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Plant Transformation Techniques

Plant tissue culture techniques allow plant cells, tissues or organs to be maintained or grown under sterile conditions. This relies on the fact that many plant cells can regenerate a whole plant from single cells or small pieces of tissue. Gene transfer techniques like Agrobacterium-mediated transformation or direct methods like particle bombardment are used to generate transgenic plants with desired traits faster than traditional breeding. The most common technique is Agrobacterium-mediated transformation, which involves Agrobacterium transferring DNA containing a gene of interest into the plant genome. Golden Rice was developed using this technique to transfer two genes into rice and address vitamin A deficiency in many parts of the world.

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Hassaan Ahmed
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132 views39 pages

Plant Transformation Techniques

Plant tissue culture techniques allow plant cells, tissues or organs to be maintained or grown under sterile conditions. This relies on the fact that many plant cells can regenerate a whole plant from single cells or small pieces of tissue. Gene transfer techniques like Agrobacterium-mediated transformation or direct methods like particle bombardment are used to generate transgenic plants with desired traits faster than traditional breeding. The most common technique is Agrobacterium-mediated transformation, which involves Agrobacterium transferring DNA containing a gene of interest into the plant genome. Golden Rice was developed using this technique to transfer two genes into rice and address vitamin A deficiency in many parts of the world.

Uploaded by

Hassaan Ahmed
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Plant transformation

techniques

Plant tissue cultureisacollectionoftechniquesusedto


maintainorgrowplantcells,tissuesororgansunder
sterileconditionsonanutrientculturemediumofknown
composition.
Planttissueculturereliesonthefactthatmanyplantcells
havetheabilitytoregenerateawholeplant(totipotency).
Singlecells,plantcellswithoutcellwalls(protoplasts),
piecesofleaves,stemsorrootscanoftenbeusedto
generateanewplantonculturemediagiventherequired
nutrientsandplanthormones

Gene Transfer in Plants


Why
Biochemical and physiological characterization
of gene, protein or cell function
Generate new plant strains for desired trait
Humans have been doing this by traditional crosses
This method is faster and allows for transgenic
(genes from different species/organisms) plants to be
created

Motivation for genetically


engineered crops
Agriculture is the biggest industrial
sector in the world
$1.3 trillion of products/year

Over past 40 years, world population


has doubled while agricultural land area
has increased by only 10%

Techniques for plant genetic


transformation
Indirect method Agrobacterium mediated gene transfer

Direct methods Particle bombardment (biolistics)


Microprojectile gun method
Electroporation

Agrobacterium tumefaciens
Gram negative bacterium which is
commonly found in soil
When plant is wounded, bacteria
enter and are pathagenic causing
Crown gall disease (plant tumor)
Only infects young dicots
DNA from bacteria is transferred
into plants where it binds to plant
histones and is integrated into plant
genome by recombination

Agrobacterium - mediated Gene Transfer

Most common method of engineering dicots, but also


used for monocots
Pioneered by J. Schell (Max-Planck Inst., Cologne)
Agrobacteria
soil bacteria, gram-negative, related to Rhizobia
species:
tumefaciens- causes crown galls on many dicots
rubi- causes small galls on a few dicots
rhizogenes- hairy root disease
radiobacter- avirulent

Agrobacterium tumefaciens
Two DNA genomic and extrachromasomal
plasmid DNA (Ti DNA) Tumor Inducing
Ti plasmid has two parts
T-DNA the portion of the plasmid inserted into the
plant cells and integrated into genome (remove host
cell gene and insert your gene of interest)
T-DNA also produces auxins and cytokinins (bacteria
producing plant hormones?!) to induce cell
proliferation (plant cancer)
And the vir region which encodes proteins for the
transfer of DNA to infected plant but proteins are not
transferred

Agrobacterium tumefaciens
Wild type Ti plasmid = 200 kb too large for cloning
LB and RB are required for insertion and recombination with plant
genome

Insertion into plant host is random


First cloned gene luciferase in
tobacco plant

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


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.

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.

