Chapter 11: Genetic

Transformation of Plants

Anon Chaulagain
Department of Biotechnology, HWIC

1
Transformation of plant cells
• Genetic transformation involves the integration of gene into
genome by means other than fusion of gametes or somatic cells

• The foreign gene (termed the "transgene") is incorporated into

the host plant genome and stably inherited through future
generations

• This plant transformation approach is being used to generate

plant processing trails, unachievable by conventional plant
breeding, especially in case where there is no source of the
desired trait in the gene pool

• In the gene of interest, the correct regulatory sequences are

incorporated i.e. promoters and terminators, and then the DNA is
transferred to the plant cell or tissue using a suitable vector
• The gene of interest is attached to a selectable marker which
allows selection for the presence of the transgene

• Confirmation for the presence of inserted genes is generally

tested by resistance to a specific antibiotic present in the medium

• Once the plant tissue has been transformed, the cells containing
the transgene are selected and regeneration back into whole
plants is carried out

• This is possible as plant cells are totipotent, which means that

they contain all the genetic sequence to control the development
of that cell into a normal plant
• Therefore, the gene of interest is present in every single plant cell;
however, where its expression is controlled by the promoter

• Plant transformation can be carried out by various ways

depending on the species of the plant

• A major method of DNA transfer in plants is Agrobacterium

mediated transformation

• Agrobacterium is a natural living soil bacteria and is capable of

infecting a wide range of plant species, causing crown gall
diseases

• It has natural transformation abilities

• When A. tumefaciens infects a plant cell, it transfers a copy of its
T-DNA, which is a small section of DNA carried on its Ti (Tumour
inducing) plasmid

• This T-DNA is flanked by two (imperfect) 25 base pair repeats

• Any DNA contained within these borders will be transferred to

the host cell when used as transformation vector
Different types of plant transformation vectors
• Plant transformation vectors comprises of plasmids that have
been purposely designed to facilitate the generation of genetically
modified plants

• The most commonly applicable plant transformation vectors are

binary vectors which have the ability to replicate in E. coli, a
common lab bacterium; as well as in Agrobacterium tumefaciens,
bacterium used to insert recombinant/customized DNA into plants

Plant transformation vectors contain three essential elements:

 Plasmids selection (creating a custom circular strand of DNA)
 Plasmids replication (so that it can be easily worked with T-DNA)
 T-DNA region (inserting the DNA into the Agrobacterium)
1. Co-integrate pTi vector:

• The genes of interest to be transferred into plants are initially

cloned in E. coli for obvious reasons of ease in the cloning
procedures

• A cointegrate vector is produced by integrating the modified E.

coli plasmid (used for cloning of and containing the gene construct
to be transferred) into a disarmed pTi

• The deletion of genes governing auxin and cytokinin production

(the oncogenes) from T-DNA of a Ti plasmid is known as disarming

• Agrobacterium containing this disarmed plasmid still transferred

this modified T-DNA into plant cells
• The cells containing the modified T-DNA were nontumorous,
produced nopaline and readily regenerated plantlets

• Since then it has been shown that only the LB and RB sequences
of T-DNA are necessary for the transfer of any DNA insert placed
between them

• The cointegration of the two plasmids is achieved within

Agrobacterium by homologous recombination

• The E. coli plasmid, e.g., pBR322, and the disarmed pTi must have
some sequences common to both for recombination to occur

• A common approach for ensuring this is to disarm a pTi by

replacing its oncogenes with sequences from the E. coli plasmid to
be used for cloning the DNA insert/gene to be transferred
• For e.g, pTiC58 was
disarmed by replacing its
oncognes with E. coli
plasmid pBR322 sequences

• This disarmed pTi is

designated as pGV3850

Figure: Homologous recombination between

a suitably disarmed pTi and a recombinant IV
(intermediate vector) containing the desired
DNA insert to produce a cointegrate vector
• The pBR322 is suitably modified to produce an intermediate
vector (IV)

• The IV must contain:

 origin for replication in E. coli

 pBR322 sequences present in the T-region of the disarmed pTi

 T-DNA (without the borders) from pTi

 appropriate selectable markers, e.g., neo gene for selection of

plant cells containing the recombinant T-DNA (T-DNA containing
the DNA insert) and kanr (kanamycin resistance) for the
selection of cointegrate vector in Agrobacterium
• The DNA inserts can be readily and conveniently inserted within
the T-DNA present in it to yield a recombinant IV

