PLANT GENETIC

TRANSFORMATION
PGT techniques are of two major types :
1. Vector mediated gene transfer
2. Direct DNA transfer (Vector less)

1. Vector Mediated Gene Transfer (VMGT) is carried out by:

a. Agrobacterium-mediated
b. Plant viruses
a. Agrobacterium tumefaciens (nicknamed as – most effective plant genetic
engineer or Natural expert of interkingdom gene transfer )
• Bacterium of family Rhizodiaceae
• Gram (-)ve
• Rod shaped
• Motile
• Phytopathogen
• Two important species :
i. A. tumefaciens-crown gall disease ( naturally evolved GE process)
ii. A. rhizogens – hairy root disease
A .tumefaciens (At)
• Infects wounded or damaged plant tissues and induce plant tumour called
crown gall
• Induce the release of some phenolic compounds by the plants
• Releases its Ti ( Tumour Inducing plasmid) into plant cell cytoplasm, leading
to crown gall disease
• T-DNA of the Ti plasmid is transferred from At to plant cell DNA to cause
disease.
• The T-DNA when integrated with host genome, it codes for protein of
auxin/cytoxin biosynthesis and some novel metabolites called Opines (amino
acid derivatives) and agropines (sugar derivatives).
• These compounds are utilized by At as source of carbon and energy.
• The growth hormones induction help in enlarged plant cell growth and
development leading to gall.
• Genetic transformation of plant genome to synthesize opines and agropines was
the base for future GT programmes.
• At causes crown gall disease in grapes, roses, store fruits etc.
•Ti plasmid - circular DNA and 200 kb in size
- independently replicate
- T DNA is 12-24 kb
•Ti plasmid origin
-Nopaline strain of At - TL- 20kb (Total) & 14kb (T-DNA)

- Octopine strain of At – TR -20kb (Total) & 7kb (T-DNA)

•Ti plasmid
-T DNA – genes for aixines (aux), cytokinin (cyt) and Opines (opc)
- T borders – 24kb on left + 24kb on right = both transferred.
- Virulence region – help in transfer of T DNA/genes for proteins of T-
DNA tumour ( 9 operons : virA ,virG, virB1, virC1, virD1, virD2 ,virD4, virE1,
virE2)

- Opine catabolism region – genes for uptake and metabolism for opine
- Ori
 T-DNA transfer and integration

a. Signal induction to At : phenolics and sugar molecules released by wounded

plant cell are signals of At infection
b.Attachment of At to plant cell : attachment through polysaccharides like
cellulose fibers of At and vir genes play important role here
c. Production of virulence proteins : virA virG ( vir genes – vir proteins)

d.Production of T DNA strand : virD1 and virD2 proteins act as promoter for
ssTDNA synthesis( ssTDNA attaches with virD2 protein)
e. Transfer of T DNA out of At complex + virG out of bacterial cell (virB helps
here)

f. Transfer of T DNA into plant cell: T DNA + virD2 goes through plasma
membrane and extends plant cell

g. Complex gets coating of vir E2 proteins which protects T-DNA from nucleus

h. vir D2 and vir E2 interact with a number of plant proteins that help in T DNA
transport and integration

i. T DNA + vir D2 + vir E2 + plant protein complexes enter nucleus through

NPC

j. In the nucleus T-DNA gets integrated into plant DNA through a process called
illegitimate recombination

k. Different from homologous recombination and do not require sequence similarity.

b. A .rhizogens (Ar)

•Cause hairy root disease/no crown gall/tumour

•The plasmids of Ar are called Ri plasmids

•Some genes are common between Ti and Ri plasmids eg :

auxin

•Instead of virulence genes, Ri has several ORFs

•They induce such proteins which help in hairy root formation

•Ri plasmids are used for GE but not common (At is preferred)

•Hairy roots are important for Plant Tissue Cultures, secondary metabolites,
pharmaceutical proteins.
Construction of Ti plasmid

• We can incorporate desired DNA sequence into T-DNA

• Ti plasmids serve as natural vectors
• There are certain limitations to Ti plasmids as vectors
-large size (200-400kb)-----trim
-absence of restriction sites-----add
-Aux and cyt genes are blocks for plant regeneration----remove
-opine production hampers plant yield---- not needed
- Ti cannot replicate in E. coli
Ti Plasmids
1. Cointegrate Vector
2. Binary vector

3. Cointegrate Vector
• Genes for hormone synthesis are removed (Receptor plasmid)
• In that place a bacterial plasmid pBR322
• Receptor plasmid (RP): T-DNA (L+R borders + vir genes )
• Intermediate Vector (IV): pBR322 sequence (Homologous to RP)+ Plant
Transformation marker (eg. Npt II, Kanamycin resistance and help in
isolation) + Bacterial resistance marker (eg res, Streptomycin resistance) +
MCS (Insertion )
•The desired gene is cloned at MCS and transforms E.coli

•Then mating of E.coli and AT is done for transfer of desired gene from
intermediate vector into AT

