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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.
Read More
FAQs on Lecture 19 - Methods of Gene Transfer - Plant Biotechnology - Botany
| 1. What are the different methods of gene transfer in botany? |  |
Ans. In botany, there are several methods of gene transfer, including Agrobacterium-mediated transformation, biolistic or particle bombardment, electroporation, protoplast fusion, and microinjection. These techniques allow scientists to introduce foreign genes into plant cells and generate genetically modified plants.
| 2. How does Agrobacterium-mediated transformation work in gene transfer? | |
Ans. Agrobacterium-mediated transformation is a commonly used method for gene transfer in botany. It involves the use of a naturally occurring soil bacterium called Agrobacterium tumefaciens. The bacterium carries a tumor-inducing (Ti) plasmid that can transfer a portion of its DNA, including the desired gene, into the plant cell. The transferred DNA integrates into the plant genome, leading to the expression of the foreign gene.
| 3. What is biolistic or particle bombardment in gene transfer? | |
Ans. Biolistic or particle bombardment is a method of gene transfer in which microscopic particles coated with the desired gene are shot into plant cells using a device called a gene gun. The high-speed particles penetrate the cell walls and deliver the foreign DNA into the plant cells. This technique is particularly useful for transforming plant species that are difficult to genetically modify using other methods.
| 4. How does electroporation work in gene transfer? | |
Ans. Electroporation is a gene transfer technique that uses brief electric pulses to create temporary pores in the cell membrane, allowing the foreign DNA to enter the plant cells. The plant tissue is exposed to an electric field, which causes the cell membranes to become permeable. The electric pulses create temporary openings through which the foreign DNA can pass into the cells. After the pulses cease, the cell membranes return to their normal state.
| 5. What is protoplast fusion in gene transfer? | |
Ans. Protoplast fusion is a method of gene transfer that involves the fusion of plant cells that have had their cell walls removed. The protoplasts from different plant species or varieties are mixed together, and under certain conditions, they can fuse to form a hybrid cell. This hybrid cell can then be regenerated into a whole plant, incorporating the genetic material from both parent cells. Protoplast fusion is commonly used in plant breeding and genetic engineering to introduce desirable traits into plants.
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