”Transformation is the genetic alteration of
a cell resulting from the direct uptake and
incorporation of exogenous genetic material
from its surroundings.”
“Integration of gene into genome by means
fusion of gametes”
Plant Transformation Methods
1.Indirect method or vectored methods
oAgro bacterium-mediated transformation.
2. Direct method.
o Protoplast electroporation.
o Protoplast polyethylene glycol method.
o Gene gun method.
“A DNA molecule used as a vehicle to carry
foreign genetic material into another cell.”
Types Of Vector:
-Plasmids. -Viral vectors.
-Cosmids. -Artificial chromosome.
Characteristics of vectors
Origin of replication
Bacterial selectable markers
Gene constructs of interest
“Small piece of DNA into which a foreign DNA
fragment is inserted for cloning purposes.”
“Also known as an expression construct, is
usually a plasmid or virus designed for
protein expression in cells.”
Plant Physiol. 2007 Dec; 145(4): 1118–1128.
Focus Issue on Vector Systems for Plant
Research and Biotechnology
Delivery of Multiple Transgenes to Plant
Mery Dafny-Yelin and Tzvi Tzfira
• Extra chromosomal DNA molecules.
•Circular & Double stranded.
•Short sequence of DNA.
• Found in prokaryotes.
Classification of plasmids
o Fertility plasmid
e.g. F plasmid of E. coli
o Col plasmid
e.g. ColE1 of E. coli
o Resistance plasmid
e.g. RP4 in Pseudomonas
o Degradative plasmid
e.g. TOL of P. putida
o Virulence plasmid
e.g. Ti plasmids of A. tumefaciens
Based on the origin or source of plasmids
Two major classes :
i) Natural plasmids:
They occur naturally in prokaryotes
ii) Artificial plasmids:
They are constructed in-vitro by re-combining
selected segments of two or more plasmids.
Nomenclature of Plasmid
322 Number given to distinguish
Occur naturally in bacteria
Have different restriction sites.
Replicate completely independent of bacteria
Genes are easily inserted into plasmids
Easily transformed into bacteria
Cannot accept large fragments
Sizes range from 10-20 kb
Standard methods of transformation are
Gram negative bacteria.
Found in soil.
Causes crown-gall disease.
Ability to introduce DNA into plant.
Curr Top Microbiol Immunol. 2014;375:155-
92. doi: 10.1007/82_2013_352. Plant viral
vectors for delivery by Agrobacterium. Gleba
YY, Tusé D, Giritch A
Colonies with recombinant plasmid are white
Colonies with non-recombinant plasmids are
For example: pUC19
Resistance to ampicilline.
Contains portion of the lacZ which codes for
“Viruses which are used as gizmo by
molecular biologists to carry genetic
material into cells” are called viral vectors.
Viral vectors are non-integrative as
compared to bacterial vectors
1.Cauliflower mosaic virus based vectors.
2.Cowpea mosaic virus
3.Bean pod mottle virus (BPMV)
4.TMV based vectors.
5.Potato virus X (PVX)
6.Bean yellow dwarf virus
7.Bacteriophage Lambda Vectors
Characteristics of viral vectors
Cell type specificity
Viruses are used in two ways
–Virus directly inserted into plant
–Virus indirectly inserted (bacteria)
Cauliflower mosaic virus
Infectious when simply rubbed on leaves
Mechanical and aphid mediated transmission
Up to 106 copies per cell within 3-4 weeks of
infection in plant.
Use of viral vectors for vaccine production in plants, M Carmen Cañizares, Liz Nicholson
and George P Lomonossoff. John Innes Centre, Norwich, UK
Small insertions (10-30 bp) in various sites
The largest insert is 256-531 bp
CaMV genome can be inserted into Ti vector
CaMV activity in plant cell
Bacteriophage Lambda Vectors
Viruses that can infect bacteria
1000 times more efficient than plasmid vectors
Clone DNA fragments in
range of 10,000 - 20,000 bps
Bacteriophage. 2012 Oct 1; 2(4): 215–224.
Soil-based systemic delivery and phyllosphere in vivo
propagation of bacteriophages
Two possible strategies for improving bacteriophage
persistence for plant disease control
Fanny B. Iriarte, Aleksa Obradović, Mine H. Wernsing, Lee
E. Jackson, Botond Balogh, Jason A. Hong, M. Timur
Momol, Jeffrey B. Jones, and Gary E. Vallad
Fast processing ,low cost, high yield
Good at targeting and entering cells
Mostly target specific types of cells
Used as virus-induced gene silencing
(VIGS) in reverse genetic studies
Virus-induced gene silencing in plants.
Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC.
Express proteins in plants for the
- Study of gene function
- Production of vaccines
- Study of metabolic engineering
- Analysis of plant-microbe interactions
Hum Vaccin. 2011 Mar;7(3):331-8. Epub 2011 Mar 1.
Geminiviral vectors based on bean yellow dwarf virus for production of vaccine
antigens and monoclonal antibodies in plants.
Chen Q, He J, Phoolcharoen W, Mason HS
Worst effects to plants by
–Producing severe disease
–Giving undesired products
–Affecting the plant adversely
(due to highest mutation rate)
Derived from bacteriophage & plasmid
Cohesive sites + plasmid = cosmid
Less used for plant transformation
Carry DNA fragments of about 40 kb
E.g. US 8298819 B2
J Virol. 2000 Oct; 74(19): 8972–8979.PMCID: PMC102093
Generation of Mutant Murine Cytomegalovirus Strains from
Overlapping Cosmid and Plasmid Clones
Mariam E. Ehsani, Tshge W. Abraha ,Cecile Netherland-
Snell,Niklaus Mueller, Meghan M. Taylor, and Barry Holwerda
Cohesive ends or sticky ends
A single-stranded end of a linear
duplex DNA molecule which can
form hydrogen-bond with a
complementary single-strand base
sequence from the end of the same
or another DNA molecule