Page 1
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 1
Lesson Prepared Under MHRD project “National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Protoplast Culture
Lesson Developer: Dr. Anupama Tiku, Department of Botany,
Ramjas College, University of Delhi
Lesson Reviewer: Dr M.K Razdan, Retired Principal ,Shyamlal College,
University of Delhi.
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi,
South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Page 2
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 1
Lesson Prepared Under MHRD project “National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Protoplast Culture
Lesson Developer: Dr. Anupama Tiku, Department of Botany,
Ramjas College, University of Delhi
Lesson Reviewer: Dr M.K Razdan, Retired Principal ,Shyamlal College,
University of Delhi.
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi,
South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 2
Chapter: Protoplasts
Table of Contents
? Introduction
? Isolation of Protoplasts
? Mechanical method
? Sequential enzymatic method
? Mixed enzymatic method
? Source of Protoplasts
? Leaf/Mesophyll
? Callus Cultures
? Cell Suspension Cultures
? Preconditioned plant material
? Culture of Protoplast
? Multiple Drop Array (MDA) Screening
? Feeder Layer Technique
? Microdrop Culture
? Co- culture of Protoplasts
? Other techniques
? Protoplast Regeneration
? Formation of Cell Wall
? Development of Callus and Regeneration of whole plant
? Protoplast Fusion
? Mechanical Fusion
? Induced fusion
? NANO
3
treatment
? High pH/Ca
++
treatment
? PEG (Polyethylene glycol) treatment
? Electrofusion
? Selection of Somatic hybrids
? Biochemical basis for complementation and selection
? Drug sensitivity
? Auxotrophic mutants
? Visual selection
Page 3
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 1
Lesson Prepared Under MHRD project “National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Protoplast Culture
Lesson Developer: Dr. Anupama Tiku, Department of Botany,
Ramjas College, University of Delhi
Lesson Reviewer: Dr M.K Razdan, Retired Principal ,Shyamlal College,
University of Delhi.
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi,
South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 2
Chapter: Protoplasts
Table of Contents
? Introduction
? Isolation of Protoplasts
? Mechanical method
? Sequential enzymatic method
? Mixed enzymatic method
? Source of Protoplasts
? Leaf/Mesophyll
? Callus Cultures
? Cell Suspension Cultures
? Preconditioned plant material
? Culture of Protoplast
? Multiple Drop Array (MDA) Screening
? Feeder Layer Technique
? Microdrop Culture
? Co- culture of Protoplasts
? Other techniques
? Protoplast Regeneration
? Formation of Cell Wall
? Development of Callus and Regeneration of whole plant
? Protoplast Fusion
? Mechanical Fusion
? Induced fusion
? NANO
3
treatment
? High pH/Ca
++
treatment
? PEG (Polyethylene glycol) treatment
? Electrofusion
? Selection of Somatic hybrids
? Biochemical basis for complementation and selection
? Drug sensitivity
? Auxotrophic mutants
? Visual selection
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 3
? Use of non – allelic albino mutants for complementation
selection
? Flow Cytometric Analysis
? Verification and Characterisation of Somatic
Hybrids/Cybrids
? Assessment of putative hybrids by studying there
morphological characters
? Isozymes fraction-1 protein Analysis
? Cytological/Chromosomal Analysis
? Molecular Analysis
? Practical Applications of Somatic hybridisation
? Means of genetic recombination in asexual or sterile plants
? Helps in overcoming barriers of sexual incompatibility
? Means of Cybrid formation for Cytoplasm gene transfer
? Limitations of Somatic Hybridisation
? Summary
? Exercises
? References
? Web links
Page 4
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 1
Lesson Prepared Under MHRD project “National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Protoplast Culture
Lesson Developer: Dr. Anupama Tiku, Department of Botany,
Ramjas College, University of Delhi
Lesson Reviewer: Dr M.K Razdan, Retired Principal ,Shyamlal College,
University of Delhi.