• The transfer of recombinant IV from E. coli into the

Agrobacterium is usually achieved by conjugation

• Since IV is nonconjugative, an E. coli strain containing a

conjugation-proficient plasmid, called helper plasmid, to mobilize
the transfer of IV is used

• An example of a conjugation-proficient helper plasmid is

pRK2013, which has the tra genes of the naturally occurring
plasmid pRK2
• The plasmid pRK2013 also does not have an origin for replication
in Agrobacterium

• Homologous recombination between the recombinant IV and the

disarmed pTi will integrate the former into the T-region (i.e.,
between the left and right borders of T-DNA) of the latter

• The resulting plasmid is a cointegrate pTi and is used as a vector

for transferring the DNA insert present in it into the plant
genomes
2. Binary vector:

• Binary vector is a combination of two vectors together present in

a single cell and T DNA present in one vector is transferred to the
host cell by the expression of number of genes of Vir region
present in another vector inside the same cell

• The vir region of Ti plasmid need not be present in the same

plasmid for an efficient transfer of T-DNA

• Vir region is essential to transfer T DNA to the host cell

• A binary vector consists of a pair of plasmids of which one

plasmid contains disarmed T-DNA sequences, while the other
contains the vir region, and ordinarily lacks the entire T- DNA
including the border
• The plasmid containing disarmed T-DNA is called mini-Ti or micro-
Ti, e.g., Bin 19, and has the origins for replication in both E. coli
and Agrobacterium

• The DNA insert is integrated within the T-region of mini-Ti, and

the recombinant mini-Ti is cloned in E. coli

• Transfer of recombinant mini-Ti from E. coli into Agrobacterium is

achieved either by conjugal transfer or direct transformation

• Mini-T Bin 19 has kanr (kanamycin resistance) gene for the

selection of Agrobacterium cells containing Bin 19, and neo gene
for the selection of transformed plant cells

• It also has a polylinker site (a DNA sequence having unique

restriction sites for several restriction endonucleases) within the α
peptide gene of E. coli lacZ locus
• Integration of a DNA insert in the polylinker site disrupts lacZ-α
enabling the selection of E. coli colonies containing the
recombinant mini-Ti as they form white colonies on X-gal + IPTG
medium as against the normal blue colonies

• The polylinker and the neo gene are placed within the LB and RB
sequences of T-DNA

• Bin 19 is capable of replication both in E. coli and Agrobacterium,

as it is based on the broad host range plasmid pRK252

• The “helper” plasmid is a Ti plasmid having a functional vir region

but lacking the T-DNA region, including the border sequences

• pAL4404 helper Ti plasmid is derived from the wild type pTiAch5

by deletion of the entire T-region
• The vir genes present in “helper” pAL44O4 induce the transfer of
T-DNA (containing the DNA insert) of the mini-Ti Bin 19 into the
plant cells

• The transformed plant cells can be selected on kanamycin

medium due to the gene neo present within the T-DNA

• The binary system avoids the transfer into plant cells/genomes of

unnecessary sequences, which occurs in the case of cointegrate
vectors

• BIBAC2 is the first such vector; it contains F-plasmid origin of

replication and transforms tobacco with high efficiency

• These vectors can also be used to transfer multiple genes, e.g.,

genes encoding sequentially acting enzymes of a metabolic
pathway
3. Plant virus vectors:

• Viruses have the following attractive features as vectors:

(i) viruses infect cells of adult plants

(ii) they produce large number of copies per cell leading to gene
amplification, and production of the recombinant protein in
large quantities

(iii) some viruses are systemic in that they spread throughout the
plant
• Plant virus genomes do not integrate into plant genome; as a
result, they cannot be used to produce stable and heritable
transformations

• But they can be used to express transgenes with a view to either

improve the phenotypic performance of host plants or to produce
large quantities of valuable proteins

• Most plant viruses have RNA genomes; two such viruses that have
great potential as vectors are, brome mosaic virus (BMV) and
tobacco mosaic virus (TMV)

• But the greatest progress has been made with the two groups of
viruses having DNA genomes, viz., caulimoviruses and gemini
viruses
3.1 Cauliflower mosaic virus (CaMV):

