Agrobacterium and plant viruses can be used as biological vectors for plant transformation. Agrobacterium mediates transformation via its tumor-inducing plasmid, using virulence genes to transfer T-DNA containing the gene of interest into the plant genome. Plant viruses can also act as gene vectors by engineering viral genomes to contain and deliver foreign genes. Retroviruses have been developed as viral vectors for animal gene transfer due to their ability to stably integrate into the host chromosome.

1. Biological method of transformation
- Agrobacterium mediated gene transfer
- Viral vector mediated gene transfer
Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to Bengaluru North University,
K. Narayanapura, Kothanur (PO)
Bengaluru 560077

2. Ti plasmid

3. Nature’s genetic engineer
1. Agrobacterium is considered as the nature’s genetic engineer.
2. Agrobacterium tumefaciens is a rod shaped, gram negative bacteria found in
the soil that causes tumorous growth termed as crown gall disease in dicot
plants.
3. The involvement of bacteria in this disease was established by Smith and
Townsend (1907).
4. Agrobacterium contains a transfer DNA (T-DNA) located in its tumor-inducing
(Ti) plasmid that is transferred into the nucleus of an infected plant cell.
5. The T-DNA gets incorporated into the plant genome and is subsequently
transcribed. The T-DNA integrated into the plant genome carries not only
oncogenic genes but also opine synthesizing genes.
Agrobacterium “Species” And Host Range
The genus Agrobacterium has been divided into a number of species on the
basis of symptoms of disease and host range.
A. radiobacter is an “avirulent” species,
A. tumefaciens causes crown gall disease,
A. rubi causes cane gall disease,
A. rhizogenes causes hairy root disease and
A. vitis causes galls on grape and a few other plant species

4. Molecular basis of Agrobacterium-mediated transformation
Ti-plasmid
The virulent strains of A. tumefaciens harbor large plasmids (140–235 kbp) known as
tumor-inducing (Ti) plasmid involving elements like
A. T-DNA – Left border and Right border, Auxin and cytokinin and opine synthesizing
genes
B. vir region- Genes in the virulence region are grouped into the operons vir ABCDEFG,
which code for the enzymes responsible for mediating conjugative transfer of T-DNA to
plant cells.
C. origin of replication,
D. region enabling conjugative transfer and
E. o-cat region (required for catabolism of opines).
•The plasmid has 196 genes that code for 195 proteins.
• The plasmid is 206,479 nucleotides long, the GC content is 56% and 81% of the material is
coding genes.
•There are no pseudogenes.

5. •The Ti plasmid is lost when Agrobacterium is grown above 28 °C.
•The modification of this plasmid is very important in the creation of
transgenic plants.
A.) T- DNA (Transfer DNA)
1. It is a small, specific segment of the plasmid, about 24kb in size
and found integrated in the plant nuclear DNA at random site.
This DNA segment is flanked by right and left borders.
2. This region has the genes for the biosynthesis of auxin (aux),
cytokinin (cyt) and opine (ocs), and is flanked by left and right
borders. It is clearly established that the right border is more
critical for T -DNA transfer.
3. The T-DNA contains two groups of genes, which possess the
ability to express in plants as follows-
i. Oncogenes for synthesis of auxins and cytokinins
(phytohormones). The overproduction of phytohormones leads
to proliferation of callus or tumour formation in plants.
ii. Opine synthesizing genes for the synthesis of opines. Thus
opines act as source of nutrient for bacterial growth, e.g.
Octopine, Nopaline.

6. The functions of T-DNA genes are listed:

7. B) Virulence genes (vir genes)
1. Virulence genes aid in the transfer of T-DNA into the host plant cell.
2. Ti plasmid contains 35 vir genes arranged in 8 operons.
3. At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir
C1, vir D1, D2, vir D4 and vir E1, E2.
4. Transfer the T-DNA to plant cell
5. Acetosyringone (AS) (a flavonoid) released by wounded plant cells activates vir genes.
6. virA,B,C,D,E,F,G (7 complementation groups, but some have multiple ORFs), span about
30 kb of Ti plasmid.
Function of vir genes
virA - transports acetosyringone (AS) into bacterium, activates virG post-translationally (by
phosphorylation)
virG - promotes transcription of other vir genes
virD2- endonuclease/integrase that cuts T-DNA at the borders but only on one strand.
virE2 - can form channels in membranes
virE1 - chaperone for virE2
virD2 & virE2 also have NLSs, gets T-DNA to the nucleus of plant cell
virB - operon of 11 proteins, gets T-DNA through bacterial membranes

