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BY-
MD SAMIYUDDIN
Jr. M.Sc. (CIB)
INTRODUCTION
 Plant genetic engineering has become one of the most
important molecular tools in the modern molecular
breeding of crops.
 Over the last decade, significant progress has been made in
the development of new and efficient transformation
methods in plants.
 Despite a variety of available DNA delivery methods,
Agrobacterium- and biolistic-mediated transformation
remain the two predominantly employed approaches.
 In particular, progress in Agrobacterium-mediated
transformation of cereals and other recalcitrant dicot
species has been quite remarkable.
 In the meantime, other transgenic-enabling technologies
have emerged, including generation of marker-free
transgenics, gene targeting, and chromosomal engineering.
 Although transformation of some plant species or elite
germplasm remains a challenge, further advancement in
transformation technology is expected because the
mechanisms of governing the regeneration and
transformation processes are now better understood and
are being creatively applied to designing improved
transformation methods or to developing new enabling
technologies.
 The genetic constitution of plants can be altered in the
laboratory by a process called transformation, whereby a
segment of DNA (deoxyribonucleic acid) is introduced
that becomes inserted in one of the plant chromosomes.
 Several methods to accomplish plant transformation have
been devised. In all these methods, single cells are
transformed and thereafter regenerated into complete,
fertile plants by tissue culture procedures.
 Some of the transformation methods can only be applied
to protoplasts.
 Nowadays, particle bombardment and the natural vector
Agrobacterium tumefaciens are preferred because they can
also cope with whole plant tissues such as roots and leaves,
which are easier to handle, more stable and require less of
the lengthy steps that are required for plant regeneration.
 Transgenes are integrated at random positions in the plant
genome, but procedures for targeted integration become
more and more efficient.
 Production of transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or
enhance gene expression
Add selectable markers
Introduce gene construct into plant
cells (transformation)
Select transformed cells or tissues
Regenerate whole plants
Plant Transformation Methods
(DIRECT)
Physical Chemical
(INDIRECT)
Biological
In Planta
•Microinjection
•Pressure
•Biolistics – gene
gun/particle
bombardment
•Electroporation
•Microinjection
•Silica/carbon fibers
•Laser mediated
•SAT
•PEG
•DEAE- dextran
•Calcium phosphate
•Artificial lipids
•Proteins
•Dendrimers
A.Tumefaciens
A. Rhizogenes
• Virus-
mediated
•Meristem
transformation
•Floral dip
method
•Pollen
transformation
Techniques for plant genetic
transformation
 Indirect method- Agrobacterium mediated gene
transfer
 Direct methods-
 Particle bombardment (biolistics)
 Microprojectile gun method
 Electroporation
 Silicon carbide fibres
 Polyethylene glycol (PEG)/protoplast fusion
 Liposome mediated gene transfer
Properties of good host
1. Easy to transform
2. Supports the replications of recombinant DNA
3. Is free from elements that interfere with replication of
recombinant DNA
4. Lacks active restriction enzymes ex: E.coli k12 substrain
HB 101
5. Does not have methylases
6. Is deficient in normal recombination function : so that
it is not altered by recombination events.
Transformation vector requirements
 Origin of replication
 Bacterial selectable marker
 Gene constructs of interest
 T-DNA borders and other Agrobacterium genes if using
Agrobacterium
 Compatible with helper plasmid if using Agrobacterium
Agrobacterium mediated gene transfer
Agrobacterium-
 Soil borne, gram negative, rod shaped, motile found in
rhizosphere
 Causative agents of “Crown gall” disease of dicoltyledones
 Have ability transfer bacterial genes to plant genome
 Attracted to wound site via chemotaxis in response to chemicals
(sugar and Phenolic molecules: acetosyringone) released from
damaged plant cells
 Contains Ti plasmid which can transfer its T-DNA region into
genome of host plants
Ti-plasmid features
 Two strains of Ti-plasmid:
-Octopine strains- contains two T-DNA region: TL (14
kb) and TR ( 7 kb)
-Nopaline strains- contain one T-DNA region(20 kb)
 Size is about 200 kb
 Has a central role in Crown-gall formation
 Contains one or more T-DNA region that is integrated
into the genome of host plants
 Contain a vir region ~ 40 kb at least 8~11 vir genes
 Has origin of replication
 Contains a region enabling conjugative transfer
 Has genes for the catabolism of opines
Nopaline Octopine
Acetosyringone
Forms of T-DNA that are found in Agrobacterium
ds circles - found only in induced bacteria, not
(apparently) in plant cells.
ds linear T-DNA - found only in induced bacteria,
not (apparently) in plant cells.
 ss linear T-DNA - found in bacteria and plant
cells.
what is not found - Ti plasmids with evidence that
T-DNA has been precisely deleted.
Process of T-DNA transfer and integration
1. Signal recognition by Agrobacterium:
-Agrobacterium perceive signals such as sugar and phenolic compounds
which are released from plants.
2. Attachment to plants cells:
Two step processes: i) initial attachment via polysaccharide ii) mesh of
cellulose fiber is produced by bacteria.