• The Cauliflower Mosaic Virus (CaMV) is a double-stranded DNA

virus (nearly 8 kb) which infects a wide range of crucifers,
especially Brassicas, such as cabbage, cauliflower, oilseed rape or
mustard

• The caulimovirus group infects a wide range of dicot crops

• In order to get itself and its DNA replicated (multiplied) within a

plant cell, the virus must trick the plant's own molecular
‘machinery' to do this task

• For this purpose the virus has two promoters (35S and 19S) in
front of its genes, which the plant cell believes to be its own
• These promoters override the plant's own regulatory system, as
they are constitutive, i.e. they are constantly switched on and
can't be regulated or switched off by the plant

• The CaMV 35S well known promoter is being used in almost all
GM crops currently grown or tested, especially GM maize

• It is the promoter of selection for plant genetic engineering, as it

is a strong and constitutive promoter

• Failure to distinguish or to ignore its capacity to be universally

active in almost any organism is irresponsible and careless and
shows a serious lack of scientific rigor and commitment to safety
The two ORF regions, one (ORFII) codes for insect transmission factor and
other (PRF VII) with unknown functions can be replaced with gene of interest
3.2 Gemini viruses:

• These viruses infect a variety of monocot and dicot plants

• They have circular single-stranded DNA genomes

• Maize streak virus (MSV) is a member of this group

• They cause yellow streaks on maize leaves, and is able to produce

infection only when transmitted by its natural insect vector (leaf-
hopper)

• MSV multiplies to a high copy number in the nuclei of dividing

cells
• MSV genome has been successfully introduced into plant cells
with the help of Agrobacterium (agroinfection)

• In this approach, MSV genome (native as well as cloned) was

inserted within the T-DNA of pTi in form of a tandem dimer

• Agrobacterium containing this recombinant pTi was used to

infect maize plants, which developed streaks on their leaves
within 2 weeks of inoculation
3.3 Tobacco mosaic virus (TMV):
• TMV have single-stranded RNA genome which also serves as
mRNA
• It encodes at least four proteins in three open reading frames
• Its genome contains 4 genes, of these the coat protein (cp) gene
seems to be nonessential and can be site of integration of
transgene
• Viral RNA promoters are successfully manipulated for the
synthesis of recombinant messenger RNAs in whole plants
• This vector consist of two steps, first, is the use of cDNA copy of
viral genome for cloning in E. coli
• Second, is in vitro transcription of the recombinant viral genome
cDNA to produce infectious RNA copies to be used for plant
infection

• In one study, the bacterial gene cat was inserted just downstream
of the cp gene initiation codon, & the RNA transcripts of this
recombinant genome were used to infect tobacco plants

• Gene cat was expressed in the infected leaves, but there was no
systemic spread of the infection

• TMV CP is produced in large amounts; hence its promoter is

suitable for an efficient expression of transgenes
3.4 Brome mosaic virus (BMV):

• BMV infects several species of Graminae, including barley

• It has three genomic segments (1, 2 and 3) each packaged into a

separate particle

• The CP gene is located on the RNA segment 3, and is the only

target site for DNA insertion, although this prevents the
formation of virus particles

• The bacterial gene cat was inserted in the cp gene of RNA 3, using
its cDNA, and its RNA transcripts were used together with RNAs 1
and 2 to infect barley protoplasts
• The infected protoplasts showed a high cat activity

• This indicates that placing a transgenes downstream to the

regulatory sequences of the cp gene of BMV will give high yields
of the protein encoded by it

• So the main steps involved in plant transformation using vectors

includes:

 Propagate vector in E. coli

 Isolate vector from E. coli and engineer (introduce a foreign

gene)

 Re-introduce engineered vector into E. coli to amplify

 Isolate engineered vector

 Introduce into Agrobacteria

 Infect plant tissue with engineered Agrobacteria (T-DNA

containing the foreign gene gets inserted into a plant cell
genome)

 In each cell T-DNA gets integrated at a different site in the

genome
Mode of Gene Delivery in Plant
• Different systems are now available for gene transfer and
successive regeneration of transgenic plants

• Most common being Agrobacterium-mediated transformation

• However, the preferred host of Agrobacterium is the dicot plants

and its efficiency to transfer genes in monocots is still
unsatisfactory

• The alternative to this, is the introduction of DNA into plants cells

without the involvement of a biological agent like,
Agrobacterium, and leading to stable transformation is known as
direct gene transfer
• The most often applied direct methods are microprojectile
bombardment or protoplast transformation