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi,
South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 2
Chapter: Protoplasts
Table of Contents
? Introduction
? Isolation of Protoplasts
? Mechanical method
? Sequential enzymatic method
? Mixed enzymatic method
? Source of Protoplasts
? Leaf/Mesophyll
? Callus Cultures
? Cell Suspension Cultures
? Preconditioned plant material
? Culture of Protoplast
? Multiple Drop Array (MDA) Screening
? Feeder Layer Technique
? Microdrop Culture
? Co- culture of Protoplasts
? Other techniques
? Protoplast Regeneration
? Formation of Cell Wall
? Development of Callus and Regeneration of whole plant
? Protoplast Fusion
? Mechanical Fusion
? Induced fusion
? NANO
3
treatment
? High pH/Ca
++
treatment
? PEG (Polyethylene glycol) treatment
? Electrofusion
? Selection of Somatic hybrids
? Biochemical basis for complementation and selection
? Drug sensitivity
? Auxotrophic mutants
? Visual selection
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 3
? Use of non – allelic albino mutants for complementation
selection
? Flow Cytometric Analysis
? Verification and Characterisation of Somatic
Hybrids/Cybrids
? Assessment of putative hybrids by studying there
morphological characters
? Isozymes fraction-1 protein Analysis
? Cytological/Chromosomal Analysis
? Molecular Analysis
? Practical Applications of Somatic hybridisation
? Means of genetic recombination in asexual or sterile plants
? Helps in overcoming barriers of sexual incompatibility
? Means of Cybrid formation for Cytoplasm gene transfer
? Limitations of Somatic Hybridisation
? Summary
? Exercises
? References
? Web links
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 4
Introduction
Protoplast is wall-less naked plant cell having nucleus and cytoplasm, after its cell wall has
been removed. Protoplasts can be isolated from different plant parts, cells or tissues growing
in vitro. If protoplasts are cultured on a suitable nutrient medium immediately after isolation,
they can re-form the cell wall and divide to form cell colonies which can further grow into
callus from which whole new plants can regenerate. Protoplast culture is not only meant for
regenerating plants but it also provides excellent opportunities for research on plant genetic
improvement through following processes:
? Somaclonal Variation
? Genetic transformation
? Somatic hybridisation and Cybridisation
Figure: Protoplasts isolated from cells of leaf of Petunia
Source:http://en.wikipedia.org/wiki/Protoplast#mediaviewer/File:Protoplasts_Petunia_sp.jpg(c
c)
Isolation of Protoplasts
There are different methods for protoplast isolation which can be classified into three main
groups:
? Mechanical (Non – Enzymatic)
? Sequential enzymatic (Two step)
? Mixed enzymatic (simultaneous) procedures
Mechanical method
Traditionally mechanical method of isolation was used in which plasmolysed tissues are cut
with a sharp – edged knife, thus releasing the protoplasts by deplasmolysis. Drawback of this
method is that cell damage is caused which affects the yield of intact protoplasts. Mechanical
Page 5
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 1
Lesson Prepared Under MHRD project “National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Protoplast Culture
Lesson Developer: Dr. Anupama Tiku, Department of Botany,
Ramjas College, University of Delhi
Lesson Reviewer: Dr M.K Razdan, Retired Principal ,Shyamlal College,
University of Delhi.
