Ppt sem i

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  • where, crossing between the plants difficult or impossible to hybridise conventionally
  • Ppt sem i

    1. 1. 5/28/2012 1
    2. 2. 5/28/2012SEMINAR-IPRESENTER:- NAME: SHILPA . V. MALAGHAN. Jr.MSc(AGRI), GPB. ID NO :PG11AGR3050 DATE:25/5/2012 2
    3. 3. 5/28/2012CONTENTS Introduction History Properties of protoplast Protocol. Applications Advantages Limitations Conclusion 3
    4. 4. 5/28/2012 INTRODUCTION Fusio Hybrid n protoplast Protoplast Somatic hybrid CellExplant 4
    5. 5. 5/28/2012 TERMINOLOGIES Protoplast Isolation plant protoplast Protoplast culture Protoplast fusion/somatic hybridization Symmetric hybrid Asymmetric hybrid (cybrid) 5
    6. 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. 7. 5/28/2012PROPERTIES 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
    8. 8. 5/28/2012SOMATIC 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 8
    9. 9. 5/28/2012PROTOPLAST ISOLATION 9
    10. 10. 5/28/2012 Isolation of Protoplast1. Mechanical Method 2. Enzymatic Method 10
    11. 11. 5/28/2012 1. MECHANICAL METHOD Plant Tissue Cells Plasmolysis Microscope Observation of cellsCutting cell with knife Release of protoplasm Collection of protoplasm 11
    12. 12. 5/28/2012CONTED., Used for vacuolated cells like onion bulb scale, radish and beet root tissues Low yield of protoplast Laborious and tedious process Low protoplast viability 12
    13. 13. 5/28/2012 2. ENZYMATIC METHOD Leaf sterlization, removal of epidermisPlasmolysed Plasmolysedcells cells Pectinase Pectinase +cellulase Release of Protoplasm isolated cells releasedProtoplasm released cellaulase Isolated Protoplasm 13
    14. 14. 5/28/2012 CONTED., Used for variety of tissues and organs Mesophyll tissue - most suitable source High yield of protoplast Easy to perform More protoplast viability 14
    15. 15. 5/28/2012PURIFICAION OF PROTOPLAST  Filtration  Centrifugation  Washing 15
    16. 16. 5/28/2012 PROTOPLAST FUSION MECHANISM OF FUSION1) Agglutination2) Membrane fusion3) Rounding off of the fused protoplast 16
    17. 17. 5/28/2012 Akym et al., 2005 17
    18. 18. 5/28/2012 Protoplast Fusion 1. Spontaneous Fusion 2. Induced Fusion MechanicalIntraspecific Intergeneric Chemofusion Electroporation Fusion 18
    19. 19. 5/28/2012 SPONTANEOUS FUSION Protoplast fuse spontaneously during isolation process mainly due to physical contact • Intraspecific produce homokaryones • Interspecific and Intergeneric have no importance 19
    20. 20. 5/28/2012INDUCED FUSION1.Chemofusion- fusion induced by chemicals • Types of chemofusogens • PEG • NaNO3 • Ca 2+ ions • Polyvinyl alcohol 20
    21. 21. 5/28/2012Concentration of Replications A Replications B Replications C MeanPEG Fusion Fusion Fusion Fusionand Time of frequency (%) frequency (%) frequency (%) frequencyincubation (%)PEG20% & 15 min 2.0 0.8 4.0 2.26PEG 20% & 20 min 1.4 1.2 3.9 2.5PEG 25% & 15 min 2.9 1.5 5.04 3.14PEG 25% & 20 min 8.06 6.56 9.1 7.1PEG 30% & 15 min 6.9 6.76 9.6 7.75PEG 30% & 20 min 8.13 5.86 13.2 9.06 Kilima et al., 2009 21
    22. 22. 5/28/2012 Kilima et al., 2009 22
    23. 23. 5/28/20122 MECHANICAL FUSION 23
    24. 24. 5/28/20123.ELECTROPORATION 24
    25. 25. 5/28/2012Akym et al., 2005 25
    26. 26. 5/28/2012 Faheem et al.,2002 Parameters Standard conditionOptimum 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