Virulence genes (chv genes) are involved in the attachment of bacterial
cells to the plants cells.
3. Vir gene induction:
VirA senses phenolics ans subsequently phosphorylating and thereby
activating VirG. VirG then induces expression of all the vir genes.
4. T-strand production:VirD1/virD2 complex recognises the LB and
RB. virD2 produces single-stranded nicks in DNA. Then virD2 attached
to ssDNA. virC may assist this process.
Process of T-DNA transfer and integration (continu)
5. Transfer of T-DNA out of bacterial cells: T-DNA/VirD2 is
exported from the bacterial cell by “T-pilus” composed of
proteins encoded by virB operon and VirD2. VirE2 and VirF are
also exported from bacterial cells.
6. Transfer of the T-DNA and Vir proteins into the plant
nuclear localization: T-DNA/VirD2 complex and other Vir
proteins cross the plasma membrane through channels formed
from VirE2. VirE2 protect T-DNA from nucleases, facilitate
nuclear localization and confer the correct conformation to the
T-DNA/virD2 complex for passage through the nuclear pore
complex (NPC). The T-DNA/VirD2/VirF2 /plant protein
complex the nucleus through nuclear pore complex. And
integrated into host chromosome.
Agrobacterium tumefaciens mediated
transforamtion in plants
Explant
{ decapitated seedlings, cells, protoplast, leaf discs}
Addition of wounding
Acetosyringone Agrobacteria
co cultivation to allow infection
antibiotics to kill bacteria
transformed and non transformed tissue
selective media to kill the non transformed tissues
i.e, addition of kanamycin and hygromycin etc
transformed tissue / callus
transformed shoots
rooted shoots
adult plants
SEEDS
Practical application of Agrobacterium-mediated
plant transformation
 1. Agrobacterium mediated transformation
methods are thought to induce less
rearrangement of the transgene.
 Lower transgene copy number that direct DNA
delivery methods.
 Successful production of transgenic plants
depends on the suitable transformation
protocols.
The basic protocol used for any Agrobacteruim
mediated transformation experiments
1. Identify a suitable explants: Suitable plant tissue is
removed and sterilized. Leaf is used for Tobacco.
2. Co-cultivate with the Agrobacterium: Leaf tissue is cut
into small pieces and placed into a culture of Agrobacterium
for about 30 mins. The explants are subsequently removed
from the bacterial culture and placed on to the MS medium
that contain no selective agent. The incubation of explants
with Agrobacterium is allowed to continue for 2 days to allow
transfer of the T-DNA transfer to the plant cells.
3. Kill the Agrobacterium with a suitable antibiotic: The
explants are removed from the medium and washed in
antibiotic (cefotaxime) solution that kill Agrobacterium cells.
The basic protocol used for any Agrobacteruim
mediated transformation experiments (Conti-)
4. Select for transformed plant cells: The explant
are transferred to fresh solid medium supplemented
with a selective agent (kanamycin). It also contains
cefotaxime. Auxin, Cytokinin are used to encourage the
regeneration of by organogenesis. High cytokinin to
auxin ratio promotes shoot formation from the
explants.
5. Regeneration of whole plant :The shoot can be
rooted by placing them on solid medium containing a
high auxin to cytokinin ratio.
Advantages of A.bacterium mediated transformation
 It is a natural mean of transfer and is perceived as more acceptable
technique to those who feel natural is best.
 It is capable of infecting intact plant cells, tissues and organs. As result
of tissue culture limitations are much less of a problem. Transformed
plants can be regenerated more rapidly.
 It is having a capable of transferring large fragments of DNA very
efficiently without substantial rearrangements.
 Integration of T-DNA is relatively precise process, it serves as an ideal
insertional mutagenesis vehicle as it introduces one to several copies
of the transferred DNA into the intact genome at one or a few loci.
 The stability of gene transferred is excellent. Integrated T- DNA gives
consistent maps and appropriate segregation ratios. Introduces traits
have been found to be stable over many generations.
 This at ability is critical to commercialization of transgenic plants.
Disadvantages
 It has limitation of host range,
 Some of the important food crops can not be infected by
this although, lately much progress has been achieved to
over come this limitation by the development of highly
virulent strains of Agrobacterium.
Sometimes, the cells in a tissue that are able to regenarate,
are difficult to transform. it might be that these are in
layers too deep to be reached by Agrobacterium, or
simply are not targets for T-DNA transfer.
Virus mediated
 Vectors based on viruses are desirable because of high efficiency
of gene transfer that can be obtained by infection and because of
the amplification of transferred gene that occurs via viral genome
replication.
 Vectors for transferring gene into plants are based on DNA or
RNA molecules, that naturally express their genetic information
in the plant cells.
 The replicating genomes of plant viruses are non-integrative
vectors, as compared to vectors based on the T-DNA of A.
Tumefaciens which are integrative gene vectors.
 The plant virus vectors do not integrate into the
plant cells or host genome , rather they spread
systematically within a plant and accumulate to
high copy number in their respective target cells.
 In most of the cases , the viral genome has modified
to accommodate the insertion of foreign sequences,
which are transferred, multiplied and expressed in
plant as part of recombinant virus genome.