• The direct DNA transfer methods have been subdivided into three
categories:

1. Chemical transformation

2. Electroporation

3. Microinjection

4. Particle bombardment method

1. Chemical transformation:

• Many chemicals such as polyamines (poly-L-ornithine and poly-L-

lysine) and dextran sulphate stimulate DNA uptake into
protoplasts

• However, they also highly reduce cell viability

• Of the many chemicals tested for their ability to stimulate DNA

uptake into protoplasts polyethylene glycol (PEG) has proved
most effective

• The optimum chemical transformation under the influence of PEG

is very simple
• The protoplasts suspended in a medium containing biologically
active DNA are treated with a relatively high concentration (28%)
of high molecular weight (4000-6000, pH 8-9) PEG

• It is recommended that DNA is added before PEG

• Treatment of protoplasts with MgCl2 (5-25 mM) significantly

improves transformation rates

• The DNA carrying the gene to be introduced should be offered to

protoplasts in linear form
2. Electroporation:

• A popular physical method for introducing new genes into

protoplasts is the use of electric field which makes the protoplasts
temporarily permeable to DNA

• In this method, electric field is playing important role

• Due to the electric field protoplast get temporarily permeable to

DNA

• In electroporation, plant cell protoplasts are kept in an ionic

solution containing the vector DNA in a small chamber that has
electrodes at opposite ends
• A pulse of high voltage is applied to the electrode which makes
the transient pores (ca. 30 nm) in the plasma membrane, allowing
the DNA to diffuse into the cell

• Immediately, the membrane reseals

• If appropriately treated, the cells can regenerate cell wall, divide

to form callus & finally, regenerate plants in suitable medium

• The critical part of the procedure is to determine conditions which

produce pores that are sufficiently large and remain open long
enough to allow for DNA diffusion

• At the same time, the conditions should make pores that are
temporary
• With a 1 cm gap between the electrodes and protoplasts of 40-
44µm diameter, 1-1.5 kVcm-2 of field strength for 10µs is required
for efficient introduction of DNA

• It was seen that presence of 13% PEG (added after DNA) during
electroporation significantly raised the transformation frequency

• The other factors which may improve the transformation

frequency by electroporation are linearizing of plasmid, use of
carrier DNA, and heat shock (45 ~ for 5 min) prior to addition of
vector, and placing on ice after pulsing

• Under optimal conditions transformation frequencies of up to 2%

have been reported

• Stably transformed cell lines and full plants of a number of cereals

have been produced through electroporation
Fig. Electroporation
• There are some parameters that can be considered when
performing in vitro electroporation:

a) Cell size:

• Cell size is inversely correlated to the size of the external field

needed to generate permeabilization

• Consequently, optimization for each cell type is essential

• Likewise, cell orientation matters for cells that are not spherical

b) Temperature:

• Plant membrane resealing is effectively temperature dependent

• Shows slow closure at low temperatures

• For DNA transfer, it has been found that cooling at the time of
permeabilization and subsequent heating in incubator increases
transfer efficacy and cell viability

c) Post-pulse manipulation:

• Cells are susceptible when in the permeabilized state

• It has been shown that waiting for 15min after electroporation in

order to allow resealing before pipetting cells, increases cell
viability
d) Composition of electrodes and pulsing medium:

• Short pulses is needed for release of metal from the standard

aluminium electrodes used in standard disposable cuvettes

• Some authors advocate the use of low conductivity or more

resistance media for DNA transfer in order to increase viability
and increase transfection efficacy
3. Microinjection:

• The microinjection technique is a direct physical approach to

inject DNA directly into the plant protoplasts or cells (specifically
into the nucleus or cytoplasm) using fine tipped (0.5-1.0 µm
diameter) capillary glass needle or micropipettes

• Through microinjection technique, the desired gene introduce

into large cells, such as oocytes, eggs, and the cells of early
embryo
4. Particle Bombardment:

• The Particle bombardment device, well known as

the gene gun, was developed to enable
penetration of the cell wall so that genetic
material containing a gene of interest can be
transferred into the cell

