Lecture 19 - Methods of Gene Transfer | Plant Biotechnology - Botany PDF Download

 Page 1


           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
         
 
 
 
Lesson Prepared Under MHRD project “ National Mission on 
Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson:Methods of Gene Transfer 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya Narender Dev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi Ahilya Vishwavidyalaya, Indore 
 
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


           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
         
 
 
 
Lesson Prepared Under MHRD project “ National Mission on 
Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson:Methods of Gene Transfer 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya Narender Dev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi Ahilya Vishwavidyalaya, Indore 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
 
Chapter: Methods of Gene Transfer  
Table of Contents  
• Introduction 
• Indirect Methods 
• Agrobacterium-mediated genetic transformation 
• Structure of Ti Plasmid 
• Use of Ti plasmid in genetic transformation 
• Steps involved in Agrobacterium-mediated genetic 
transformation of plants by ‘Wounded explant’ 
method  
• Direct Method 
• Microprojectile/particle Bombardment (biolistics) 
• Electroporation 
• Microinjection 
• Chemical mediated gene transfer 
• Liposome mediated gene transfer 
• Silicon carbide method 
• Selection of transformants 
• Selectable marker 
• Screening marker 
• Summary 
• Exercise/practice 
• Glossary 
• References/Bibliography/Further Reading 
• Web links 
 
 
 
 
Page 3


           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
         
 
 
 
Lesson Prepared Under MHRD project “ National Mission on 
Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson:Methods of Gene Transfer 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya Narender Dev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi Ahilya Vishwavidyalaya, Indore 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
 
Chapter: Methods of Gene Transfer  
Table of Contents  
• Introduction 
• Indirect Methods 
• Agrobacterium-mediated genetic transformation 
• Structure of Ti Plasmid 
• Use of Ti plasmid in genetic transformation 
• Steps involved in Agrobacterium-mediated genetic 
transformation of plants by ‘Wounded explant’ 
method  
• Direct Method 
• Microprojectile/particle Bombardment (biolistics) 
• Electroporation 
• Microinjection 
• Chemical mediated gene transfer 
• Liposome mediated gene transfer 
• Silicon carbide method 
• Selection of transformants 
• Selectable marker 
• Screening marker 
• Summary 
• Exercise/practice 
• Glossary 
• References/Bibliography/Further Reading 
• Web links 
 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
Foreign genes are introducedartificially into crops by overcoming the fertility barriers.This 
process, also known asgenetic transformation,is a very important step in genetic engineering. 
Horizontal vs vertical gene transfer 
The natural transfer of genetic material from one organism to another is referred to as 
horizontal gene transfer or the lateral gene transfer. The foreign DNA is either randomly 
inserted into the host genome or recombines if there is sequence homology between the two 
genomes. This is different from the vertical gene transfer where the genetic material is 
transferred from the parents to the offsprings, through sexual reproduction.  
 
Horizontal gene transfer is facilitated by various mechanisms. In prokaryotes mainly 
transformation (intake of genetic material from surrounding), conjugation (exchange of 
genetic material with the physical union of two cells) and transduction (transmission of DNA 
through bacteriophages from one cell to another) are responsible for the transfer of the gene 
within organisms. In eukaryotes, the presence of the outer cell membrane and the nuclear 
membrane makes transfer of DNA difficult between organisms. Horizontal gene transfer plays 
important role in evolution of both prokaryotes and eukaryotes. 
Page 4


           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
         
 
 
 
Lesson Prepared Under MHRD project “ National Mission on 
Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson:Methods of Gene Transfer 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya Narender Dev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi Ahilya Vishwavidyalaya, Indore 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
 
Chapter: Methods of Gene Transfer  
Table of Contents  
• Introduction 
• Indirect Methods 
• Agrobacterium-mediated genetic transformation 
• Structure of Ti Plasmid 
• Use of Ti plasmid in genetic transformation 
• Steps involved in Agrobacterium-mediated genetic 
transformation of plants by ‘Wounded explant’ 
method  
• Direct Method 
• Microprojectile/particle Bombardment (biolistics) 
• Electroporation 
• Microinjection 
• Chemical mediated gene transfer 
• Liposome mediated gene transfer 
• Silicon carbide method 
• Selection of transformants 
• Selectable marker 
• Screening marker 
• Summary 
• Exercise/practice 
• Glossary 
• References/Bibliography/Further Reading 
• Web links 
 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
Foreign genes are introducedartificially into crops by overcoming the fertility barriers.This 
process, also known asgenetic transformation,is a very important step in genetic engineering. 
Horizontal vs vertical gene transfer 
The natural transfer of genetic material from one organism to another is referred to as 
horizontal gene transfer or the lateral gene transfer. The foreign DNA is either randomly 
inserted into the host genome or recombines if there is sequence homology between the two 
genomes. This is different from the vertical gene transfer where the genetic material is 
transferred from the parents to the offsprings, through sexual reproduction.  
 
