CHAPTER 3

Gene transfer methods in plants

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 What do mean gene transfer?

Gene transfer is introduction of new genetic material/DNA in to a cell

Introduction of additional DNA into plant cells or tissues is termed as plant

transformation

Gene transfer is a very important step in genetic engineering

 Genetic engineering transfer genes from verities of organisms in to

plants.

 Gene transfer method is a technique used to efficiently and stably

introduce foreign genes into the genome of target cells.

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A DNA of any source can be used for plant transformation.

These transformation process has become a very important technique for

both scientific and commercial purpose.

These transgenic plants, which have a new set of DNA, aid

• in the understanding of plant gene regulation and

• also for identification and evaluation of useful traits in plants.

 Plant transformation is used for the introduction of these useful traits into
other plants.

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The two main methods that are used to transfer foreign genes into plants

1. Indirect method

In this case vector is needed for insertion of the foreign DNA into the host
genome.

2. Direct methods

This method is vector independent.

The DNA is directly inserted into the host genome

The result of gene transfer is called transgenic organism.

 Transgenic organism is an organism whose genome has been altered by introducing one or more
DNA sequences from another species by artificial means.

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 Vector-mediated or indirect gene transfer
Indirect gene transfer method is also known as Agrobacterium-mediated
genetic transformation

Among the various vectors used in plant transformation, the Ti plasmid of

Agrobacterium tumefaciens has been commonly used

This bacterium is known as “natural genetic engineer” of plants

 because these bacteria have natural ability to transfer T-DNA of their

plasmids into plant genome upon infection of cells at the wound site
and cause an unorganized growth of a cell mass known as crown gall.

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Wounded plants secrete a sap with cells to form crown gall growth.
high content of phenolic compounds
which serve as chemical attractants
for Agrobacteria and stimulate
expression of vir genes

It results in infection of plant by

Agrobacterium, insertion of T-DNA
region at a random site in host
genome and proliferation of plant

Crown gall disease caused by Agrobacterium

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Ti plasmids
• Tumour inducing (Ti) plasmids are used as gene vectors for delivering
useful foreign genes into target plant cells and tissues.

 In transformation experiments, vector is the genetic carrier needed to

transport the gene of interest, promoter, terminator and selectable marker
genes to DNA of host plant.

• The foreign gene is cloned in the transfer DNA (T-DNA) region of Ti-
plasmid in place of unwanted sequences

 Virulence of Agrobacterium is conferred by Ti plasmid having genes for

tumor-induction, T-DNA integration and synthesis of plant hormones and
opines.
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Organization of Ti plasmid

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T-DNA region:

This region has the genes for the biosynthesis of auxin (aux), cytokinin
(cyt) and opine (ocs), and

is flanked by left and right borders.

These three genes-aux, cyto and ocs are referred to as oncogenes,

as they are the determinants of the tumor phenotype

Virulence region:

 The genes responsible for the transfer of T-DNA from Agrobacteria to

plant cells are located outside T-DNA and the region is referred to as vir or
virulence region.

 Vir region codes for proteins involved in T-DNA transfer 9

Opine catabolism region:

This region codes for proteins involved in the uptake and metabolisms of
opines

The growth hormones cause plant cells to proliferate and form the gall
while opines are utilized by A. tumefaciens as sources of carbon and
energy

In addition to T-DNA , Virulence and Opine catabolism regions there is

ORI region in Ti plasmid that is responsible for the origin of DNA
replication which permits the Ti plasmid to be stably maintained in A.
tumefaciens.

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Ti plasmid vectors are mainly composed of the following components:

1. The right border sequence of T-DNA which is absolutely required for T-

DNA integration into plant cell DNA.

2. A multiple cloning site (poly-linker DNA) that promotes the insertion of

cloned gene into the region between T-DNA borders.

3. An origin of DNA replication that allows the plasmids to multiply

4. A selectable marker gene for appropriate selection of the transformed

cells.

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 To use Ti-plasmids a vector its T-DNA region is removed excepting the border
regions and the vir genes.

 Within Agrobacterium, T-DNA is part of a small circular DNA sequence called the
Ti plasmid

 The transgene is then inserted between the T-DNA regions, where it is

transferred to plant cell and becomes integrated into the plant‟s chromosome

 Agrobacterium Ti plasmid is preferred over all other vectors, because of wide

host range of this bacterial system and the capacity to transfer genes due to the
presence of T - DNA border sequences

Researchers replace the Agrobacterium genes on T-DNA with the gene they are
studying, and the Agrobacterium is used to infect the plant tissues. 12
 Steps involved in Agrobacterium-mediated genetic transformation of
plants by „Wounded explant‟ method

To transform plants,

1. leaf discs (in case of dicots) or embryogenic callus (in case of monocots)
are collected and infected with Agrobacterium carrying recombinant Ti-
plasmid vector.

2. The infected tissue is then cultured (co-cultivation) on shoot regeneration

medium for 2-3 days during which time the transfer of T-DNA along with
foreign genes takes place.