    27. 27. 5/28/20121 Parental types/unfused protoplast2 Homokaryons3 Heterokaryons 27
    28. 28. 5/28/2012CONTI…… = chloroplast = mitochondria Fusion = nucleus heterokaryoncybrid hybrid cybrid hybrid 28
    29. 29. 5/28/2012 visual markers Florescent dye staining Complementation Transgenic selectable marker Mass culture Selective markers Chromosomal analysis Molecular markers 29
    30. 30. 5/28/2012CULTURE OF THE HYBRID CELLS Hybrid cells are cultured on suitable medium provided with the appropriate culture conditions. culture in liquid medium 30
    31. 31. 5/28/2012CULTURE ON SOLID MEDIA 31
    32. 32. 5/28/2012CULTURE IN SEMI SOLID MEDIUM/IN SOLID MEDIUM 32
    33. 33. 5/28/2012ALGINATE BEADS CULTURE 33
    34. 34. 5/28/2012FEEDER LAYER CULTURE 34
    35. 35. 5/28/2012CULTURE ON FREE CONDITIONED MEDIA 35
    36. 36. 5/28/2012NURSE CULTURE 36
    37. 37. 5/28/2012NURSE CULTURE WITH MICRO CELL 37
    38. 38. 5/28/2012REGENERATION OF CULTURED HYBRIDCELLS 38
    39. 39. 5/28/2012 Hirosi et al., 2006 39
    40. 40. 5/28/2012CHARACTERISATION OF HYBRID  Morphological  Cytological  Biochemical  Molecular biology technique 40
    41. 41. 5/28/2012Patel et al.,2011 41
    42. 42. 5/28/2012 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. 42
    43. 43. 5/28/2012Overcoming sexual incompatible barriers & Production of auto and allopolyploids. In case of jute Saha et al., 2001 43
    44. 44. 5/28/2012CONTI…. For cpDNA For nuclear DNA Suthern bloting Peroxidase isozyme pattern44
    45. 45. 5/28/2012Production of novel intergeneric hybridsFig. 1 (a) M. incana; (b) A somatic hybrid plant (pot cultivation); (c) B. oleracea; (d) Leavesof (from left to right) M. incana (M), hybrids (H1 and H3), nonfusion plant(R1), and B. oleracea(K) Xiaoguang et al., 2008 45
    46. 46. 5/28/2012Characterisation of somatic hybrids with molecular markersBrassica oleracea=K and Matthiola incana=M RAPD RAPD Xiaoguang et al., 2008 46 SRAP
    47. 47. 5/28/20121. T1 +T2 +S12. T1 +T2 +S2 Fusion combinations3. T1 +T2 +S34. 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 47
    48. 48. 5/28/2012CONT…. Xiang et.al., 2004 48
    49. 49. 5/28/2012TRANSFER OF DESIRABLE GENES Transfer of PLRV resistance gene from Solanum verrucosum to potato (S. tuberosum L.) Carrasco et al.,2000 49
    50. 50. clone A405nm ResponseS. tuberosum (ver R3064) 0.5675 0.03 SS. 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
    51. 51. 5/28/2012Genotype Tuber Tuber Mean Maturitya Fertility (%) yield (g) number tuber weight (g)Kennebec 3760 27.2 138.2 7.0 91.0R3064 1000 27.2 36.8 5.5 91.5S. 172 13.2 13.3 3.5 65890verrucosumVR1 890 20.8 43.1 5.5 56.4VR2 1330 20.8 64.7 5.5 55.6VR3 2290 41.2 55.7 5.5 66.1VR4 2920 31.2 91.4 5.5 19.0VR5 1400 23.2 60.4 5.5 25.2VR6 1560 38.8 40.2 5.5 35.3 51
    52. 52. 5/28/2012CONT….Genotype Tuber Tuber Mean Maturity Fertility yield number tuber weightVR7 710 10.8 62.5 4.5 25.1VR8 1030 21.6 47.8 5.5 42.2VR9 1800 46.0 39.2 5.0 65.5VR10 1170 26.0 45.6 5.5 76.8VR11 980 15.2 65.0 5.5 45.0VR12 1880 39.6 47.4 5.5 32.5VR14 1090 24.4 45.3 4.5 25.8VR16 1120 25.2 44.3 4.5 32.6VR17 1260 28.8 43.9 5.5 21.7VR(Mean) 1429 27.6 53.1 5.3 41.6VR(CV) 41.0 36.3 26.1 7.9 44.1 Carrasco et al.,2000 52
    53. 53. 5/28/2012 Deval et al., 2011 53
