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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 
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FAQs on Tissue culture media and sterilization techniques - Plant Biotechnology - Botany

1. What is tissue culture media and why is it important in botany?
Ans. Tissue culture media refers to the nutrient-rich substances used to grow and maintain plant cells, tissues, or organs in a laboratory setting. It provides essential nutrients, vitamins, sugars, and growth regulators necessary for the growth and development of plant tissues outside their natural environment. Tissue culture media is important in botany as it allows researchers to study plant growth, development, and behavior under controlled conditions, facilitating the production of disease-free plants, genetic engineering, and conservation of rare plant species.
2. What are the different types of tissue culture media used in botany?
Ans. There are several types of tissue culture media used in botany, including: - Murashige and Skoog (MS) medium: This is the most commonly used medium and supports the growth of a wide range of plant species. - Gamborg's B5 medium: It is suitable for the growth of many crop plants and has a lower concentration of macronutrients compared to MS medium. - Woody Plant Medium (WPM): This medium is specifically formulated to support the growth of woody plant species. - Schenk and Hildebrandt (SH) medium: It is commonly used for callus induction and root cultures. - White's medium: This medium is used for the growth of orchids and other delicate plant species.
3. What are the sterilization techniques used in tissue culture?
Ans. Sterilization is a crucial step in tissue culture to prevent contamination and ensure the growth of healthy plant tissues. The commonly used sterilization techniques in tissue culture include: - Autoclaving: This involves subjecting the culture media, containers, and tools to high-pressure steam at around 121°C for a specific duration, effectively killing microorganisms. - Filtration: The culture media can be sterilized by passing it through a filter with a pore size small enough to trap microorganisms while allowing the passage of nutrients. - Chemical sterilization: Some culture media components, such as plant hormones, cannot withstand autoclaving. In such cases, chemical sterilization using disinfectants like sodium hypochlorite or ethanol is employed. - Flame sterilization: Tools and implements used in tissue culture, such as scalpels and forceps, can be sterilized by heating them over a flame until red-hot, effectively killing any adhering microorganisms.
4. How often should tissue culture media be replaced?
Ans. The frequency of media replacement in tissue culture depends on several factors, including the type of plant species, growth stage, and purpose of the culture. In general, tissue culture media should be replaced every 2-4 weeks to ensure the continuous availability of nutrients, growth regulators, and to prevent the accumulation of toxic metabolites. However, for specific applications like long-term maintenance of cultures or slow-growing species, media replacement intervals may be extended up to several months.
5. Can tissue culture media be prepared at home or is it commercially available?
Ans. Tissue culture media can be prepared at home by individuals with the necessary expertise, equipment, and ingredients. However, due to the precise formulation and sterility requirements, it is more commonly purchased commercially from specialized suppliers. Commercially available tissue culture media are pre-packaged and quality-controlled to ensure consistent and reliable results. Additionally, these media are often available in different formulations specifically designed for various plant species and growth stages, providing convenience and saving time for researchers and plant tissue culture enthusiasts.
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