• This physical direct gene transfer method, gene

gun is used for genetic transformation of several
organisms to introduce a diverse range of
desirable traits
Fig. A gene gun
• Plant transformation using particle bombardment apparatus
follows the same steps as in Agrobacterium
mediated transformation method
 Isolation of desired genes from the source organism
 To develop a functional transgenic construct including the
selected gene of interest; promoters to drive expression;
modification of codon, if needed, to increase successful protein
production; and marker genes to facilitate tracking of the
introduced genes in the host plant
 Insertion of transgenic construct into a useful plasmid
 Introduce the transgenes into plant cells
 Regenerate the plants cells
 Test the performance of traits or gene expression under in vitro,
greenhouse and field conditions
• In particle bombardment method, 1-2 µm tungsten or gold
particles (called micro-projectiles) coated with genetically
engineered DNA are accelerated with air pressure at high
velocities and shot into plant tissues on a Petri-plate

• This is the second most widely used method, after Agrobacterium

mediated transformation, for plant genetic transformation

• The device accelerates particles in one of the two ways:

 by means of pressurized helium gas or

 by the electrostatic energy released by a droplet of water

exposed to high voltage
Fig. Diagrammatic illustration of gene transfer using Gene Gun method
• The earlier devices used blank cartridges in a modified firing
mechanism to provide the energy for particle acceleration, and
thus, the name particle gun

• It is also called Biolistics, Ballistics or Bioblaster

• The microcarriers (or microprojectiles), the tungsten or gold

particles coated with DNA, are carried by macrocarriers (macro
projectiles) which are then inserted into the apparatus and
pushed downward at high velocities

• The Macro-projectile is stopped by a perforated plate, while

allowing the microprojectiles to propelled at a high speed into the
plant cells on the other side
• As the micro-projectiles enter the plant cells, the transgenes are
free from the particle surface and may inserted into the
chromosomal DNA of the plant cells

• Selectable markers help in identifying those cells that take up the

transgene or are transformed

• The transformed plant cells are then regenerated and developed

into whole plants by using tissue culture technique

• The technique has many advantages and can be used to deliver

DNA into virtually all the tissues, like immature and mature
embryos, shoot-apical meristem, leaves, roots etc.

• Particle bombardment methods are also useful in the

transformation of organelles, such as chloroplasts
• This enables engineering of organelle-encoded herbicide or
pesticide resistance in crops & to study photosynthetic processes

• Limitations to the particle bombardment method, compared to

Agrobacterium-mediated transformation, include:
 frequent incorporation of multiple copies of the transgene at a
single insertion site
 rearrangement of the inserted genes
 insertion of the transgene at multiple insertion sites
 these multiple copies can be associated with silencing of the
transgene in subsequent progeny
 the target tissue may often get damaged due to lack of control
of bombardment velocity
Agrobacterium mediated gene transfer
• Agrobacterium tumefaciens and Agrobacterium rhizogenes are
common gram-negative soil borne bacteria causing induction of
“crown gall” and “hairy root” diseases

• These bacteria naturally insert their genes into the genome of

higher plants

• The studies on crown gall formation revealed that the virulent

strains of bacteria introduce a part of their genetic material into
the infected cells where it gets integrated randomly with the
genetic material of the host cell
• The bacterial genes are able to replicate along with the plant
genome and uses the machinery of plants to express their genes
in terms of the synthesis of a special class of compounds, called
opines, which the bacterium uses as nutrients for its growth but
are useless to the host cells

• In the process, Agrobacterium causes plant tumors (gall

formation) commonly seen near the junction of the root and the
stem and is called “crown gall disease”

• A. tumefaciens attracted to the wound site via chemotaxis, in

response to chemicals (sugars and phenolic molecules) released
from the damaged plant cells
• The disease afflicts a great range of
dicotyledonous plants, which constitute
one of the major groups of flowering plants

• Tumorous plant cells were found to contain

DNA of bacterial origin integrated in their
genome

• Furthermore, the transferred DNA (named

T-DNA) was originally part of a small
molecule of DNA located outside the
chromosome of the bacterium

• This DNA molecule was called Ti (tumor-

inducing) plasmid
Plant gene structure
• Plant ribosomal RNA genes and a number of other structural
genes from a variety of species have now been analyzed in
considerable detail

• In common with many animal genes, some plant gene sequences

have been found to have their coding sequences interrupted by
introns or intervening sequences