    54. 54. 5/28/2012PCR products generated by primer OPB07 54 Deval et al., 2011
    55. 55. 5/28/2012 Atrazin resistance from S. nigrum to tomato(VF36) Fusion combinations1. S. nigrum + tomato(VF36)2. UV irradiated S. nigrum + tomato3. UV irradiated S. nigrum + iodoacetate treated tomato4. Back fusion of tomato with somatic hybrid Jain et al.(1988) 55
    56. 56. 5/28/2012Nicotiana tabacum N. repanda 56 Sun et al. (2005)
    57. 57. 5/28/2012 Sun et al. (2005) 57
    58. 58. 5/28/2012 B. napus + Orychophragmus violaceus B. napus + Xinjiang low erucic acid containing somatic hybrids Qiong et al., 2009 58
    59. 59. 5/28/2012 Feng et al (2006) 59
    60. 60. 5/28/2012 60
    61. 61. 5/28/2012 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 61
    62. 62. 5/28/2012 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
    63. 63. 5/28/2012Species Useful traits transferred ReferenceBrassica 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 etviolaceus 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. 64. Solanum 5/28/2012S. 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 SzczerbakowaS. tuberosum (+) S. nigrum (Phytophthora infestans)† et al. (2003)S. tuberosum (+) S. Resistance to bacterial wilt (R. Fock et al.stenotomum solanacearum)* (2001)CitrusC. amblycarpa (+) Citroncirus Improved rootstock for Mexican Medina-Urrutiawebberri 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 plantsC. reticulata cv. Blanco (+) C. Liu and Deng tolerant to citrus exocortis virusparadisi (2002) (CEV)†C. reticulata cv. Blanco (+) C. Tolerance to citrus blight, tristeza Costa et al. 64
    65. 65. 5/28/2012C. reticulata cv. Blanco (+) Resistance to CEV† Guo et al. (2002)Poncirus trifoliataC. 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)lansiumC. unshiu cv. Guoqing No. 1 (+) Generation of seedless cybrids† Guo et al. (2004)C. grandis cv. Buntan PinkC. unshiu cv. Guoqing No. 1 (+) Generation of seedless cybrids† Guo et al. (2004)C. reticulata cv. BlancoC. unshiu cv. Guoqing No. 1 (+) Generation of seedless cybrids† Guo et al. (2004)C. reticulata×C. sinensis Michael et al., 200565
    66. 66. 5/28/2012 ReferenceSpecies Application Gene recognition mechanisms Leister and KatagiriArabidopsis thaliana involved in plant pathogenicity (2000) Elucidation of plant signalA. thaliana/Zea mays Sheen (2001) transduction mechanisms Electrophysiological studies ofBrassica 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 theHelianthus annuus Cormeau et al. (2002) plasma membraneHibiscus cannabinus Viral replication processes Liang et al. (2002) Comparison of stress mechanisms in 66Hordeum vulgare Zhou et al. (2000)
    67. 67. Nicotiana Shanka 5/28/2012 et al. Viral recombination and replicationbenthamiana (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 membranesOryzasativa/Pisumsativum/Sorghu Ishikawa et al. Membrane permeability and tolerance to Al3+m (2001)bicolor/Triticumvulgare/Z. maysPhaseolus 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 electronVicia faba Goh et al. (2002) transport Intracellular responses to drought and salinityVigna radiata Kim et al. (2004) stress Vermeer et al.V. unguiculata Studies on plasma membrane organisation Shapka et al., 200467 (2004)
    68. 68. 5/28/2012 68
    69. 69. 5/28/2012 69
    70. 70. 5/28/2012 70

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