 Plant viruses as vectors
 Caulimoviruses – ds DNA – CaMV
 Geminiviruses - 2ss DNA – maize streak virus
 RNA plant viruses - TMV
Vectorless or Direct DNA transfer
 The trem “Direct gene transfer” is used to discriminate
between the methods of plant transformation that rely on
Agrobacterium (indirect method) and those that do not (direct
methods). Direct gene transfer methods all rely on the delivery
of large amount of naked DNA whilst plant is transiently
permeabilised.
Direct methods-
Particle bombardment (biolistics)
Microprojectile gun method
Electroporation
Silicon carbide fibres
Polyethylene glycol (PEG)/protoplast
fusion
Liposome mediated gene transfer
Physical methods
 DMGT: DNA Mediated Gene Transfer
A general scheme for production of transgenic plants using
various direct DNA delivery methods have been
commercialized.
 Electroporation
 Particle bombardment / Micro projectile / Biolistics
 Electro injection
 Micro injection
 macro injection
 Liposome mediated
 Silicon carbide fibre mediated
 ultra sound mediated
 DNA transfer via pollen
Electroporation technique.
 Is the process where by electrical impulses of high
field strength are used to reversibly permeabalize cell
membrane to facilitates uptake of large molecules,
including DNA.
 It has been used for long time for transient and
integrative transformation of protoplasts.
 1 to 1.5 k V. so uses low capacitance hence short
decay time .
Electroporation
 It can be used to deliver DNA into plant cells and protoplasts.
 The genes of interest require plant regulatory sequence.
 Plant materials is incubated in a buffer solution containing
DNA and subjected to high-voltage electric pulse.
 The DNA then migrates through high-voltage-induced pores in
the plasma membrane and integrates into the genome.
 It can be used to transform all the major cereals particularly
rice, wheat, maize.
 Advantages and disadvantages:
 Both intact cells and tissue can be transformed.
 The efficiency of transformation depends upon the plant
materials, electroporation and tissue treatment conditions used
for transformation.
 ~40 to 50% incubated cells receive DNA
 ~50% of the transformed cells can survive

Duracell
DNA containing
the gene of interest
Plant cell
Protoplast
Electroporation Technique
Power supply
DNA inside the
plant cell
The plant cell with
the new gene
Biolistic/Particle bombardment
 High velocity micro projectile were utilize to
deliver nucleic acids into loving cells.
 Advantages :
Transformation of organized tissue
Universal delivery system
Transformation of recalcitrant spp
Study of basic plant development processes.
Particle bombardment
 Why Biolistics or Biolistic bombardment?
 Is the most powerful method for introducing nucleic acids
into plants, because the helium pressure can drive
microcarriers through cell walls
 Is much easier and less time consuming than microinjecting
nucleic acids into plant cells or embryos
 Allows transformation of animal cells that have unique
growth requirements and that are not amenable to gene
transfer using any other method
 Requires less DNA and fewer cells than other methods, and
can be used for either transient or stable transformation
Principle
 The gold or tungsten particles are coated with the DNA that
is used to be transform the plant tissue.
 The particles are propelled at high speed into the target
plant material where the DNA is released within then cell
and can integrate into the genome.
 Two types of plant tissues are used for particle
bombardment:
 a) Primary explants that are bombarded and then induced
to become embryogenic
 b) Proliferating embryonic cultures that are bombarded
and then allowed to proliferate further and subsequently
regenerate.
PDS-1000/He bombardment System
Fig: Schematic representation of the PDS-1000/He
system upon activation. The arrows indicate the
direction of helium flow
Fig: The PDS-1000/He system, shown here
with magnified view of the Hepta adaptor.
How the PDS-1000/He System Works
 The sample to be transformed is placed in the bombardment chamber,
which is evacuated to subatmospheric pressure
 The instrument is fired; helium flows into the gas acceleration tube and is
held until the specific pressure of the rupture disk is reached
 The disk bursts, and the ensuing helium shock wave drives the
macrocarrier disk (which carries the coated microparticles) a short
distance toward the stopping screen
 The stopping screen retains the macrocarrier, while the microparticles
pass through the screen into the bombardment chamber and penetrate
the target cells
 The launch velocity of microcarriers depends on a number of adjustable
parameters: the helium pressure (rupture disk selection, 450–2,200 psi),
the amount of vacuum, the distance from the rupture disk to the
macrocarrier, the distance from the microcarrier launch assembly to the
stopping screen, and the distance between the stopping screen and target
cells. Adjusting these parameters allows you to produce a range of
velocities to optimally transform many different cell types.
The Helium Gas Gun – Circa 2000
Particle Gun
Microinjection
 Under a microscope, a cell is manipulated to a blunt
capillary. Gentle suction holds the cell in place.
 With a micromanipulator, a very fine tipped pipet is
inserted into the cytoplasm or nucleus.
 DNA or RNA is injected directly into the nucleus or
cytoplasm.
 Microinjection has been successfully used with large frog
eggs, cultured mammalian cells, mammalian embryos,
and plant protoplasts and tissues
Microinjection
Microinjection techniques for plant protoplasts utilize a holding
pipette for immobilizing the protoplast while an injection pipette is
utilized to inject the macromolecule.