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3. After this, the transformed tissues (leaf discs) are transferred to plant
regeneration medium supplemented with usually lethal concentration of an
antibiotic to selectively eliminate non-transformed tissues

4. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred
to root-inducing medium,

5. After another 3-4 weeks, complete plants are transferred to soil following
the hardening (acclimatization) of regenerated plants

In the process of transgenic plant development cultured cells and

protoplasts can be used for gene transfer followed by regeneration leading
to the production of transgenic plants

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Diagram illustrating Agrobacterium-mediated transformation
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Generally in Agrobacterium mediated gene transfer:

1. Isolate genes of interest from the source organism.

2. Insert the transgene into the Ti-plasmid.

3. Introduce the T-DNA containing-plasmid into Agrobacterium

4. Co-culture of explants with Agrobacterium.

5. Killing of Agrobacterium with a suitable antibiotic

6. Selection of transformed plant cells.

7. Regeneration of whole plants.

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 Limitations to use Ti plasmids as a vector

 A major limitation of the Ti plasmid is that it works only in those plants that
the Agrobacterium normally infects.

 The need for long tissue culture periods to recover transgenic plants

 Requirements for sterile conditions for plant regeneration

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 Vectorless or direct gene transfer

 The term direct transfer of gene is used when the foreign DNA is directly
introduced into the plant genome.

 Direct DNA transfer methods rely on the delivery DNA into the plant cells.

 In the direct gene transfer methods, the foreign gene of interest is

delivered into the host plant cell without the help of a vector

 Direct methods are those methods which do not use bacteria as mediators
for integration of DNA into host genome. These methods include micro-
projectile bombardment, electroporation and microinjection.
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 Majority of the direct DNA transfer methods are simple and effective.

 Particle (or micro projectile) bombardment is the most effective method for
gene transfer, and creation of transgenic plants.

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Electroporation:
 Electroporation is an efficient method for the incorporation of foreign DNA
into protoplasts

 It is a method of DNA introduction into plant cells by making minute pores

in the plant cell membrane

 This method is based on the use of short electrical impulses of high field
strength.

 These impulses increase the permeability of protoplast membrane and

facilitate entry of DNA molecules into the cells

 The electric field causes holes to form in the plasma membrane allowing
DNA to be taken up by the cell
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 Treated protoplasts are then cultured to obtain cell colonies and plants
 Plant cell electroporation generally utilizes the protoplast because thick
plant cell walls restrict macromolecule movement
 Electroporation as a transformation method is fast, convenient, simple, and
inexpensive and has low cell toxicity.
 The disadvantage associated with this technique is difficulty in
regenerating plants from protoplasts, if protoplast is used for
electroporation.
 It has been achieved on a variety of species and tissue types.
 drawback: requires protoplast regeneration which is difficult.

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Microinjection

• Microinjection is a direct physical method involving the mechanical

insertion of the desirable DNA into a target cell.

• The target cell may be the one identified from intact cells, protoplasts,
callus, embryos, meristems, etc

• The technique of microinjection involves the transfer of the gene through a

micropipette into the cytoplasm/nucleus of a plant cell or protoplast.

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As the process of microinjection is complete, the transformed cell is
cultured and grown to develop into a transgenic plant.

In fact, transgenic tobacco and Brassica napus have been developed by
this approach.

The major limitations of microinjection are that it is slow, expensive, and

has to be performed by trained and skilled personnel.

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Illustration of microprojectile method

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Particle Bombardment
 It is also known as biolistics or gene gun method of gene transfer

 Particle bombardment is the most important and effective direct gene transfer method.

 The delivery of DNA into cells of intact plant organs or cultured cells is done by a process
called Projectile Bombardment.

 A biolistic particle delivery system is a device for plant transformation where cells are
bombarded with heavy metal particles coated with DNA

 Agrobacterium-mediated genetic transformation system works well for dicotyledonous

plants but has low efficiency for monocots.

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Were as Biolistic particle delivery system provides an effective and

versatile way to transform almost all type of cells.

Particle bombardment is the most effective method for gene transfer, and

creation of transgenic plants.

Important crop plants like, wheat, rice and maize have now been

transformed by this method.

This method is especially important for monocots, for which efficiency of

other transformation methods is not satisfactory.

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Plant transformation process using particle bombardment includes the
following steps:

(1) Isolate protoplasts from leaf tissues.

(2) Inject DNA-coated particles into the protoplasts using particle gun.

(3) Regenerate into whole plants.

(4) Acclimate the transgenic plants in a greenhouse

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Particle bombardment method of Plant transformation
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 Advantages of particle bombardment over Agrobacterium-mediated DNA
transfer
This system is species independent and can been used successfully for a wide
range of organisms.

Many species which are recalcitrant to other direct transfer methods or are not
readily amenable to Agrobacterium-mediated transformation have been
transformed by this technique.

Introduced DNA does not need sequences necessary for T-DNA replication and
transfer as complex interaction between bacterium and plant tissue does not take
place.

Transformation of organelle DNA (mitochondria and chloroplasts) has also been

achieved by this method.

Multiple genes can be introduced in a single plant.

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Limitations of particle bombardment method :

 Limited regeneration capacity of tissue being bombarded

Insertion of multiple copies of the gene

Damage to the cellular tissue.

Specialized and expensive equipment's are required

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 Group Assignment
1.Cosmids (Group 5)
2. bacteriophages, (Group1)
3. bacterial artificial chromosomes (BACs), (Group4)
4. yeast artificial chromosomes (YACs) (Group2)
5. Expression vectors(Group3)

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 CHAPTER 4

Selection and Regeneration of transgenic plants

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