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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 fromRead More
1. What is tissue culture media and why is it important in botany? |
2. What are the different types of tissue culture media used in botany? |
3. What are the sterilization techniques used in tissue culture? |
4. How often should tissue culture media be replaced? |
5. Can tissue culture media be prepared at home or is it commercially available? |
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