 In order to manipulate the protoplasts without damage, the
protoplasts are cultured for from about 1 to 5 days before the
injection is performed to allow for partial regeneration of the cell
wall.
It was found that injection through the partially regenerated cell
wall could still be accomplished and particular compartments of the
cell could be targeted.
 The methods are particularly useful for transformation of plant
protoplasts with exogenous genes.
Microinjection of GOI
Silicon carbide fibres-Whiskers
 Plant materials (Cells in suspension culture, embryos and
embryo-derived callus) is introduced into a buffer containing
DNA and the silicon fibers which is then vortexed.
 The fibers (0.3-0.6 μm in diameter and 10-100μm long)
penetrate the cell wall and plasma membrane, allowing the
DNA to gain access to the inside of the cells.
 Disadvantages and advantages
 The drawbacks of this technique relate to the availability of
suitable plant material and the inherent dangers of the fibers,
which require careful handing.
 Many cereals, produce embryonic callus that is hard and
compact and not easily transformed with this technique.
 Despite the some disadvantages, this method is recently used
for successful transformation of wheat, baerly, and maize
without the need to cell suspension.
Silicon carbide fibre mediated
 0.6 micro m dia, 10 – 80 micro m lth.
 vortexing in an eppendorf tube a mixture of plasmid DNA encoding
a selectable or screen able marker gene, si fibre and the explants
tissue.
 DNA delivery in his system is presumably due to cell wall penetration
by DNA coated si C F.
 cellular penetration is likely enhanced by frequent and forceful
intercellular collisions encountered in the votrexed millue.
 DNA bound to the fibre surface is carried into the penetrated cell and
becomes integrated into the nuclear genome and stably transform
plant cells with plant regeneration capacity will unable to the
application of Si c f mediated.
Silicon carbide fiber
Ultrasound mediated
 Immersion of explant in sonication buffer containing plasmid
DNA and is then sonicated with an ultra sonic pulse
regenerater at 0.5 w/cm2 aucostic intensity for 30 mins.
Then the samples are rinsed in buffer soln, and then cultured
for Gth and deferentiation.
 Advantage:
 Being simple method
 Inexpensive and multi functional equipment.
 There is no need of tissue culturists.
DNA transfer via pollen
 Y pollen as v?
 Fertilization of zygotic embryogenesis, and ovules are
difficult to isolate and injection of DNA into embryo sac in
insitu seemed to be too tedious and unpredictable..
 Ex: DNA treated pollen as vector for pollinating fertile
plants of Maize by ohta.
 Gene transfer through pollens
Chemical gene transfer method
 PEG mediated
 Calcium phosphate Co-precipitation
 The polycation DMSO technique
 DEAE dextran procedure
Calcium phosphate Co-precipitation
 DNA is mixed with CaCl2 soln & isotonic buffer to form
DNA CaPO4 ppt. this ppt is allowed to react with actively
dividing cells for several hours, washed and then incubated
in fresh culture medium.
 The efficiency of transformation though low can be increased
by giving them a physiological shock with DMSO.
 relative success depends upon high DNA concentration and
its apparent protection in the ppt.
The polycation DMSO technique
 Involves polycation, polybrene to increase the
adsorption of DNA to the surface followed by a
brief treatment with 25-30% of DMSO to increase
the membrane permeability and enhance uptake.
 Advantage :
 Less toxic than other polycations,
 High transformation efficiency and allows very
small quantities of plasmid DNA to be used.
DEAE dextran procedure
 Transformation of cells with DNA complexed to the high molecular
weight polymer Di Ethyl Amino Ethyl dextran is used obtain
efficient transient expression. The efficiency increases to 80% when
DMSO shock id given, but it wont produce stable transformants.
 Advantage:
 Direct gene transfer method
 Ex: cereals
 Disadvantage
 After transformation the further improvement of plant tissue
culture is not good so..
 It show unpredictable pattern of foreign DNA integration
 During their passage into the nucleus , the DNA are subjected to
nucleolytic cleavage resulting in truncation, recombination,
rearrangement or silencing of DNA occurs.
Tomatoes comes in many
varieties, colors and shapes
Transgenic tomatoes -
expressing different malarial
antigens
Normal and mutant tomato fruit
high-pigment 1 (hp1/hp1), high-pigment
2 (hp2/hp2), Never-ripe (Nr/Nr),
Green-ripe (Gr/Gr), Colorless non-
ripening (Cnr/Cnr) &
ripening-inhibitor (rin/rin) mutations
Delivery of a corn-based edible
vaccine
Transgenic corn kernels (a)
Corn snack (b) or
Embryo or germ cells (c)
Dr Eady
Crop & Food Research in New Zealand and
his collaborators in Japan
Tearless Onion
http://www.dailymail.co.uk/news/article-514799/The-orange-purple-
green-cauliflowers-scientists-claim-healthier-you.html
High anthocyanin purple tomato and red
wild-type tomato
Purple tomatoes high in anthocyanin
http://news.bbc.co.uk/2/hi/heal
th/7688310.stm
http://www.seedquest.com/News/releases/2008/october/24091.htm
Anthocyanins offer
protection against certain
cancers, cardiovascular disease
and age-related degenerative
diseases. Anthocyanins also
have anti-inflammatory activity,
promote visual acuity and hinder
obesity and diabetes.
