Tissue culture media and sterilization techniques | Plant Biotechnology - Botany PDF Download

 Page 1


 
Institute of Lifelong Learning, University of Delhi                                                                        1 
 
 
                                                                                                           
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Tissue culture media and sterilization techniques 
Lesson Developer: Namrata Dhaka 
Department/College: University of Delhi, South Campus 
Lesson Reviewer: Dr Parul Agarwal 
Department/College: Department of Genetics, University of Delhi South 
Campus 
Language Editor: Vinee Khanna 
Department/College: University of Delhi, South Campus 
Lesson Editor: Dr. Rama Sisodia, Fellow in Botany ILLL 
 
Page 2


 
Institute of Lifelong Learning, University of Delhi                                                                        1 
 
 
                                                                                                           
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Tissue culture media and sterilization techniques 
Lesson Developer: Namrata Dhaka 
Department/College: University of Delhi, South Campus 
Lesson Reviewer: Dr Parul Agarwal 
Department/College: Department of Genetics, University of Delhi South 
Campus 
Language Editor: Vinee Khanna 
Department/College: University of Delhi, South Campus 
Lesson Editor: Dr. Rama Sisodia, Fellow in Botany ILLL 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         2 
 
Learning outcomes 
After reading this chapter, the reader should be able to: 
? Understand the nutritional requirements of plants growing in vitro. 
? Explain the different components of tissue culture media. 
? Carry out calculations for making culture media with any given concentration. 
? Understand the importance of sterilization and the methods of sterilization. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Page 3


 
Institute of Lifelong Learning, University of Delhi                                                                        1 
 
 
                                                                                                           
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Tissue culture media and sterilization techniques 
Lesson Developer: Namrata Dhaka 
Department/College: University of Delhi, South Campus 
Lesson Reviewer: Dr Parul Agarwal 
Department/College: Department of Genetics, University of Delhi South 
Campus 
Language Editor: Vinee Khanna 
Department/College: University of Delhi, South Campus 
Lesson Editor: Dr. Rama Sisodia, Fellow in Botany ILLL 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         2 
 
Learning outcomes 
After reading this chapter, the reader should be able to: 
? Understand the nutritional requirements of plants growing in vitro. 
? Explain the different components of tissue culture media. 
? Carry out calculations for making culture media with any given concentration. 
? Understand the importance of sterilization and the methods of sterilization. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         3 
 
Table of contents 
Chapter: Tissue culture media and sterilization techniques 
? Introduction 
? Historical perspective 
? Components of plant tissue culture media 
? Water 
? Organic compounds 
? Carbon source 
? Vitamins 
? Amino acids 
? Complex organic compounds 
? Inorganic nutrients 
? Macronutrients 
? Micronutrients  
? Iron Source 
? Plant Growth Regulators 
? Auxins 
? Cytokinins 
? Gibberellins 
? Abscisic acid 
? Gelling agents 
? Agar 
? Agarose 
? Gellan gums 
? pH of tissue culture media 
? Procedure for making tissue culture medium 
? Arrangement of the equipments and apparatus 
? Preparation of stock solutions of MS medium 
? Stock solution for MS macronutrients I 
Page 4


 
Institute of Lifelong Learning, University of Delhi                                                                        1 
 
 
                                                                                                           
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Tissue culture media and sterilization techniques 
Lesson Developer: Namrata Dhaka 
Department/College: University of Delhi, South Campus 
Lesson Reviewer: Dr Parul Agarwal 
Department/College: Department of Genetics, University of Delhi South 
Campus 
Language Editor: Vinee Khanna 
Department/College: University of Delhi, South Campus 
Lesson Editor: Dr. Rama Sisodia, Fellow in Botany ILLL 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         2 
 
Learning outcomes 
After reading this chapter, the reader should be able to: 
? Understand the nutritional requirements of plants growing in vitro. 
? Explain the different components of tissue culture media. 
? Carry out calculations for making culture media with any given concentration. 
? Understand the importance of sterilization and the methods of sterilization. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         3 
 
Table of contents 
Chapter: Tissue culture media and sterilization techniques 
? Introduction 
? Historical perspective 
? Components of plant tissue culture media 
? Water 
? Organic compounds 
? Carbon source 
? Vitamins 
? Amino acids 
? Complex organic compounds 
? Inorganic nutrients 
? Macronutrients 
? Micronutrients  
? Iron Source 
? Plant Growth Regulators 
? Auxins 
? Cytokinins 
? Gibberellins 
? Abscisic acid 
? Gelling agents 
? Agar 
? Agarose 
? Gellan gums 
? pH of tissue culture media 
? Procedure for making tissue culture medium 
? Arrangement of the equipments and apparatus 
? Preparation of stock solutions of MS medium 
? Stock solution for MS macronutrients I 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         4 
 
