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PLANT GENETIC TRANSFORMATION

METHODES
by
SUGIYONO

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INTRODUCTION

All stable transformation methods


consist of three steps:
1)
2)
3)

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Delivery/transfer of DNA into a single plant cell.


Integration of the DNA into the plant cell genome.
Conversion of the transformed cell into a whole plant.

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Introduction of DNA into cells :


1) Direct DNA transfer
a)

Particle bombardment
b) Electroporation
c) Microinjection
d) Chemical induction
e) Protoplasts fusion

2) Indirect DNA Transfer

Agrobacterium-mediated transformation

Agroinfiltration

Stable transformation using Agrobacterium (&


Floral dipping)

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The only known natural example of


inter-kingdom DNA transfer

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Overall, Agrobacterium can transfer T-DNA to a broad


group of plants.
Yet, individual Agrobacterium strains hve a limited host
range.
The molecular basis for the strain-specific host range is
unknown.
Many monocot plants can be transformed (now),
although they do not form crown gall tumors.
Under lab conditions, T-DNA can be transferred to
yeast, other fungi, and even animal and human cells.

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T-DNA of A. tumefaciens is excised and integrates into


the plant genome as part of the natural infection
process.
Any foreign DNA inserted into the T-DNA will also
be integrated.

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The T-DNA element is defined by its borders


but not the sequences within. So researchers
can substitute the T-DNA coding region with
any DNA sequence without any effect on its
transfer from Agrobacterium into the plant.

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1. Cytokinins
(plant hormone for cell plant division and tumorous growth)
2. Enzymes for indoleacetic acid (auxin) synthesis

Another plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and
preventing aging)

3. Enzymes for synthesis and release of novel plant metabolites:


the opines (uniques amino acid derivatives)
the agrocinopines (phosphorylated sugar derivatives) .

Nopaline

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Opines and agrocinopines are NUTRIENTS for A.tumefacies.


They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
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Plant hormones (auxin, cytokinin)


Opines (octopine, nopaline)

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Wounded plants secrete sap with acidic pH (5.0


to 5.8) and a high content of various phenolic
compounds (lignin, flavonoid precursors)
serving as chemical attractants to agrobacteria
and stimulants for virgene expression.
Among these phenolic compounds,
acetosyringone (AS) is the most effective.

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The existence and orientation of right border is


absolutely required for Agrobacterium
pathogenicity but not the left border.
Transfer of the T-DNA is polar from right to
left.

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Although right border and left border are


required to delimit the transferred segments,
the T-DNA content itself has no effect on the
efficiency of transfer.
Therefore, researchers replace most of the TDNA with DNA of interest, making
Agrobacterium a vector for genetic
transformation of plants.

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Constructing transgenes
The minimal requirements for the transgene are a
promoter, coding region, and terminator
General structure:
5---Promoter ..Coding region.terminator---3

Example: Bt gene with 35S promoter, nptII selectable


marker, and Tnos terminator sequence
5 --P35SBtTnos--//--P35SnptIITnos---3

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Promoter: DNA sequence controlling spatial and/or temporal


level of transgene expression. Promoters can be

1. Constitutive
CaMV 35S: from Cauliflower mosaic virus 35S rRNA
gene
2. Tissue specific
Glutelin GT1: Endosperm specific
3. Inducible
Cis-Jasmone: Arabidopsis genes induced by
stress/wounding

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Termination sequence: DNA sequence signaling end of


the gene during transcription.
TNOS: Nopaline synthase gene from Agrobacterium

tumefaciens

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Selectable Marker: Encodes a protein (enzyme) that

allows the transformed cells to grow while the growth of the


non-transformed cells is inhibited. Examples include

1. Antibiotic resistance
2. Herbicide resistance

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Selectable Marker: Encodes a protein (enzyme) that

allows the transformed cells to grow while the growth of the


non-transformed cells is inhibited. Examples include
2. Herbicide resistance
..Members of the genus Streptomyces produce bialaphos,
which leads to the production of glufosiante, ultimately
inhibiting glutamine synthetase. The biochemical and
toxicological characteristics of glufosinate have made it a
popular, nonselective herbicide, which has been commercialized
under the names Basta, Buster and Liberty by Bayer
Crop Science. Other Streptomyces spp. can detoxify glufosinate
by producing an acetylating enzyme via production of a
phosphinothricin acetyl transferase (PAT) enzyme encoded by
the bar (bialaphos resistance) gene. Treatment of genetically
modified plants carrying a bar gene with glufosinate or
bialaphos provides a very efficient means of selection in genetic
transformation protocols.
http://www.patentlens.net/daisy/Phosph/g2/710.html
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Reporter genes: Genes that, upon expression in the

transgenic plants, provide a clear indication that genetic


transformation did occur, and indicate the location and the
level of expression.
A. Glucuronidase (GUS)
B. Luciferase, green fluorescent protein (GFP)

