What is Gene transfer? methods used in gene transfer in plant breeding.

1. Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani
College of Agriculture, Parbhani
GP-601
Department:- Agricultural Botany
Plant Genetic Resources And Pre-breeding
T
I
T
L
E:-Gene transfer tools and techniques into
cultivated species
Course incharge :-
Dr. R.R Dhutmal
Associate Professor.
Department of Agril. Botany
Presented by:-
Deshmukh S.S.
Ph.D. Scholar
2021A/5P

2. Gene transfer tools and techniques
What is Gene transfer?
Insertion of copies of a gene into living cells in order to induce
synthesis of the gene's product.
Why Gene transfer?
To add a desired trait to a crop, a foreign gene (transgene) encoding
the trait must be inserted into plant cells, along with a “cassette” of
additional genetic material. The cassette includes a DNA sequence
called a “promoter,” which determines where and when the foreign
gene is expressed in the host, and a “marker gene” that allows
breeders to determine which plants contain the inserted gene by
screening or selection.

3. Methods of Gene Transfer
Vector-Mediated Gene Transfer
Agrobacterium mediated Plant viral vectors
 Crown gall disease
(A.tumefaciens)
 Hairy root
disease(A.rhizogenes))
 Caulimo viruses as
Vectors:
eg.Cauliflower
mosaic virus (CaMV)
 Gemini viruses
 RNA plant viruses
 Use of cDNA
Direct or Vector-less DNA Transfer
Physical chemical
 Electroporation
 Gene gun method
 Microinjection
 Silicon carbide fibres
Liposome
PEG mediated
 DEAE

4. Virus mediated gene transfer
 Plant viruses are considered as efficient gene transfer agents as they can
infect the intact plants and amplify the transferred genes through viral
genome replication. Viruses are natural vectors for genetic engineering.
They can introduce the desirable genes into almost all the plant cells since
the viral infections are mostly systemic.
 Plant viruses are non-integrative vectors i.e. the viral genomes are
suitably modified by introducing desired foreign genes. These
recombinant viruses are transferred, multiplied and expressed in plant
cells. They spread systemically within the host plant where the new
genetic material is expressed.
 Various viruses used for gene transfer are: Gemini virus, Cauliflower
mossaic virus(CaMV), RNA plant virus, Complementary DNA, etc.

5. Agrobacterium mediated gene transfer
 The Agrobacterium system was first successful plant transformation system.
 This method is also known as vector mediated gene transfer or biological method
of gene transfer.
 Agrobacterium is naturally occurring gram negative soil bacterium with two
species.
 A. Tumifacience induces tumors called crown gall (Ti plasmid) and A. rihzogenes
causes hairy root inducing (Ri plasmid).
 The Ti plasmid has an innate ability to transmit bacterial DNA into plant
cell.
 The gene of a donor organism can be introduced into the Ti plasmid at the
T-DNA region.
 This plasmid now becomes a recombinant plasmid.
 By Agrobacterium infection, the donor genes can transferred from the
recombinant Ti-plasmid and integrated into the genotype of the host
plant.

6. • The T-DNA region of any Ti plasmid
is defined by the presence of the
right and the left border sequences.
• These border sequences are 24bp
imperfect repeats.
• Any DNA between the borders will
be transferred into the genome of the
plant
• The vir (virulence) region of the Ti
plasmid contains the gene required
for the T-DNA transfer process.
• The gene in this region encode the
DNA processing enzymes required
for excision, transfer and integration
of the T-DNA segment.

7.  Like A. tumefaciens, A. rhizogenes also infect plants.
 But, unlike A. tumefaciens, it causes infection in hairy root giving
rise to hairy root disease.
 As A. tumefaciens is tumor inducing, A. rhizogenes is Root
inducing hence the plasmids isolated are referred to as Ri
plasmids(Root inducing).
 The products of these genes are involved in the metabolism of
plant growth regulators which gets sensitized to auxin and leads
to root formation.