8. C) Opines:
• are a class of carbohydrate derivatives that serve as a nutrient source for the
agrobacteria,
• Derivatives of amino acids synthesized by T-DNA.
• This region codes for proteins involved in the uptake and metabolisms of opines.
Ti plasmids can be classified according to the opines produced :
1. Nopaline plasmids - carry gene for synthesizing nopaline in the plant and for
utilization (catabolism) inthe bacteria.
2. Octopine plasmids - carry genes to synthesize octopine in the plant and catabolism in
the bacteria.
3. Agropine plasmids - carry genes for agropine synthesis and catabolism.
Opine genes can be used as marker genes-
For screening of transformants or
recombinants

10. DNA transfer into the plant genome
Ti plasmid
Ri Plasmid

11. Ti plasmid based vectors:
1. Disarmed Ti-plasmid derivatives as plant vectors
2. Binary Vectors
3. C0-integrate vectors
1. Disarmed Ti vector
1. Ti plasmid is a natural vector for genetically engineering plant cells due to its ability to
transfer T-DNA from the bacterium to the plant genome.
2. But wild-type Ti plasmids are not suitable as vectors due to the presence of oncogenes
in T-DNA that cause tumor growth in the recipient plant cells.
3. For efficient plant regeneration, vectors with disarmed T-DNA are used by making it
non-oncogenic by deleting all of its oncogenes.
4. The foreign DNA is inserted between the RB and LB and then integrated into the plant
genome without causing tumors.

12. Construction: Structure of the Ti-plasmid pGV3850 with disarmed
T-DNA
1. Zambryski et al. (1983) substituted pBR322 sequences in the T-DNA of pTiC58, without
disturbing the left and right border regions and the nos gene.
2. The resulting construct was called pGV3850. No tumour cell formation takes place
when modified T-DNA is transferred from Agrobacterium carrying pGV3850 plasmid.
3. The evidence of transfer is done by screening the cells for nopaline production.
Drawbacks
Several drawbacks are associated with
disarmed Ti- vector systems are:
• Necessity to carry out enzymatic assays on
all potential transformants.
• Not convenient as experimental gene
vectors due to large size.
• Difficulty in in vitro manipulation and
• Absence of unique restriction sites in the
T-DNA.

13. 2. Binary vector
• Binary vector was developed by Hoekma et al (1983) and Bevan in (1984).
• It utilizes the trans- acting functions of the vir genes of the Ti-plasmid and can act on any T-
DNA sequence present in the same cell.
• Binary vector contains transfer apparatus-helper plasmid (contains the vir genes) and the
disarmed T-DNA containing the transgene on separate plasmids.
Binary vector system
Binary vector consists of a pair of plasmids:
1) A disarmed Ti plasmid: This plasmid has T-DNA containing LB and RB with gene of interest +
ori for both E. coli and Agrobacterium. Also called as mini-Ti or micro Ti plasmid eg: Bin 19.
2) Helper Ti plasmid has virulence region that mediates transfer of T-DNA in micro Ti plasmid to
the plant.
Plasmid with disarmed
T-DNA but without
vir gene
Plasmid with vir region but
without G0I

14. A binary vector system
Plasmid with disarmed T-
DNA but without vir gene
Plasmid with vir region but
without G0I

15. Binary vector Cloning Strategy:
1. Move T-DNA onto a separate, small
plasmid.
2. Remove aux and cyt genes.
3. Insert selectable marker (kanamycin
resistance) gene in T-DNA.
4. Vir genes are retained on a separate
plasmid.
5. Put foreign gene between T-DNA
borders.
6. Co-transform Agrobacterium with both
plasmids.
7. Infect plant with the transformed
bacteria.
Examples of Binary vector system
pBIN19- one of the first binary vectors developed in 1980s and was widely used.
pGreen- A newly developed vector with advanced features than pBIN19.