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GFP: So many ways to be green

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Plasmid map

bar nos
35S gusA-intron
ter
(monocot)

ampR

ColE1 ori

BX EH

35S

35S

crtI

RBR

35S
ter

XhoI

ssu
ter

XBE

XhoI

nptII

BX

XhoI

nos

XBE

XhoI

XB E B

LBR

lox

lox

ColE1 ori

spc/sptr

RK2

pBECKS2000.4

35S gusA-intron
(monocot)

XBE

ampR

35S

ColE1 ori

spc/sptr

RBR

crtI 35

35S

S
ter

lox
ColE1 ori

BX EH

XhoI

hph

BX

XhoI

nos
ter

BE

XhoI

nptII

E XH

XhoI

nos

XBX

nos

LBR

lox

RK2

pBECKS2000.6

E=EcoRI; X=XbaI; H=HindIII; B=BamHI

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Every induced
Agrobacterium cell
produces one T-strand.
VirD1 and VirD2 are
involved in the initial Tstrand processing,
acting as site-and
strand-specific
endonucleases.

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The T-complex is
composed of at least
three components:
one T-strand DNA
molecule, one VirD2
protein, and around
600 VirE2 proteins.

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Transport of the Tcomplex into the host


cell most likely occurs
through a type IV
secretion system.
In Agrobacterium, the
type IV transporter
(called T-pilus)
comprises proteins
encoded by virD4 and
by the 11 open reading
frames of the virB
operon.

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Because the large size of


T-complex (50,000 kD,
~13nm in diameter), the
nuclear import of Tcomplex requires active
nuclear import.
The T-complex nuclear
import is presumably
mediated by the Tcomplex proteins, VirD2
and VirE2. Both of them
have nuclear-localizing
activities.

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Flanking sequence tags (FSTs) analysis showed


no obvious site preference for integration
throughout the genome.
About 40% of the integrations are in genes and
more of them are in introns.

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RICE TRANSFORMATION PROTOCOL


Vector Construction
Bacterial Culture and maintenance
3)
Agrobacterium infection
Calli-agrobacterium co-cultivation
5)
Agrobacterium removal
6)
Calli selection
7)
Calli multiplication
8)
Calli regeneration
9)
Shoot development
10) Analysis of transgenic plants.
11) Root induction
12) Acclimatisation
13) Plant growth and maintenance
14) Progeny test
1)

2)

4)

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UC-Davis (BS & PhD)


Univ Wash (MS)
At Monsanto for nearly 20
yearsdeveloped methods
to specically target our genetic
engineering tool, Agrobacterium,
to the right cells at the right
time.
Produced Roundup Ready
Soybean
Now Chief Technology Officer-ArborGen

Floral dip transformation of Arabidopsis

Seems to transform ovule


Not easily conducive for other species

Most species: using organogenesis or


embryogenesis-based tissue culture methods to
regenerate transgenic plants

Floral dipping Arabidopsis

From the following


article
Agrobacterium-mediated
transformation of
Arabidopsis thaliana using
the floral dip method
Xiuren Zhang, Rossana
Henriques, Shih-Shun Lin,
Qi-Wen Niu and Nam-Hai
Chua
Nature Protocols 1, 641 646 (2006)
doi:10.1038/nprot.2006.97

: www.plantmethods.com/content/2/1/16/figure/F1

http://wwww.cirad.fr/presentation/programmes/biotrop/resultats/images/agrobac.gif

Infection (cocultivation) and DNA transfer


Agrobacterium strain and acetosyringone
Kill off unwanted Agrobacterium after gene
transfer
Selection methods to prevent escapes
Plant regeneration

Agrobacterium-mediated transformation of
monocotyledons can be achieved with the same efficiency
as dicotyledons
The T-DNA is transferred from Agrobacterium to
dicotyledons and monocotyledons by an identical
molecular mechanism and with similar efficiency
The majority of plants produced by this method are free
of morphological aberrations
The fertility of the transgenic plants was found to be
similar to seed-grown plants, and the segregation ratio for
the transgene was exactly 3 : 1, which supports the
conclusion that the transgene has been integrated into the
genome

Molecular analysis of genomic DNA from engineered


plants indicated the presence of vector sequences outside
the T-DNA borders.
Agrobacterium was also found to persist on the surface and
within tissues of transformed plants in soil-grown plants
up to 12 months after transformation (Christou, 1997)