8. It consist of the following four main steps-
1) Gene Cloning - Used to make multiple identical copies of a gene
• Plasmids of Agrobacterium contain tumour producing genes.
• The foreign gene which has to be used for genetic transformation is incorporated in the
plasmid for cloning.
2) Genetic transformation- The process of transfer, integration and expression of
transgene in the host is known as genetic transformation.
• For genetic transformation bacteria with cloned gene mixed in the cell culture or
protoplast culture of the host plant for a day or two.
• Out of several thousand of cells in the culture medium only few are transferred.
3) Identification of transformed cells- Selectable marker, PCR, ELISA, Southern,
northern and western blot techniques are used for detection of presence of transformed cells.
• Selectable markers is widely used they are attached to the target gene for easy
identification.
• The kanamycin resistant gene is used for identification of transformed cells.
• The lethal conc. is put in the plasmid mixed cell suspension the non-transformed cells
will die and only transformed cells will survive.
4) Regeneration of transformed cells- The transformed cells are transferred in to
culture medium for transformation into whole plant.

9. Limitations-:
• Host specificity- This method has limited range of host. It can not
infest monocots especially cereals.
• Somaclonal variation- Lots of variation is induced in the tissue
culture which poses problems in the identification of transformed
cells.
• Slow regeneration- Regeneration of cells into whole plant takes
more time than regeneration from meristem and organ culture.

10. Direct or Vector-less DNA transfer
• Introduction of DNA into plant cells without involvement of
biological agents such as Agrobacterium and leading to stable
transformation is called direct gene transfer.
• There are various methods of direct gene transfer -
1. Gene gun method
2. Electroporation
3. Micro-injection
4. Macro-injection
5. Chemical method
6. Pollen transformation
7. Delivery via growing pollen gene
8. Laser induced transformation
9. Fibre mediated gene transfer
10. Lipofection

11. Electroporation
.
• This method involves electroporation or chemical fusagens such
as PEG (poly ethyl glycol) with calcium phosphate.
• A suspension of protoplast with desired DNA is prepared then
high voltage current is applied through the protoplast.
• The electric current leads to formation of small temporary holes in
the protoplast membrane through which the DNA can pass.
• After entry into the cell the foreign DNA gets incorporated with
the host genome resulting in genetic transformation
• Then protoplast are then cultured into whole part.

12. • It is used with protoplast culture.
• This is cheaper method of foreign gene transfer.
• This is tedious than particle bombardment approach.
• It exhibits host specificity.

15. Advantages of electroporation
1. Method is fast.
2. Less costly.
3. Applied for a number of cell types.
4. Simultaneously a large number of cell can be treated.
5. High percentage of stable transformants can be produced.

16. Thiselectroporator is for low-current applications suchasthose using small electrodes

17. BIOLISTICS OR MICROPROJECTILES OR PARTICLE GUN
• First described as a method of gene transfer into plants by John Sanford at Cornell
university in 1987.
• This method is known by various names such as Particle bombardment method,
micro-projectile method and Particle acceleration method.
• Foreign DNA is delivered into host plant cells using high velocity metal particles
through gene gun.
• In this method regenerable tissues such as meristems, immature embryos and
embryogenic callus are used for bombardment .
• The gold or tungsten particles (1-1.5 milli macron size) are used for bambardment.
• The DNA to be transformed into the cells is coated onto microscopic beads made of
either gold or tungsten. Beads are carefully coated with DNA.

18. • The transformed tissues are identified by PCR.
• The transformed tissues are regenerated in culture medium into whole
plant.

20. • The gene gun has played a pivotal role in the production of
transgenic plants, allowing the transformation of species that were
previously difficult to transform. Examples includes maize,
soybean, wheat, barley, oat and rice. The first transgenic maize
and soybean varieties to be commercialized were produced by
particle bombardment.
• Another important application is the delivery of genes to
organelles. The finding that the chloroplast of Chlamydomonas
can be transformed by microprojectile bombardment has been
extended to higher plant.
• In addition to delivering DNA, the gene gun should be a
useful tool for delivering other molecules, such as RNA into cells.
GENERAL APPLICATIONS OF BIOLISTICS

21. Advantages and Disadvantages of biolistics
1. This method does not exhibit host specificity.
2. There is no need of isolation of protoplast.
3. This method is simple than Agrobacterium mediated method
of gene transfer.
Disadvantages
1. There is no control over the copy number and site of
integration of foreign gene.
2. Cost of equipment is high.