World's First Blue Roses On Display In Japan
The Blue Rose was developed by Suntory
Flowers
October 31, 2008,
Thank you
„Urs Sami

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Plant transformation methods

  • 2. INTRODUCTION  Plant genetic engineering has become one of the most important molecular tools in the modern molecular breeding of crops.  Over the last decade, significant progress has been made in the development of new and efficient transformation methods in plants.  Despite a variety of available DNA delivery methods, Agrobacterium- and biolistic-mediated transformation remain the two predominantly employed approaches.
  • 3.  In particular, progress in Agrobacterium-mediated transformation of cereals and other recalcitrant dicot species has been quite remarkable.  In the meantime, other transgenic-enabling technologies have emerged, including generation of marker-free transgenics, gene targeting, and chromosomal engineering.
  • 4.  Although transformation of some plant species or elite germplasm remains a challenge, further advancement in transformation technology is expected because the mechanisms of governing the regeneration and transformation processes are now better understood and are being creatively applied to designing improved transformation methods or to developing new enabling technologies.
  • 5.  The genetic constitution of plants can be altered in the laboratory by a process called transformation, whereby a segment of DNA (deoxyribonucleic acid) is introduced that becomes inserted in one of the plant chromosomes.  Several methods to accomplish plant transformation have been devised. In all these methods, single cells are transformed and thereafter regenerated into complete, fertile plants by tissue culture procedures.  Some of the transformation methods can only be applied to protoplasts.
  • 6.  Nowadays, particle bombardment and the natural vector Agrobacterium tumefaciens are preferred because they can also cope with whole plant tissues such as roots and leaves, which are easier to handle, more stable and require less of the lengthy steps that are required for plant regeneration.  Transgenes are integrated at random positions in the plant genome, but procedures for targeted integration become more and more efficient.
  • 7.  Production of transgenic plants Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants
  • 8. Plant Transformation Methods (DIRECT) Physical Chemical (INDIRECT) Biological In Planta •Microinjection •Pressure •Biolistics – gene gun/particle bombardment •Electroporation •Microinjection •Silica/carbon fibers •Laser mediated •SAT •PEG •DEAE- dextran •Calcium phosphate •Artificial lipids •Proteins •Dendrimers A.Tumefaciens A. Rhizogenes • Virus- mediated •Meristem transformation •Floral dip method •Pollen transformation
  • 9. Techniques for plant genetic transformation  Indirect method- Agrobacterium mediated gene transfer  Direct methods-  Particle bombardment (biolistics)  Microprojectile gun method  Electroporation  Silicon carbide fibres  Polyethylene glycol (PEG)/protoplast fusion  Liposome mediated gene transfer
  • 10. Properties of good host 1. Easy to transform 2. Supports the replications of recombinant DNA 3. Is free from elements that interfere with replication of recombinant DNA 4. Lacks active restriction enzymes ex: E.coli k12 substrain HB 101 5. Does not have methylases 6. Is deficient in normal recombination function : so that it is not altered by recombination events.
  • 11. Transformation vector requirements  Origin of replication  Bacterial selectable marker  Gene constructs of interest  T-DNA borders and other Agrobacterium genes if using Agrobacterium  Compatible with helper plasmid if using Agrobacterium
  • 12. Agrobacterium mediated gene transfer Agrobacterium-  Soil borne, gram negative, rod shaped, motile found in rhizosphere  Causative agents of “Crown gall” disease of dicoltyledones  Have ability transfer bacterial genes to plant genome  Attracted to wound site via chemotaxis in response to chemicals (sugar and Phenolic molecules: acetosyringone) released from damaged plant cells  Contains Ti plasmid which can transfer its T-DNA region into genome of host plants
  • 13. Ti-plasmid features  Two strains of Ti-plasmid: -Octopine strains- contains two T-DNA region: TL (14 kb) and TR ( 7 kb) -Nopaline strains- contain one T-DNA region(20 kb)  Size is about 200 kb  Has a central role in Crown-gall formation  Contains one or more T-DNA region that is integrated into the genome of host plants  Contain a vir region ~ 40 kb at least 8~11 vir genes  Has origin of replication  Contains a region enabling conjugative transfer  Has genes for the catabolism of opines
  • 15.
  • 16. Forms of T-DNA that are found in Agrobacterium ds circles - found only in induced bacteria, not (apparently) in plant cells. ds linear T-DNA - found only in induced bacteria, not (apparently) in plant cells.  ss linear T-DNA - found in bacteria and plant cells. what is not found - Ti plasmids with evidence that T-DNA has been precisely deleted.