16. 3. Co- integrate vectors
Transfer is achieved using a ‘triparental mating’ in which three bacterial strains are mixed
together:
(i) An E. coli strain carrying a helper plasmid able to mobilize the intermediate vector in
trans;
(ii) The E. coli strain carrying the recombinant intermediate vector;
(iii) A. tumefaciens carrying the Ti plasmid
1. In first E.coli strain: The DNA to be introduced into the plant transformation vector is
sub cloned in a conventional Escherichia coli plasmid vector for easy manipulation,
producing a so-called intermediate vector. These vectors are incapable of replication
in A. tumefaciens and also lack conjugation functions.
2. --- An another E. coli strain carrying a helper plasmid able to mobilize the
intermediate vector .
3. Conjugation between the two E. coli strains transfers the helper plasmid to the
carrier of the intermediate vector, which in turn is mobilized and transferred to the
recipient Agrobacterium.
4. Homologous recombination between the T-DNA sequences of the Ti plasmid and
intermediate vector forms a large co- integrate plasmid resulting in the transfer of
recombinant T-DNA to the plant genome.

17. Construction of a Co-integrate vector (foreign gene cloned into an appropriate plasmid is
integrated with a disarmed Ti-plasmid through homologous recombination).
1. At first; an intermediate vector is made using E. coli plasmid + origin of replication +
pBR322 sequences + some markers + gene of interest.
2. -Second vector is a disarmed pTi vector = Left and right borders+ some markers +
pBR322 sequences + vir region.
3. -Both intermediate vector and disarmed pTi has some sequences in common (pBR322
sequences).
4. -Therefore by homologous recombination, co-integration of two plasmids will take
place within Agrobacterium.
5. Now we have a co-integrate vector that has both T-DNA with our gene of interest with
in the T-DNA borders and vir region. This complete vector is used for transformation in
plant cells. eg: pGV2260.
in
Agrobac
terium
tumefac
iens

18. Vector systems based on Ti Plasmids

19. Vector systems based on Ti Plasmids
Co-integration/exchange vector systems:
Genes of interest (goi) are exchanged into the T-DNA region of a Ti-plasmid (either
oncogenic or disarmed) via homologous recombination. Following the exchange, the
exchange/co-integration vector can be cured (removed) from the Agrobacterium cell.
Binary Vector systems:
Genes of interest are maintained within the T-DNA region of a binary vector. Vir proteins
encoded by genes on a separate replicon (vir helper) mediate T-DNA processing from the
binary vector and T-DNA transfer from the bacterium to the host cell. The selection
marker is used to indicate successful plant transformation. ori, Origin of replication; Abr,
antibiotic-resistance gene used to select for the presence of the T-DNA binary vector in E.
coli (during the initial stages of gene cassette construction) or in Agrobacterium.

20. Viral vectors
Plant Viral vectors
Animal Viral vectors

21. Plant Viral vectors
 Plant viruses can be used to engineer viral vectors, tools commonly used to
deliver genetic material into plant cells.
Plant viruses are considered as efficient gene transfer agents as they infect the intact
plants and amplify the transferred genes through viral genome replication
 Tobacco mosaic virus (TMV) is the first virus to be discovered.
Plant viruses have been engineered to express vaccines, monoclonal antibodies, and
other therapeutic proteins.
They are non integrative vectors Eg. Pepper mint mottle virus, Leaf curl virus.
The virus is an important source of gene regulatory elements, used exclusively in the
genetic manipulation of plants and significantly impact on plant virology and plant
molecular biology.
Criteria needed:
1. The virus must be capable of spreading from cell to cell through plasmodesmata
2. The viral genome should be able to replicate in the absence of viral coat protein
and spreads from cell to cell
3. Elicit little or no disease symptoms
4. Should have broad range of host

22. Plant viral vectors
Well studied and widely
used vector:

23. Plant viruses are also used as expression vectors.
DNA Vectors
1. Cauliflower mosaic virus (CMV)
2. Gemini viruses
3. Mastreviruses
4. Begomoviruses
RNA Vectors
5. Tobacco mosaic virus (TMV)
6. Brome mosaic virus (BMV)
7. Hordeiviruses
8. Potexviruses
9. Comoviruses