• These introns are transcribed but not represented in mature

mRNA and hence, are not translated

• No introns have been found in rRNA genes but they have been
demonstrated in a number of other plant structural genes
A typical plant gene has the following region beginning with 5'end:

 Promoter: For transcription initiation

 Enhancer/silencer: Concerned with regulation of gene
 Transcriptional start site or cap site: From here initiation of transcription
take place
 Leader sequence: It is untranslated region
 Initiation codon
 Exons
 Introns
 The stop codon
 A second untranslated region
 Poly A tail
• Promoter is a region of DNA sequence which helps in the
transcription of a particular gene

• This contains specific DNA sequences as well as response

elements which provide a secure initial binding site for RNA
polymerase

• These proteins called transcription factors that recruit RNA

polymerase

• The CAAT and TATA boxes represent consensus sequences within

promoter for RNA polymerase II
• ATG (AUG in mRNA) is initiation codon for mRNA translation, and
mark the beginning of coding sequence of the gene

• A sequence between the cap site and ATG is not translated and
form the 5'-leader sequence of mRNA

• Codon TAG/TAA/TGA are chain terminating codon and it is

followed by a stretch of nontranslated region

• At the end, poly-adenylation site is present which denotes the

end of transcription
Organization of T-DNA
• The transfer DNA (T-DNA) is the transferred DNA of the tumour
inducing plasmid (pTi) of some Agrobacterium species of bacteria

• T-DNA has both its side 24 kb direct repeat border sequence and
contains the gene for tumor/hairy root induction and also for
opines biosynthesis

• pTi has three genes, two of these genes (iaaM and iaaH) encode

enzymes which together convert tryptophane into IAA (Indol-3-
acetic acid) a type of auxin
• If these two genes are deleted then shooty crown gall will
produce

• Therefore, the locus was earlier called “shooty locus” and the
genes were designated as tms 1 (tumour with shoots) and tms 2

• The third gene, ipt, encodes an enzyme which produces Zeatin-

type cytokinin isopentenyl adenine

• The deletion of ipt, causes rooty crown galls and the region was
earlier designated as “rooty locus” and denoted by tmr (tumour
having roots)

• In addition to these, another locus called tml and the deletion of

which results in large tumours

• Besides, T-DNA also contains genes involved in opine

biosysnthesis which are located near the right border of T-DNA
• NOS gene is also present which involves in the synthesis of
enzyme nopaline synthase (nopaline type Ti)

• In case of octopine type Ti, it contains OCS gene for octopine

synthase

• All the genes present in T-DNA contain eukaryotic regulatory

sequences

• As a result, these genes are expressed only in plants and not

expressed in Agrobacterium

• Transfer is initiated at the right border and terminated at the left

border and requires the vir genes of the Ti plasmid to transfer T
DNA into plant cell
T-DNA transfer and integration
• The steps involved in T-DNA transfer and integration into the
plant genome are explained in figure:
• Wounded plant cell releases phenolics substances and sugars (1);
which are sensed by vir A, vir A activates vir G, vir G induces
expression of vir gene of Ti-plasmid (2); vir gene produce all
the vir-protein (3); vir D1 and vir D2 are involve in ssT-DNA
production from Ti-plasmid and its export (4) and (5); the ssT-DNA
(with associated vir D1 and vir D2) with vir E2 are exported through
transfer apparatus vir B (6); in plant cell, T-DNA coated
with vir E2 (7); various plant proteins influence the transfer of T-

DNA + vir D1 + vir D2 + vir E2 complex and integration of T-DNA to

plant nuclear DNA (8); (LB= left border; RB= Right border; pTi = Ti
plasmid, NPC = nuclear pore complex)
Process
1. Signal recognition by Agrobacterium spp.:

• The wounded plant cells release certain chemicals, such as

phenolics and sugars

• These chemicals are recognized by Agrobacterium as signals

• This in turn results in a sequence of biochemical events in

Agrobacterium that helps in transfer of T-DNA of Ti plasmid

2. Attachment to plant cell:

• Attachment of this bacterium to plant cells is a two step process

• It involves an initial attachment via a polysaccharides (the product

of att R locus)
• Subsequently, a mesh of cellulose fibres is produced by
Agrobacterium