  • 17. Process of T-DNA transfer and integration 1. Signal recognition by Agrobacterium: -Agrobacterium perceive signals such as sugar and phenolic compounds which are released from plants. 2. Attachment to plants cells: Two step processes: i) initial attachment via polysaccharide ii) mesh of cellulose fiber is produced by bacteria. Virulence genes (chv genes) are involved in the attachment of bacterial cells to the plants cells. 3. Vir gene induction: VirA senses phenolics ans subsequently phosphorylating and thereby activating VirG. VirG then induces expression of all the vir genes. 4. T-strand production:VirD1/virD2 complex recognises the LB and RB. virD2 produces single-stranded nicks in DNA. Then virD2 attached to ssDNA. virC may assist this process.
  • 18. Process of T-DNA transfer and integration (continu) 5. Transfer of T-DNA out of bacterial cells: T-DNA/VirD2 is exported from the bacterial cell by “T-pilus” composed of proteins encoded by virB operon and VirD2. VirE2 and VirF are also exported from bacterial cells. 6. Transfer of the T-DNA and Vir proteins into the plant nuclear localization: T-DNA/VirD2 complex and other Vir proteins cross the plasma membrane through channels formed from VirE2. VirE2 protect T-DNA from nucleases, facilitate nuclear localization and confer the correct conformation to the T-DNA/virD2 complex for passage through the nuclear pore complex (NPC). The T-DNA/VirD2/VirF2 /plant protein complex the nucleus through nuclear pore complex. And integrated into host chromosome.
  • 19. Agrobacterium tumefaciens mediated transforamtion in plants Explant { decapitated seedlings, cells, protoplast, leaf discs} Addition of wounding Acetosyringone Agrobacteria co cultivation to allow infection antibiotics to kill bacteria transformed and non transformed tissue selective media to kill the non transformed tissues i.e, addition of kanamycin and hygromycin etc transformed tissue / callus transformed shoots rooted shoots adult plants SEEDS
  • 20. Practical application of Agrobacterium-mediated plant transformation  1. Agrobacterium mediated transformation methods are thought to induce less rearrangement of the transgene.  Lower transgene copy number that direct DNA delivery methods.  Successful production of transgenic plants depends on the suitable transformation protocols.
  • 21.
  • 22.
  • 23. The basic protocol used for any Agrobacteruim mediated transformation experiments 1. Identify a suitable explants: Suitable plant tissue is removed and sterilized. Leaf is used for Tobacco. 2. Co-cultivate with the Agrobacterium: Leaf tissue is cut into small pieces and placed into a culture of Agrobacterium for about 30 mins. The explants are subsequently removed from the bacterial culture and placed on to the MS medium that contain no selective agent. The incubation of explants with Agrobacterium is allowed to continue for 2 days to allow transfer of the T-DNA transfer to the plant cells. 3. Kill the Agrobacterium with a suitable antibiotic: The explants are removed from the medium and washed in antibiotic (cefotaxime) solution that kill Agrobacterium cells.
  • 24. The basic protocol used for any Agrobacteruim mediated transformation experiments (Conti-) 4. Select for transformed plant cells: The explant are transferred to fresh solid medium supplemented with a selective agent (kanamycin). It also contains cefotaxime. Auxin, Cytokinin are used to encourage the regeneration of by organogenesis. High cytokinin to auxin ratio promotes shoot formation from the explants. 5. Regeneration of whole plant :The shoot can be rooted by placing them on solid medium containing a high auxin to cytokinin ratio.
  • 25.
  • 26. Advantages of A.bacterium mediated transformation  It is a natural mean of transfer and is perceived as more acceptable technique to those who feel natural is best.  It is capable of infecting intact plant cells, tissues and organs. As result of tissue culture limitations are much less of a problem. Transformed plants can be regenerated more rapidly.  It is having a capable of transferring large fragments of DNA very efficiently without substantial rearrangements.  Integration of T-DNA is relatively precise process, it serves as an ideal insertional mutagenesis vehicle as it introduces one to several copies of the transferred DNA into the intact genome at one or a few loci.  The stability of gene transferred is excellent. Integrated T- DNA gives consistent maps and appropriate segregation ratios. Introduces traits have been found to be stable over many generations.  This at ability is critical to commercialization of transgenic plants.
  • 27. Disadvantages  It has limitation of host range,  Some of the important food crops can not be infected by this although, lately much progress has been achieved to over come this limitation by the development of highly virulent strains of Agrobacterium. Sometimes, the cells in a tissue that are able to regenarate, are difficult to transform. it might be that these are in layers too deep to be reached by Agrobacterium, or simply are not targets for T-DNA transfer.
  • 28. Virus mediated  Vectors based on viruses are desirable because of high efficiency of gene transfer that can be obtained by infection and because of the amplification of transferred gene that occurs via viral genome replication.  Vectors for transferring gene into plants are based on DNA or RNA molecules, that naturally express their genetic information in the plant cells.  The replicating genomes of plant viruses are non-integrative vectors, as compared to vectors based on the T-DNA of A. Tumefaciens which are integrative gene vectors.
  • 29.  The plant virus vectors do not integrate into the plant cells or host genome , rather they spread systematically within a plant and accumulate to high copy number in their respective target cells.  In most of the cases , the viral genome has modified to accommodate the insertion of foreign sequences, which are transferred, multiplied and expressed in plant as part of recombinant virus genome.