Language Editor: Namrata Dhaka
Department/College: Department of Genetics, University of Delhi,
South Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 2
Chapter: Protoplasts
Table of Contents
? Introduction
? Isolation of Protoplasts
? Mechanical method
? Sequential enzymatic method
? Mixed enzymatic method
? Source of Protoplasts
? Leaf/Mesophyll
? Callus Cultures
? Cell Suspension Cultures
? Preconditioned plant material
? Culture of Protoplast
? Multiple Drop Array (MDA) Screening
? Feeder Layer Technique
? Microdrop Culture
? Co- culture of Protoplasts
? Other techniques
? Protoplast Regeneration
? Formation of Cell Wall
? Development of Callus and Regeneration of whole plant
? Protoplast Fusion
? Mechanical Fusion
? Induced fusion
? NANO
3
treatment
? High pH/Ca
++
treatment
? PEG (Polyethylene glycol) treatment
? Electrofusion
? Selection of Somatic hybrids
? Biochemical basis for complementation and selection
? Drug sensitivity
? Auxotrophic mutants
? Visual selection
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 3
? Use of non – allelic albino mutants for complementation
selection
? Flow Cytometric Analysis
? Verification and Characterisation of Somatic
Hybrids/Cybrids
? Assessment of putative hybrids by studying there
morphological characters
? Isozymes fraction-1 protein Analysis
? Cytological/Chromosomal Analysis
? Molecular Analysis
? Practical Applications of Somatic hybridisation
? Means of genetic recombination in asexual or sterile plants
? Helps in overcoming barriers of sexual incompatibility
? Means of Cybrid formation for Cytoplasm gene transfer
? Limitations of Somatic Hybridisation
? Summary
? Exercises
? References
? Web links
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 4
Introduction
Protoplast is wall-less naked plant cell having nucleus and cytoplasm, after its cell wall has
been removed. Protoplasts can be isolated from different plant parts, cells or tissues growing
in vitro. If protoplasts are cultured on a suitable nutrient medium immediately after isolation,
they can re-form the cell wall and divide to form cell colonies which can further grow into
callus from which whole new plants can regenerate. Protoplast culture is not only meant for
regenerating plants but it also provides excellent opportunities for research on plant genetic
improvement through following processes:
? Somaclonal Variation
? Genetic transformation
? Somatic hybridisation and Cybridisation
Figure: Protoplasts isolated from cells of leaf of Petunia
Source:http://en.wikipedia.org/wiki/Protoplast#mediaviewer/File:Protoplasts_Petunia_sp.jpg(c
c)
Isolation of Protoplasts
There are different methods for protoplast isolation which can be classified into three main
groups:
? Mechanical (Non – Enzymatic)
? Sequential enzymatic (Two step)
? Mixed enzymatic (simultaneous) procedures
Mechanical method
Traditionally mechanical method of isolation was used in which plasmolysed tissues are cut
with a sharp – edged knife, thus releasing the protoplasts by deplasmolysis. Drawback of this
method is that cell damage is caused which affects the yield of intact protoplasts. Mechanical
Protoplast Culure
Institute of Lifelong Learning, University of Delhi, South Campus 5
isolation of protoplast from higher plants was first attempted by Klercker (1892) from highly
vacuolated cells of storage tissues (onion bulbs, scales, radish root, mesocarp of cucumber and
beet root).
Sequential enzymatic method
E.C. Cocking (1960) was the first to use enzymatic method for isolation of protoplasts by using
concentrated solutions of cellulase enzyme (prepared from cultures of fungus Myrothecium
verrucaria) to degrade cell walls and demonstrated large- scale protoplast isolation from the
cells of higher plants. Takebe et. al. (1968) used sequential or two – step procedure for
isolating mesophyll protoplasts by using commercially prepared Cellulase and Macerozyme
enzymes. This process involves incubation of macerated plant tissues with pectinases followed
by final digestion of cell wall and release of protoplasts by cellulase treatment.
Figure: Vacuolated protoplasts as observed by E. C. Cocking using a phase contrast
microscope.
Source: http://www.nature.com/nature/journal/v187/n4741/abs/187962a0.html
Cocking, E. C. "A method for the isolation of plant protoplasts and vacuoles." (1960): 962-
963.
Mixed enzymatic method
Power and Cocking (1968) followed mixed enzymatic (simultaneous procedure) approach by
plasmolysing the plant tissues in a mixture of pectinase and cellulase to induce concomitant
separation of cells and degradation of their walls to release protoplasts directly in single step.
This method is widely accepted as it consumes less time and reduces the chances of microbial
contamination by eliminating few steps.
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