•The transformed AT with RP + IV are cultivated on minimal medium +

streptomycin and transformants are isolated

•Within the AT, IV + RP integrate to develop cointegrate plasmids

•Now such cointegrated plasmids are used to transform plant cells

•The transformed plant cells are isolated against kanamycin resistance

•Several advantages of cointegrate vector like easy cloning, MCS and easy transfer
from E.coli- AT-plant cell.
2. Binary Vectors (BV)
•T DNA with LB + RB + PTM (plant transformation marker) +MCS + RES
(Bacterial resistance marker) eg: tetracycline resistance +OriT (E.coli to AT) +RK 2
(Replication in broad host range)
•BV involves only the transfer of BV, but not integrated (as opposed to CV) ; more
frequently used
•Genetic transformation steps are as in cointegrated vector system
• Plant transformation using AT
•Most widely used technique
•Prepare the explant source which MUST produce phenolics for activation of
virulence genes
•Transformed cells must be able to regenerate
PROTOCOL

•Development of AT with CV or BV

•Identification of suitable explant material (cells /tissue /callus /protoplasts/

organs)

•Co-culture of explants with AT

•Killing AT with suitable antibiotic without harming plant cells

•Selection of transformant cells

•Regeneration of whole plants

 Plus points Negative points

1. Natural gene transfer 1. PTC

2. Wide range infection 2. Easy to culture but not easy
3. Large genes can be transferred to transform 
4. Transgene stability
5. Effective regeneration
2. Virus mediated gene transfer

•Efficient gene transfer agents since they can infect wide range of plants and
multiply them in a systematic manner
•Viruses are nonintegrative vector with small genomes
•Selection of appropriate virus for GE
 Should be able to spread through plasmodesmata
 Should be able to replicate and spread systematically
 Coat protein should not be mandatory for infection
 Should not show disease in plant cells
 Broad host range is must for GE of diverse crops
 DNA viruses, not RNA, are preferred
Viruses for GE :
i. Caulimoviruses
ii. Geminiviruses
iii. RNA viruses
i. Caulimoviruses as vectores :
• Circular dsDNA
• Widely distributed
• Wide host range
• ~15 viruses are known
• CaMV is important
• Carnation etched virus
• Dahlia mosaic virus
• Mirabilis mosaic virus
• Strawberry vein binding virus
•For effective transformayion, the foreign DNA must be coated with viral proteins
•The inserted DNA should not interfere with host DNA
•No introns in CaMV DNA/only gene II and VII are available
•Gene II has been proved more useful

Plus points Negative points

1. Cruciferae, datura etc. 1. Limited space for insertion

2. Systemic infection 2. Looses infectivity if large DNA inserted
3. Large quantitative infection 3. Integration often difficult
4. Genome size 8kb
5. 6major + 2 minor coding regions
(genes II and VII are not
essential) for infection
ii. Gemini viruses
•Gemini = twins, GMV = twin capsid structures
•1 or 2 ssDNAs

ds
•Ss DNA ss DNA
Replication

•Infect wide range of plants (monocots & dicots)

•Large no. of Replicative forms of DNA form inside the cell
•Transfer of viral DNA into plant cells is very difficult
•May be AT is way out
• Eg : curlytop virus (CTV), Bean Golden mosaic virus (BGMV)
RNA plant viruses for PGT :
•ssRNA viruses
 monopartite – large/undivided genome/TMV
 multipartite – small/divided/in the same virus body or not
•plant viruses
 high level gene expression
 high infection efficiency
 but as vector not useful (integration difficult)
• cDNA of viruses are also attempted but with much little success
 
eg : chloramphenical resistance gene
 
BMV
 
 
Protoplast no expression in plant
 useful only in veg.propagated plants
 Direct gene transfer methods for PGT

1. Physical methods
a. Electroporation
b. Particle bombardment/ biolistics
c. Microinjection
d. Liposome – mediated gene transfer
e. Silicon carbide fibre mediated gene transfer
2. Chemical methods
f. Polyethylene glycol method
g. Calcium phosphate co-precipitation method
h. DEAE dextran method
a. Electroporation
•It uses high field strength “electric impulses to reversibly permialize the cell
membranes” for the uptake of DNA
• Plant cells/tissues/protoplasts + buffer containing target DNA

High voltage electric impulses

  Formation of porous membrane due to thinning effect
 
Transfer of DNA through pores
•Methodology
 Expression vector preparation
 Preparing the DNA for transfer
 Preparing the protoplast for receiving DNA
 Electroporation
 Regeneration of plantlet
•Several types of electroporation devices but ideal is the one which can give
1kV/cm field strength and pulse length 1-40ms

•It can be fabricated indigenously, but not advised

•Plasmid DNA must be purified using gene clean kits

•Osmoticum of the culture medium during and after electroporation should be

maintained by sucrose rather than others (eg:manitol)

•Proper selection of EFS ( electric field strength ) and a compatible capacitor is a

must ( 500-800 v/cm is ideal )