  • 30.  Plant viruses as vectors  Caulimoviruses – ds DNA – CaMV  Geminiviruses - 2ss DNA – maize streak virus  RNA plant viruses - TMV
  • 31. Vectorless or Direct DNA transfer  The trem “Direct gene transfer” is used to discriminate between the methods of plant transformation that rely on Agrobacterium (indirect method) and those that do not (direct methods). Direct gene transfer methods all rely on the delivery of large amount of naked DNA whilst plant is transiently permeabilised. Direct methods- Particle bombardment (biolistics) Microprojectile gun method Electroporation Silicon carbide fibres Polyethylene glycol (PEG)/protoplast fusion Liposome mediated gene transfer
  • 32. Physical methods  DMGT: DNA Mediated Gene Transfer A general scheme for production of transgenic plants using various direct DNA delivery methods have been commercialized.  Electroporation  Particle bombardment / Micro projectile / Biolistics  Electro injection  Micro injection  macro injection  Liposome mediated  Silicon carbide fibre mediated  ultra sound mediated  DNA transfer via pollen
  • 33. Electroporation technique.  Is the process where by electrical impulses of high field strength are used to reversibly permeabalize cell membrane to facilitates uptake of large molecules, including DNA.  It has been used for long time for transient and integrative transformation of protoplasts.  1 to 1.5 k V. so uses low capacitance hence short decay time .
  • 34. Electroporation  It can be used to deliver DNA into plant cells and protoplasts.  The genes of interest require plant regulatory sequence.  Plant materials is incubated in a buffer solution containing DNA and subjected to high-voltage electric pulse.  The DNA then migrates through high-voltage-induced pores in the plasma membrane and integrates into the genome.  It can be used to transform all the major cereals particularly rice, wheat, maize.  Advantages and disadvantages:  Both intact cells and tissue can be transformed.  The efficiency of transformation depends upon the plant materials, electroporation and tissue treatment conditions used for transformation.  ~40 to 50% incubated cells receive DNA  ~50% of the transformed cells can survive 
  • 35. Duracell DNA containing the gene of interest Plant cell Protoplast Electroporation Technique Power supply DNA inside the plant cell The plant cell with the new gene
  • 36. Biolistic/Particle bombardment  High velocity micro projectile were utilize to deliver nucleic acids into loving cells.  Advantages : Transformation of organized tissue Universal delivery system Transformation of recalcitrant spp Study of basic plant development processes.
  • 37. Particle bombardment  Why Biolistics or Biolistic bombardment?  Is the most powerful method for introducing nucleic acids into plants, because the helium pressure can drive microcarriers through cell walls  Is much easier and less time consuming than microinjecting nucleic acids into plant cells or embryos  Allows transformation of animal cells that have unique growth requirements and that are not amenable to gene transfer using any other method  Requires less DNA and fewer cells than other methods, and can be used for either transient or stable transformation
  • 38. Principle  The gold or tungsten particles are coated with the DNA that is used to be transform the plant tissue.  The particles are propelled at high speed into the target plant material where the DNA is released within then cell and can integrate into the genome.  Two types of plant tissues are used for particle bombardment:  a) Primary explants that are bombarded and then induced to become embryogenic  b) Proliferating embryonic cultures that are bombarded and then allowed to proliferate further and subsequently regenerate.
  • 39. PDS-1000/He bombardment System Fig: Schematic representation of the PDS-1000/He system upon activation. The arrows indicate the direction of helium flow Fig: The PDS-1000/He system, shown here with magnified view of the Hepta adaptor.
  • 40. How the PDS-1000/He System Works  The sample to be transformed is placed in the bombardment chamber, which is evacuated to subatmospheric pressure  The instrument is fired; helium flows into the gas acceleration tube and is held until the specific pressure of the rupture disk is reached  The disk bursts, and the ensuing helium shock wave drives the macrocarrier disk (which carries the coated microparticles) a short distance toward the stopping screen  The stopping screen retains the macrocarrier, while the microparticles pass through the screen into the bombardment chamber and penetrate the target cells  The launch velocity of microcarriers depends on a number of adjustable parameters: the helium pressure (rupture disk selection, 450–2,200 psi), the amount of vacuum, the distance from the rupture disk to the macrocarrier, the distance from the microcarrier launch assembly to the stopping screen, and the distance between the stopping screen and target cells. Adjusting these parameters allows you to produce a range of velocities to optimally transform many different cell types.
  • 41. The Helium Gas Gun – Circa 2000 Particle Gun
  • 42. Microinjection  Under a microscope, a cell is manipulated to a blunt capillary. Gentle suction holds the cell in place.  With a micromanipulator, a very fine tipped pipet is inserted into the cytoplasm or nucleus.  DNA or RNA is injected directly into the nucleus or cytoplasm.  Microinjection has been successfully used with large frog eggs, cultured mammalian cells, mammalian embryos, and plant protoplasts and tissues
  • 43. Microinjection Microinjection techniques for plant protoplasts utilize a holding pipette for immobilizing the protoplast while an injection pipette is utilized to inject the macromolecule.  In order to manipulate the protoplasts without damage, the protoplasts are cultured for from about 1 to 5 days before the injection is performed to allow for partial regeneration of the cell wall. It was found that injection through the partially regenerated cell wall could still be accomplished and particular compartments of the cell could be targeted.  The methods are particularly useful for transformation of plant protoplasts with exogenous genes.