• Several chromosomal virulence genes (chv genes) are involved in

attachment of bacterial cells to the plant cells

3. Induction of virulence gene:

• vir A (a membrane-linked sensor kinase) senses phenolics (such as

acetosyringone) and autophosphorylates, subsequently
phosphorylating and, thereby, activating vir G

• This activated vir G induces expression of virulence gene of Ti

plasmid to produce the corresponding virulence proteins (D, D2,
E2, B)

• It has been also identified that certain sugars (e.g. glucose,

galactose, xylose etc.) also induce virulence gene
Table. Agrobacterium virulence protein function
4. Production of T-DNA strand:

• The right and left border sequence of T-DNA are identified by vir
D1/vir D2 protein complex and vir D2 produces single stranded
DNA (ss-T-DNA)

• After nicking, vir D2 becomes covalently attached to the 5'end of

ss-T-DNA strand & protect & export the ss-T-DNA to plant cells

5. Transfer of T-DNA out the bacterial cell:

• The ss-T-DNA – vir D2 complex in association with vir E2 is

exported from bacterial cell by a “T-pilus” (a membrane channel
secretary system)
6. Transfer T-DNA into plant cell and integration:

• The single stranded T-DNA – vir D2 complex and other vir

proteins cross the plant plasma membrane

• In the plant cells, T-DNA gets covered with vir E2

• This covering of vir E2 helps in protection of ss-T-DNA from

degradation by nucleases

• vir D2 and vir E2 interact with variety of plant proteins which

influence the T-DNA transport and integration

• The T-DNA – Vir D2 – Vir E2 – plant proteins complex enters the

nucleus through nuclear pore complex (NPC)
• In the nucleus, T-DNA gets integrated into the plant genome by a
process referred to as “illegitimate recombination”

• This process is unlike homologous recombination as it does not

depend on extensive region of sequence similarity
Ti and Ri Plasmids
• Agrobacterium species harboring tumor-inducing (Ti) or hairy
root-inducing (Ri) plasmids cause crown gall or hairy root
diseases, respectively in plants

• Agrobacterium tumefaciens is a plant pathogen that induces

tumor on a wide variety of dicotyledonous plants and the disease
is caused by tumor-inducing plasmid (pTi)

• Similarly, Agrobacterium rhizogenes is a plant pathogen that

induces hairy roots on a wide variety of dicotyledonous plants and
the disease is caused by root-inducing plasmid (pRi)

• Virulence (vir) genes of Ri as well as of Ti plasmids are essential

for the T-DNA transfer into plant chromosomes
• These natural plasmids provide the basis for vectors to make
transgenic plants

• The plasmids are approximately 200 kbp in size

• Both pTi and pRi are unique in two respects:

(i) they contain some genes, located within their T-DNA, which
have regulatory sequences recognized by plant cells while their
remaining genes have prokaryotic regulatory sequences

(ii) both plasmids naturally transfer a part of their DNA, the T-DNA,
into the host genome, which makes Agrobacterium a natural
genetic engineer
• Complete sequence analysis confirms that the pathogenic
plasmids contain gene clusters for DNA replication, virulence, T-
DNA, opine utilization and conjugation

• T-DNA genes have lower GC content, which is presumably

suitable for expression in host plant cells

• Besides these genes, each plasmid has a large no. of unique genes

• Even plasmids of the same opine type differ considerably in gene

content and are highly chimeric in structures

• The plasmids seem to interact with each other and with plasmids
of other members of the Rhizobiaceae and are likely to shuffle
genes of infection between Ti and Ri plasmids

• Plasmid stability genes are talked about, which are important for
plasmid evolution and construction of useful strains
The Ti plasmid
• The Ti plasmid contains all the genes which required for tumor
formation

• Virulence genes (vir genes) are also located on the Ti plasmid

• The vir genes encode a set of proteins responsible for the

excision, transfer and integration of the T-DNA into the plant
nuclear genome

• The basic elements of the vectors designed for Agrobacterium-

mediated transformation that were taken from the native Ti-
plasmid

 The T-DNA border sequences, at least the right border, which

initiates integration of the T-DNA region into the plant genome
 The vir genes, which are required for transfer of the T-DNA region
to the plant

 A modified T-DNA region of the Ti plasmid, in which the genes

responsible for tumor formation are removed by genetic
engineering and replaced by foreign genes of diverse origin, e.g.,
from plants, bacteria, virus; when these genes are removed,
transformed plant tissues or cells regenerate into normal-
appearing plants and, in most cases, fertile plants