•Upto 3 electric pulses can increase yield of transformants proportionately

•Post-electroporation protoplasts are more fragile/ needs careful handling

Some scientists also use terms like :

• Microprojectile

• MP bombardment

• Particle bombardment

• Particle acceleration

• Ballastics etc.etc.
b. Particle bombardment / biolistics

•It employs high velocity microprojectile for delivery of substance into cells

•DNA is coated onto micron sized gold or tungsten particles by precipitation

with calcium chloride and spormidine

•Inside the cell, the DNA elutes of particles

•It is followed by integration and expression

•Biolistics = biology + ballastics

•Also known as gene guns, particle guns and shot gun techniques
Methodology
• Culture and preparation of plant cells
• Sterilization of macro carriers and holders
• Sterilization of gold particles
• Mixing by vortex
• Coating of gold particles with DNA
• Particle bombardment
• Post bombardment modification of culture medium ( reduced osmoticum
etc. )
• Analysis of transient and long term gene expression
• Callusing/embryogenesis and regeneration
•Embryonic cultures are ideal for bombardment

•Addition of osmotic (mannitol and sorbitol) to bombardment medium yield

stable transformation

•Gold particle size 0.6 to 1 µm

•Tungsten size variable

•DNA to be integrated must be ultra pure.use of gene clear technologies/kits

•Biolistics is a term work and require extra skills/care

•Different steps to be done by different workers in tandem

•Subculture of transformants / screening for bacterial growth

•Reported and marker genes must express transiently in cultures

•Appropriate quantity of DNA must be used

•Most popular technique for PGT and more than 70% of transgenic crops are
developed by this technique
•Maize with Bt-toxin gene is produced by this tech.
•Many environmental factors influence PBT (temp./humidity/photoperiod)
•PDS-1000/HC from biorad is commonly used

Demerits

i. High copy number of transgene

Gene silencing
ii. Damage of target tissue
iii. Chimeric plants
c. Micro-injection
•Direct physical method
•Target may be cells/protoplasts/callus/embryos/meristems etc.
•Even cell organelles can be transferred
•The technique uses a micropipette (0.5 to 10 µm tip )
•During transfer, the target cell is immobilized by agarose embedding
•The cell is also held by suction holding pipette
•After transformation, the cell is cultured
•For eg : Brassica napus and tobacco for herbicide disease resistance
Negative point
i. Slow process
ii. Require high order of skills
d. Liposome – mediated gene transfer technique :
•Liposomes are artificially created lipid vesicles
•Lipid vesicles are made up of phospholipid membranes
•Conventionally, they are used in drug delivery system
•Liposomes enclosing target DNA can be employed to fuse with protoplasts
and transfer the genes
•It is recommended to use this technique along with PEG method
•The technique involves
 Adhesion of liposomes to protoplast surface
 Fusion at the sight of attachment
 Release of plasmid inside cell
•This technique is advantageous because the encapsulate transgene has protection
from outside factors/damage
•More stable transformants in wide range of plant cells
•Regeneration is the main problem
e. Silicon carbide fibre mediated transformation
• SCFs are about 0.3 to 0.6 µm in diameter / 10-100 µm in length
• SCFs are available with trade name “whiskers”
• SCFs have the ability to penetrate cell wall and plasma membrane
• DNA is coated on SCFs and mixed with cells / protoplasts in suspension
medium
• High speed vortex mixture done
• During this process, the SCFs penetrates the cell wall / plasma membrane
and the coated DNA is delivered
•It is a simple and cost effective technique
Negative points
 SCFs are carcinogenic and needs careful handling
 Embryogenic plant cells with hard cell wall can resist the penetration of
SCFs
 2. Chemical methods :

a. PEG mediated transformation

•PEG destabilizes the plasma membrane of protoplasts in presence of
divalent cations (Ca2+)
•Makes the plasma membrane permeable for naked DNA
•40% PEG dissolved in mannitol and calcium nitrate solution added slowly
to a mixture of
•Protoplasts in suspension medium + Plasmid DNA and incubation at 25°C
•Protoplasts get transformed
•Main advantage is a large number of protoplasts can be transformed
 Negative points
i. DNA is susceptible for degradation
ii. DNA may even mutate by methylation
iii. Random integration
iv. Regeneration problems

b. DEAE dextran mediated PGT

• Diethyl amino ethyl dextran polymer of high molecular weight
• Mixed with DNA and incubate cells / protoplasts for transformation
• Not much reliable method
c. Calcium phosphate co – precipitation method
•DNA is mixed with CaCl2 solution + isotonic phosphate buffer

DNA – calcium phosphate (ppt.)

• The actively dividing cells / protoplasts are incubated with this ppt. for
several hours – unstable ppt.
•Cells get transformed
•High concentration of DNA + protection of complex ppt. (from
degradation)
•Addition of dimethyl sulfoxide (DMSO) increase the efficiency of
transformation
e. Direct imbibitions methods
•No physical and chemical method involved
•Not successful