? Stock solution for MS macronutrients II 
(calcium chloride solution) 
? Stock solution for MS macronutrients III 
(Iron source) 
? Stock solution for MS micronutrients 
? Stock solution for vitamins 
? Stock solution for PGRs 
? Preparation of MS medium 
? Dispensing of medium 
? Storage of media 
? Sterilization techniques 
? Sterilization of media 
? Autoclaving 
? Filter sterilization 
? Sterilization of instruments and glassware 
? Sterilization of plant material 
? Sterile transfer area 
? Procedure for starting tissue culture 
experiments inside laminar air flow 
chamber 
? Precautions to be taken while carrying out 
tissue culture experiments inside the 
laminar hood 
? Summary 
? Glossary 
? References 
? Exercise 
? Web links 
 
Page 5


 
Institute of Lifelong Learning, University of Delhi                                                                        1 
 
 
                                                                                                           
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Tissue culture media and sterilization techniques 
Lesson Developer: Namrata Dhaka 
Department/College: University of Delhi, South Campus 
Lesson Reviewer: Dr Parul Agarwal 
Department/College: Department of Genetics, University of Delhi South 
Campus 
Language Editor: Vinee Khanna 
Department/College: University of Delhi, South Campus 
Lesson Editor: Dr. Rama Sisodia, Fellow in Botany ILLL 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         2 
 
Learning outcomes 
After reading this chapter, the reader should be able to: 
? Understand the nutritional requirements of plants growing in vitro. 
? Explain the different components of tissue culture media. 
? Carry out calculations for making culture media with any given concentration. 
? Understand the importance of sterilization and the methods of sterilization. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         3 
 
Table of contents 
Chapter: Tissue culture media and sterilization techniques 
? Introduction 
? Historical perspective 
? Components of plant tissue culture media 
? Water 
? Organic compounds 
? Carbon source 
? Vitamins 
? Amino acids 
? Complex organic compounds 
? Inorganic nutrients 
? Macronutrients 
? Micronutrients  
? Iron Source 
? Plant Growth Regulators 
? Auxins 
? Cytokinins 
? Gibberellins 
? Abscisic acid 
? Gelling agents 
? Agar 
? Agarose 
? Gellan gums 
? pH of tissue culture media 
? Procedure for making tissue culture medium 
? Arrangement of the equipments and apparatus 
? Preparation of stock solutions of MS medium 
? Stock solution for MS macronutrients I 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         4 
 
? Stock solution for MS macronutrients II 
(calcium chloride solution) 
? Stock solution for MS macronutrients III 
(Iron source) 
? Stock solution for MS micronutrients 
? Stock solution for vitamins 
? Stock solution for PGRs 
? Preparation of MS medium 
? Dispensing of medium 
? Storage of media 
? Sterilization techniques 
? Sterilization of media 
? Autoclaving 
? Filter sterilization 
? Sterilization of instruments and glassware 
? Sterilization of plant material 
? Sterile transfer area 
? Procedure for starting tissue culture 
experiments inside laminar air flow 
chamber 
? Precautions to be taken while carrying out 
tissue culture experiments inside the 
laminar hood 
? Summary 
? Glossary 
? References 
? Exercise 
? Web links 
 
Tissue culture media and sterilization techniques 
 
 
Institute of Lifelong learning, University of Delhi                                         5 
 
Introduction 
In the natural environment, plants grow in soil which contains various inorganic nutrients 
and they utilize the light energy from sun, to synthesize the organic compounds which 
provide energy. The other essential compounds like vitamins and amino acids are also 
synthesized by plant cells. Thus, their nutritional requirements are met in part by the 
nutrients that are available in the soil and in part through their own biosynthetic machinery. 
In tissue culture, plants are raised in an aseptic environment in laboratories. So how are 
their nutritional requirements met under in vitro conditions? The nutrition to the explants 
growing in vitro is provided through ‘culture medium’, which contains all the organic and 
inorganic components required for the growth of plants. 
 
Figure: An overview of the steps involved in tissue culture. A number of tissues can be 
used as explants and used for in vitro culture to regenerate whole plants by various 
methods of tissue culture. 
Source: ILLL in-house 
In this Chapter, we shall discuss the components of the tissue culture media and the 
method of preparing the media. 
Historical perspective 
The chemical formulations of tissue culture media that are used today were developed 
based on empirical evidences from numerous early experiments. The first insights for 
formulation of tissue culture medium for example, Gautheret’s medium (1939), came from