22. MICRO-INJECTION
• The injection of plasmid DNA is delivered into cells by microscopic needles
known as micro-injection.
• It is used with protoplast culture.
• The DNA solution is injected directly inside the cell using capillary glass
micropipettes with the help of micromanipulators of a microinjection

23. Applications of microinjection
 Process is applicable for plant cell as well as animal cell but more common for
animal cells.
 Technique is ideally useful for producing transgenic animal
quickly.
 Procedure is important for gene transfer to embryonic cells.
 Applied to inject DNA into plantnuclei.
 Method has been successfully used with cells and protoplast of tobacco, alfalfa etc.
 Microinjection is potentially a useful method for simultaneous introduction of
multiple bioactive compounds such as antibodies, peptides, RNAs, plasmids,
diffusion markers, elicitors, Ca2+ as well as nucleus and artificial micro or Nano
particles containing those chemicals into the same target single-cells.

24. • This technique does not require protoplast.
• Instrument will be simple and cheap.
• Methods may prove useful for gene transfer into cereals which do not
regenerate from cultured cell easily.
• Technically simple.
Advantages-:
Disadvantages-:
• The regeneration from protoplast is generally slow.
• In many plant species regeneration from protoplast is difficult.
• It is highly technical and time consuming.

25. LIPOSOME MEDIATED GENE TRANSFER
• This technique is found very successful in the transfection of plant
protoplasts and animal host cells.
• Liposomes are microscopic vesicles developed in a laboratory
environment.
• Each liposome is a spherical ball like structure made up of
phospholipid bilayers with a hollow central space, allowing
liposomes to interact directly with cells.
• A liposome can fuse with the cell membrane of the taken host cell and
can deliver its content to it. The recombinant DNA enclosed in the
liposome vesicles penetrates into the protoplast of the host cell.

26. • Lipofection Procedure :In this technique the recombinant
DNA, which is negatively charged at a near neutral pH
because of its phosphodiester backbone, is mixed with the lipid
molecules with positively charged (cationic) head groups.
• The lipid molecules form a bilayer around the recombinant
DNA molecules. This results in the formation of liposomes
which are further mixed with the host cells.
• Most eukaryotic cells are negatively charged at their surface,
so the positively charged liposomes interact with the cells.
• Cells take up the lipid-recombinant DNA complexes, and some
of the transfected DNA enters the nucleus

28. CALCIUM PHOSPHATE MEDIATED DNA TRANSFER
• It was the first chemical transfection method to be used with animal
cells.
• Calcium phosphate is probably the most widely used transfection
method.
• This is a simple, reliable method applicable to many cultured cell
lines, and the reagents are inexpensive.
• It can be used both for transient and stable transformation.
• The principle of the technique is that DNA in a buffered phosphate
solution is mixed gently with calcium chloride, which causes the
formation of a fine DNA-calcium phosphate coprecipitate.

29. • The precipitate settles onto the cells and some of the particles are taken
up by endocytosis. The most efficient transfection occurs in cells
growing as a monolayer, because these cells become evenly coated with
the precipitate.
• The procedure was developed in 1973 by Graham and van der Erb for
the introduction of adenovirus DNA into rat cells.
• In a report by Szybalska and Szybalski published in 1962 the presence
of calcium was shown to be responsible for the successful
transformation of human cells with genomic DNA.
• The first mammalian cell lines stably transfected with plasmid DNA
were also produced by calcium phosphate transfection, in 1978.

30. Limitations of calcium phosphate mediated DNA transfer
• Frequency is very low.
• Integrated genes undergo substantial modification.
• Many cells do not like having the solid precipitate adhering to them and
the surface of their culture vessel.
• Due to above limitations transfection applied to somatic gene therapy is
limited.

31. POLYETHYLENE GLYCOL MEDIATED TRANSFECTION
 This method is utilized for protoplast only. Polyethylene glycol stimulates
endocytosis and therefore DNA uptake occurs.
 Protoplasts are kept in the solution containing PEG.
 Calcium chloride is added and sucrose and glucose acts as osmotic
buffering agent.
 After exposure of the protoplast to exogenous DNA in presence of PEG
and other chemicals, PEG is allowed to get removed. Intact surviving
protoplasts are then cultured to form cells with walls and colonies in turn.
 After several passages in selectable medium frequency of transformation
is calculated. PEG based vehicles were less toxic and more resistant to
nonspecific protein adsorption making them an attractive alternative for
non-viral gene delivery.

32. • Validation of transferred genes detected by
several methods-
• Selectable marker
• Southern blot techniques
• Northern blot techniques
• Western blot techniques

34. Thank You