Protoplast Culture | Plant Biotechnology - Botany PDF Download

 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.