24. Animal viral vectors
Animal viruses can be divided into DNA and RNA viruses, depending on the
nature of their genomes.
Animal viruses have to recognize a specific host cellular receptor for entry during
infection. Host receptor binding is the initial step of virus life cycle and could be an
effective target for preventing virus infection.
SV40 vector
Retroviral vector
Bovine Papillomavirus DNA Vectors

26. Retrovirus has been considered to be an ideal viral vector for gene therapy, since the
viral genome becomes integrated into the chromosome and maintained stably upon cell
division.
Retroviral vectors are widely used for transgene expression in many laboratories.
Two kinds of retroviral vectors are available:
(1) murine leukemia virus (MLV)-derived vector and
(2) (2) HIV-derived lentivirus vector.

27. Retroviral vector
• Single stranded RNA genome- typically between 7 to 12 kb long in size
• Has two copies of the genome, which resemble eukaryotic mRNAs.
• The viral genome is reverse transcribed by reverse transcriptase into a DNA double-
strand copy inside the host cells. This process is called as Reverse transcription.
Features of RV vector
o Contains gene for replication, expression and packaging (ψ sequences).
o Gene of interest may BE inserted in the nonessential coding region or it may replace
some essential gene (gag).
o genomes are used as vectors, generally as shuttle vectors.
-Retrovirus genomes commonly contain these three open reading frames that encode for
proteins that can be found in the mature virus.
•Group-specific antigen (gag) codes for core and structural proteins of the virus,
•polymerase (pol) codes for reverse transcriptase, protease and integrase and
•envelope (env) codes for the retroviral coat proteins.

28. During the process of reverse transcription, sequences from the termini of viral RNA are
duplicated to generate long terminal repeats(LTRs).
These long terminal repeats contain both the promoter and the polyadenylation signal
for the transcription of viral mRNAs.
The specificity of proviral DNA integration is also determined by the long terminal
repeats.
Although retroviruses can integrate at many sites within the cellular genome, integrative
recombination always occurs at particular sites at the ends of the LTRs.
The sequences appropriately inserted between the two LTRs will be integrated intact.
After integration, the DNA genome behaves
like any cellular gene being transcribed and
replicating only if the cell replicates. Thus
the cell expresses the therapeutic gene and
no further viral replication is possible.

30. 1. Retroviral vectors are
created by removal
of the retroviral gag,
pol, and env genes.
2. Retroviral vectors
resemble their
parent virus except
that the genome
encodes as a
therapeutic gene or
gene of interest
instead of the viral
structural proteins.
Cloning and Packaging
Cloning and Packaging

31. 1. The packaging cell line has two separate
retroviral gene regions on its chromosomes;
one contains the gag gene, and the other
contains the pol and env genes.
2. In each of these inserts, transcription is
driven by sequences within the 5' long
terminal repeat (5' -LTR) region.
3. Both virus DNA segments lack the
encapsidation sequence (ψ) that is required
for packaging a retroviral genome into a viral
capsid.
4. The packaging cell line synthesizes viral
proteins, but because there is no
encapsidation (Δψ) sequence within either
of the retroviral mRNAs, empty viral capsids
are produced.
5. The viral proteins continue to be
synthesized after the transfection of a
packaging cell line with a full-length retroviral
vector carrying a remedial (therapeutic) gene
(Gene X) and a selectable marker gene
(Neor).
6. The full-length RNAs from the retrovirus
vector sequence are replicated, and because
they have an encapsidation region (ψ), they
are packaged into viral capsids.
7. The released viral particles are
replication defective because they do
not have a pol gene.

32. Thus, To generate the recombinant virus, the vector plasmid containing the transgene is
transfected into packaging cells, where Gag, Pol, and Env proteins are constitutively
expressed.
In packaging cells, the viral RNA encoding the transgene is transcribed from 5′ LTR
promoter, and the viral RNA is then packaged into the retroviral vector via the
recognition of the packaging signal (Ψ).
Such generated recombinant retrovirus retains the ability to infect target cells and to
express the transgene.
Upon infection of target cells, the viral RNA will be reverse transcribed and integrated
into the chromosome as a proviral DNA.
In target cells, the viral RNA encoding the transgene will be transcribed, and the
transgene will be expressed.