/2012
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CONTENTS
 Introduction
 History
 Properties of protoplast
 Protocol.
 Applications
 Advantages
 Limitations
 Conclusion
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INTRODUCTION
Fusio Hybrid
n protoplast
Protoplast Somatic hybrid
Cell
Explant
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TERMINOLOGIES
 Protoplast
 Isolation plant protoplast
 Protoplast culture
 Protoplast fusion/somatic hybridization
 Symmetric hybrid
 Asymmetric hybrid (cybrid)
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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

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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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PROTOPLAST ISOLATION
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Isolation of Protoplast
1. Mechanical Method 2. Enzymatic Method
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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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PURIFICAION OF PROTOPLAST
 Filtration
 Centrifugation
 Washing
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PROTOPLAST FUSION
MECHANISM OF FUSION
1) Agglutination
2) Membrane fusion
3) Rounding off of the fused protoplast
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Akym et al., 2005
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Protoplast Fusion
1. Spontaneous Fusion 2. Induced Fusion
Mechanical
Intraspecific Intergeneric Chemofusion Electroporation
Fusion
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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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INDUCED FUSION
1.Chemofusion- fusion induced by chemicals
• Types of chemofusogens
• PEG
• NaNO3
• Ca 2+ ions
• Polyvinyl alcohol
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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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Kilima et al., 2009
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2 MECHANICAL FUSION
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3.ELECTROPORATION
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Akym et al., 2005 25

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Faheem et al.,2002
Parameters Standard condition
Optimum protoplast density 2.5 x lo5 p.ml-'
Electrofusion buffer 13% mannitol and 0.1 % MES; pH
5.8.
Suitable fusion chamber SSH-C03
Chamber capacity 0.8 ml
Electrode spacing 2.0 mm
Chamber constant 0.05
Frequency 1 MHz
Volts AC (primary) 40 volts
Initial time 30 seconds
Pulse width 20 pS
Volts DC 400 volts
Field strength 2.0 KV cm-I
Repeat interval 5 seconds
n = number of DC pulses 4
VDC decreasing rate 80%
VAC decreasing rate 70% 26

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1 Parental types/unfused protoplast
2 Homokaryons
3 Heterokaryons
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CONTI……
= chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon
cybrid hybrid cybrid
hybrid
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 visual markers
 Florescent dye staining
 Complementation
 Transgenic selectable marker
 Mass culture
 Selective markers
 Chromosomal analysis
 Molecular markers
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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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CULTURE ON SOLID MEDIA
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CULTURE IN SEMI SOLID MEDIUM/IN
SOLID MEDIUM
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ALGINATE BEADS CULTURE
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FEEDER LAYER CULTURE
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CULTURE ON FREE CONDITIONED MEDIA
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NURSE CULTURE
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NURSE CULTURE WITH MICRO CELL
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REGENERATION OF CULTURED HYBRID
CELLS
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Hirosi et al., 2006
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CHARACTERISATION OF HYBRID
 Morphological
 Cytological
 Biochemical
 Molecular biology
technique
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Patel et al.,2011
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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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Overcoming sexual incompatible barriers &
Production of auto and allopolyploids.
In case of jute
Saha et al., 2001
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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
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Characterisation of somatic hybrids with molecular
markers
Brassica oleracea=K and Matthiola incana=M
RAPD
RAPD
Xiaoguang et al., 2008
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SRAP

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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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CONT….
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
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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
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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
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Uninoculated Cntrol 0.115 0.001 ………
Carrasco et al.,2000

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Genotype Tuber Tuber Mean Maturitya Fertility (%)
yield (g) number tuber
weight (g)
Kennebec 3760 27.2 138.2 7.0 91.0
R3064 1000 27.2 36.8 5.5 91.5
S. 172 13.2 13.3 3.5 65890
verrucosum
VR1 890 20.8 43.1 5.5 56.4
VR2 1330 20.8 64.7 5.5 55.6
VR3 2290 41.2 55.7 5.5 66.1
VR4 2920 31.2 91.4 5.5 19.0
VR5 1400 23.2 60.4 5.5 25.2
VR6 1560 38.8 40.2 5.5 35.3
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CONT….
Genotype Tuber Tuber Mean Maturity Fertility
yield number tuber
weight
VR7 710 10.8 62.5 4.5 25.1
VR8 1030 21.6 47.8 5.5 42.2
VR9 1800 46.0 39.2 5.0 65.5
VR10 1170 26.0 45.6 5.5 76.8
VR11 980 15.2 65.0 5.5 45.0
VR12 1880 39.6 47.4 5.5 32.5
VR14 1090 24.4 45.3 4.5 25.8
VR16 1120 25.2 44.3 4.5 32.6
VR17 1260 28.8 43.9 5.5 21.7
VR(Mean) 1429 27.6 53.1 5.3 41.6
VR(CV) 41.0 36.3 26.1 7.9 44.1
Carrasco et al.,2000
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Deval et al., 2011 53

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PCR products generated by primer OPB07
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Deval et al., 2011

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 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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Nicotiana tabacum N. repanda
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Sun et al. (2005)

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Sun et al. (2005)
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 B. napus + Orychophragmus violaceus
 B. napus + Xinjiang
low erucic acid containing somatic hybrids
Qiong et al., 2009 58

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Feng et al (2006)
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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

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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)

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