• The T-DNA region genes are responsible for the tumorigenic

process

• Some of them control the production of plant growth hormones

that cause proliferation of the transformed plant cells
• The T-DNA region is flanked at both ends by 24 base pairs (bp)
direct repeat border sequence called T-DNA borders

• The T-DNA left border is not essential, but the right border is
indispensable for T-DNA transfer

• Ti plasmid is grouped into two general categories:

• i) Nopaline type pTi

• ii) Octopine type pTi

Fig. the Ti plasmid: (A) nopaline type pTi; (B) Octopine type pTi
Ri plasmid
• Agrobacterium rhizogenes is a soil born gram negative bacterium

• It causes hairy root disease of many dicotyledonous plants

• The ability of A. rhizogenes to incite hairy root disease is

confirmed by a virulence plasmid, which is similar to that found in
A. tumefaciens which causes crown gall tumors of plants

• The virulence plasmid of A. rhizogenes is commonly known as the

Ri-plasmid (pRi)

• The pRi have extensive functional homology with the pTi

• The pRi contains distinct segment(s) of DNA, which is transferred

to plant genome during infection
• The transfer T-DNA to the plant genome is mediated by another
segment on the plasmid known as the virulence (vir) region

• All strains of A. rhizogenes are known to produce agrocinopine

Fig. Ri plasmid
Selection and Screening of Transformations
• Genetic selection of transformed cells is a significant step of any
plant transformation

• Screening of transformed cells or plants for gene integration and

expression in transformed cells or plants is a process that involves
several techniques, including DNA and RNA blot hybridization
analysis, PCR, ELISA analysis

• In the absence of a correct selection system one would face with

the option of screening every shoot that regenerates in a
transformation experiment

• In cases where transformation frequency is high this may be

possible but for plant species that transform with low frequencies
this would be a laborious, if not impossible, task
• Therefore, a selectable marker gene is incorporated into the plant
transformation vectors and an appropriate selecting agent is
added to the culture medium which favors the growth of only
transformed cells

• The genes used as selectable markers are dominant and typically

of bacterial origin

• For successful selection, the target plant cells must be susceptible

to moderately low concentrations of the selecting agent in a non-
leaky way

• The compound that inhibits the growth but does not kill the wild
type cells is preferred as a selecting agent in plant transformation

• The concentration of the selecting agent used varies widely

depending on sensitivity of the plant species and explant source
Table: Selectable marker genes used in plant transformation
• A screening can also be possible by screening or scorable or
reporter gene, incorporated into the transformation vectors,
which allows for the detection of transformed cells, tissues or
plants

• The essential features of an ideal reporter gene are:

i. An efficient and easy detection with high sensitivity

ii. Lack of endogenous activity in plant cells

iii. A relatively rapid degradation of the enzyme

Table: Screenable marker genes used in plant transformation
• The screening markers presently used are mostly derived from
bacterial genes coding for an enzyme that is readily detected by
the use of

 Chromogenic

 Fluorigenic

 photon emitting or radioactive substrates

• A screening marker gene is functional only if an enzyme with

comparable activity is not present in non-transformed cells

• The utility of any particular gene construct as a transformation

marker varies depending on plant species & the tissue involved
• The kanamycin resistance gene is probably the most extensively
used selectable marker phenotype and Uid A gene (also referred
to as gus), which encodes β-glucuronidase, is the most versatile
reporter gene
• The screened cells and the plants regenerated from
transformation; further subjected to biochemical analyses, s.a.
 Southern hybridization
 PCR
 Northern hybridization
• The former determines the presence and the number of copies of
the introduced gene while the latter demonstrates the presence
of transcripts of the transgene
Application of Genetic Engineering

1. Protection of plants against viruses

2. Protection of plants against insects

3. Production of herbicide-tolerant plants

4. Protection of plants against pathogenic bacteria & fungi

5. Protection against environmental stress

6. Control of senescence and fruit ripening

1. Protection of plant against viruses:
• Plant viruses can cause severe damage to crops by substantially
reducing vigor, yield, and product quality
• Virus resistance is achieved usually through the antiviral
pathways of RNA silencing, a natural defense mechanism of
plants against viruses