Horizontal gene transfer is facilitated by various mechanisms. In prokaryotes mainly 
transformation (intake of genetic material from surrounding), conjugation (exchange of 
genetic material with the physical union of two cells) and transduction (transmission of DNA 
through bacteriophages from one cell to another) are responsible for the transfer of the gene 
within organisms. In eukaryotes, the presence of the outer cell membrane and the nuclear 
membrane makes transfer of DNA difficult between organisms. Horizontal gene transfer plays 
important role in evolution of both prokaryotes and eukaryotes. 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
Video: Horizontal vs Vertical gene transfer https://www.youtube.com/watch?v=EHTEFdSadXM 
 
Plant transformation systems generally include following steps, 
• Introduction of a DNA segment into totipotent cells. 
• Its integration into host cells genome. 
• Subsequent regeneration from transformed cellto produce whole plant.  
Plant transformation methods therefore require an efficient wayto introduce DNA into cell and 
the regeneration of the transformed cells or tissues into whole plants.  
TheDNA segment which is introduced in this process contains the gene of interestanda 
cassette containing additional genetic material. Additional genetic material includes 
• A promoter which determines the site and timing of expression of the introduced gene 
• A terminator to identify the end of transcription and  
• Amarker gene which allows selection of plants having the introduced gene. 
 Various desirable traits have been efficiently introduced and stably expressed in almost 150 
plant species. 
Different methods are available to achieve genetic transformation of plants i.e. the delivery of 
the foreign DNA into the host plant. These are divided into two main groups 
• Indirect methods: In this case vector is needed for insertion of the foreign DNA into the 
host genome. 
• Direct methods: This method is vector independent. The DNA is directly inserted into the 
host genome. 
 
Page 5


           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
         
 
 
 
Lesson Prepared Under MHRD project “ National Mission on 
Education Through ICT” 
 
Discipline: Botany 
Paper: Plant Biotechnology 
 
National Coordinator: Prof. S.C. Bhatla 
 
Lesson:Methods of Gene Transfer 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya Narender Dev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi Ahilya Vishwavidyalaya, Indore 
 
Language Editor: Namrata Dhaka 
Department/College: Department of Genetics, University of Delhi, 
South Campus 
 
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
 
Chapter: Methods of Gene Transfer  
Table of Contents  
• Introduction 
• Indirect Methods 
• Agrobacterium-mediated genetic transformation 
• Structure of Ti Plasmid 
• Use of Ti plasmid in genetic transformation 
• Steps involved in Agrobacterium-mediated genetic 
transformation of plants by ‘Wounded explant’ 
method  
• Direct Method 
• Microprojectile/particle Bombardment (biolistics) 
• Electroporation 
• Microinjection 
• Chemical mediated gene transfer 
• Liposome mediated gene transfer 
• Silicon carbide method 
• Selection of transformants 
• Selectable marker 
• Screening marker 
• Summary 
• Exercise/practice 
• Glossary 
• References/Bibliography/Further Reading 
• Web links 
 
 
 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
Foreign genes are introducedartificially into crops by overcoming the fertility barriers.This 
process, also known asgenetic transformation,is a very important step in genetic engineering. 
Horizontal vs vertical gene transfer 
The natural transfer of genetic material from one organism to another is referred to as 
horizontal gene transfer or the lateral gene transfer. The foreign DNA is either randomly 
inserted into the host genome or recombines if there is sequence homology between the two 
genomes. This is different from the vertical gene transfer where the genetic material is 
transferred from the parents to the offsprings, through sexual reproduction.  
 
Horizontal gene transfer is facilitated by various mechanisms. In prokaryotes mainly 
transformation (intake of genetic material from surrounding), conjugation (exchange of 
genetic material with the physical union of two cells) and transduction (transmission of DNA 
through bacteriophages from one cell to another) are responsible for the transfer of the gene 
within organisms. In eukaryotes, the presence of the outer cell membrane and the nuclear 
membrane makes transfer of DNA difficult between organisms. Horizontal gene transfer plays 
important role in evolution of both prokaryotes and eukaryotes. 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
Video: Horizontal vs Vertical gene transfer https://www.youtube.com/watch?v=EHTEFdSadXM 
 
Plant transformation systems generally include following steps, 
• Introduction of a DNA segment into totipotent cells. 
• Its integration into host cells genome. 
• Subsequent regeneration from transformed cellto produce whole plant.  
Plant transformation methods therefore require an efficient wayto introduce DNA into cell and 
the regeneration of the transformed cells or tissues into whole plants.  
TheDNA segment which is introduced in this process contains the gene of interestanda 
cassette containing additional genetic material. Additional genetic material includes 
• A promoter which determines the site and timing of expression of the introduced gene 
• A terminator to identify the end of transcription and  
• Amarker gene which allows selection of plants having the introduced gene. 
 Various desirable traits have been efficiently introduced and stably expressed in almost 150 
plant species. 
Different methods are available to achieve genetic transformation of plants i.e. the delivery of 
the foreign DNA into the host plant. These are divided into two main groups 
• Indirect methods: In this case vector is needed for insertion of the foreign DNA into the 
host genome. 
• Direct methods: This method is vector independent. The DNA is directly inserted into the 
host genome. 
 
           Methods of Gene Transfer  
Institute of Lifelong Learning, University of Delhi 
 
 
 
Figure: Types of methods for gene transfer in plants 
Source: Namrata Dhaka, Research Scholar, Department of Genetics, University of Delhi, 
South Campus. 
 
In-direct Methods  
Agrobacterium-mediated genetic transformation 
The method of genetictransformation, which employs bacteria as a vector to introduce the 
gene construct into the target cell,isknown as indirect method. Thismethod uses 
Agrobacterium (a gram-negative soil bacteria which causes crown gall disease in many 
plants) for the plant transformation experiments.  
The most commonly studied species of this genus is Agrobacterium tumefaciens, which forms 
an efficient delivery system for genetic transformationin plants.These bacteria harbor a large 
plasmid called Ti plasmid (tumour inducing) having tumor-inducing genes (T-DNA) and other 
genes involved in integration of T-DNA into host genome.Wounded plants secrete a sap with 
high content of phenolic compounds which serve as chemical attractants for Agrobacteria and 
stimulateexpression of virgenes. It results ininfection of plantby Agrobacterium, insertion of 
T-DNA region at a random site inhost genome and proliferation of plant cells to form crown 
gall growth.