Agrobacterium tumefaciens
n

Methods of Agrobacterium Delivery


Vacuum Infiltration
Plant leaf disks are placed in a suspension of bacteria
and vacuum pulled
Air is release like a sponge being squeezed
Vacuum is released and solution floods tissue
Plant disk is cultured

Methods of Agrobacterium Delivery


Floral Dip
Simple submersion of plant into bacterium
suspension
No vacuum is needed
Conducted with plants grown until just
flowering
Progeny seeds are harvested and
germinated using selective antibiotic

Methods of Agrobacterium Delivery

Cultured cells
Callus or protoplasts
Easy screen for positive using selection
New plants can be cloned using plant
tissue culture

Direct gene transfer methods


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 methodsParticle bombardment (biolistics)


Microprojectile gun method
Electroporation

Advantages and disadvantages of direct


gene transfer

Adv
Widespread use of transformation of cereal
crops that initially proved difficult to
transformation with Agrobacterium.

Disadv
They tend to lead higher frequency of
transgene rearrangement and higher copy
number. This can lead to high frequency of
gene silencing.

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: The PDS-1000/He system, shown here


with magnified view of the Hepta adaptor.

Fig: Schematic representation of the PDS-1000/He


system upon activation. The arrows indicate the
direction of helium flow

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

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

Table

Why genetically engineer


plants?
To improve the agricultural or horticultural value
of plants
To serve as living bioreactors for the production
of economically important proteins or
metabolites
To provide a renewable source of energy
(biofuels)
To provide a powerful means for studying the
biological action of genes and gene products

Reasons for Plant Gene


Transfer
Golden Rice
Grains such as rice, produce all but two of the enzymes
needed to produce beta carotene (vit A precursor)
Rice feeds half the worlds population
Vit A deficiencies are associated with blindness, night
blindness, diabetes, anemia and easy infections
WHO estimates 220 mil women and children affected by
preventable vit A-deficiency night blindness 400 mil
world wide people are affected by beta carotene
deficiency
1 million children/year dye from related diseases

Reasons for Plant Gene


Transfer

Golden Rice
Two genes - phytoene synthase
(psy) and phytoene desaturase
(crt I), are transformed into rice
via bacterial transmission

Golden Rice Synthesis


Two
Two Daffodil
Daffodil genes
genes and
and one
one bacterial
bacterial gene
gene
Erwinia
Erwinia uredovora
uredovora were
were cloned
cloned into
into
agrobacterium
agrobacterium T
T DNA
DNA and
and inserted
inserted into
into rice
rice
genome
genome to
to generate
generate needed
needed enzymes
enzymes
Phytoenesynthase&
Lycopene--cyclase

T DNA

Carotenedesaturase

Germ-line transformation with


agrobacterium
X
Cross
T-formed rice with genes

T-formed rice with gene

Progeny rice plant with complete carotene pathway

Reasons for Plant Gene


Transfer
Spring Canola production is limited to frost in
northern climes (US and Canada)
Protoplasts were exposed to UV light to generate
mutations in genomic DNA
Cells were cultured into mature plants where defense
signaling for cold tolerance was elevated (salicylic acid,
jasmonic acid
329 mutant embryos were screened 74 were selected
for further development with increased cold tolerance
Another approach is to clone and over express
antifreeze genes

Antisense molecules

Insecticide biotechnology
The gene coding for the
crystalline protein (Cry) was
transferred from the bacterium
to the plant.
Now the plant produces the
toxin protein, so dont need to
inoculate with bacterium
This strategy kills the pest before
it kills the plant

Reasons for Plant Gene


Transfer
Drought resistant crops
Insect resistant
Increase growth rate and higher oil
producing plants for biofuels
Production of pharmacuticals
Vaccine in bannana against diarrhea
(immune stimulating disease against
cholera bacteria)

Advantages
Theproductionofexactcopiesofplantswithgood
desirabletraits
Toquicklyproducematureplants.
Theproductionofmultiplesofplantsintheabsence
ofseeds
TheregenerationGMplants
Theproductionofdiseasefreeplantsinsterile
Theproductionofplantsfromseedsthatotherwisehave
verylowchancesofgerminatingandgrowing
Tocleanplantsparticularlyfromviralandotherinfections
andtoquicklymultiplytheseplantsas'cleanedstock'
forhorticultureandagriculture.

Concerns about genetically


modified foods
Human health
Unsuspected allergens
What other issues are there?

Environment
Messing up the gene pool of non-target
species in the environment
Lateral gene transfer in nature
Still poorly understood in nature

Confirmed: DNA From Genetically Modified Crops Can


Be Transferred Into Humans Who Eat Them

In a new study published in the


peer reviewed Public Library of
Science
(PLOS), researchers emphasiz
e that there is sufficient
evidence that meal-derived
DNA fragments carry complete
genes that can enter into the
human circulation system
through an unknown
mechanism

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