  • 44.
  • 46. Silicon carbide fibres-Whiskers  Plant materials (Cells in suspension culture, embryos and embryo-derived callus) is introduced into a buffer containing DNA and the silicon fibers which is then vortexed.  The fibers (0.3-0.6 μm in diameter and 10-100μm long) penetrate the cell wall and plasma membrane, allowing the DNA to gain access to the inside of the cells.  Disadvantages and advantages  The drawbacks of this technique relate to the availability of suitable plant material and the inherent dangers of the fibers, which require careful handing.  Many cereals, produce embryonic callus that is hard and compact and not easily transformed with this technique.  Despite the some disadvantages, this method is recently used for successful transformation of wheat, baerly, and maize without the need to cell suspension.
  • 47. Silicon carbide fibre mediated  0.6 micro m dia, 10 – 80 micro m lth.  vortexing in an eppendorf tube a mixture of plasmid DNA encoding a selectable or screen able marker gene, si fibre and the explants tissue.  DNA delivery in his system is presumably due to cell wall penetration by DNA coated si C F.  cellular penetration is likely enhanced by frequent and forceful intercellular collisions encountered in the votrexed millue.  DNA bound to the fibre surface is carried into the penetrated cell and becomes integrated into the nuclear genome and stably transform plant cells with plant regeneration capacity will unable to the application of Si c f mediated.
  • 49. Ultrasound mediated  Immersion of explant in sonication buffer containing plasmid DNA and is then sonicated with an ultra sonic pulse regenerater at 0.5 w/cm2 aucostic intensity for 30 mins. Then the samples are rinsed in buffer soln, and then cultured for Gth and deferentiation.  Advantage:  Being simple method  Inexpensive and multi functional equipment.  There is no need of tissue culturists.
  • 50. DNA transfer via pollen  Y pollen as v?  Fertilization of zygotic embryogenesis, and ovules are difficult to isolate and injection of DNA into embryo sac in insitu seemed to be too tedious and unpredictable..  Ex: DNA treated pollen as vector for pollinating fertile plants of Maize by ohta.
  • 51.  Gene transfer through pollens
  • 52. Chemical gene transfer method  PEG mediated  Calcium phosphate Co-precipitation  The polycation DMSO technique  DEAE dextran procedure
  • 53. Calcium phosphate Co-precipitation  DNA is mixed with CaCl2 soln & isotonic buffer to form DNA CaPO4 ppt. this ppt is allowed to react with actively dividing cells for several hours, washed and then incubated in fresh culture medium.  The efficiency of transformation though low can be increased by giving them a physiological shock with DMSO.  relative success depends upon high DNA concentration and its apparent protection in the ppt.
  • 54. The polycation DMSO technique  Involves polycation, polybrene to increase the adsorption of DNA to the surface followed by a brief treatment with 25-30% of DMSO to increase the membrane permeability and enhance uptake.  Advantage :  Less toxic than other polycations,  High transformation efficiency and allows very small quantities of plasmid DNA to be used.
  • 55. DEAE dextran procedure  Transformation of cells with DNA complexed to the high molecular weight polymer Di Ethyl Amino Ethyl dextran is used obtain efficient transient expression. The efficiency increases to 80% when DMSO shock id given, but it wont produce stable transformants.  Advantage:  Direct gene transfer method  Ex: cereals  Disadvantage  After transformation the further improvement of plant tissue culture is not good so..  It show unpredictable pattern of foreign DNA integration  During their passage into the nucleus , the DNA are subjected to nucleolytic cleavage resulting in truncation, recombination, rearrangement or silencing of DNA occurs.
  • 56. Tomatoes comes in many varieties, colors and shapes Transgenic tomatoes - expressing different malarial antigens
  • 57. Normal and mutant tomato fruit high-pigment 1 (hp1/hp1), high-pigment 2 (hp2/hp2), Never-ripe (Nr/Nr), Green-ripe (Gr/Gr), Colorless non- ripening (Cnr/Cnr) & ripening-inhibitor (rin/rin) mutations
  • 58. Delivery of a corn-based edible vaccine Transgenic corn kernels (a) Corn snack (b) or Embryo or germ cells (c)
  • 59. Dr Eady Crop & Food Research in New Zealand and his collaborators in Japan Tearless Onion
  • 61. High anthocyanin purple tomato and red wild-type tomato Purple tomatoes high in anthocyanin http://news.bbc.co.uk/2/hi/heal th/7688310.stm
  • 62. http://www.seedquest.com/News/releases/2008/october/24091.htm Anthocyanins offer protection against certain cancers, cardiovascular disease and age-related degenerative diseases. Anthocyanins also have anti-inflammatory activity, promote visual acuity and hinder obesity and diabetes.
  • 63. World's First Blue Roses On Display In Japan The Blue Rose was developed by Suntory Flowers October 31, 2008,