PLANT GENETIC TRANSFORMATION

METHODES
by
SUGIYONO

11/9/2019 Creating a better future 1

 INTRODUCTION

All stable transformation methods

consist of three steps:
1) Delivery/transfer of DNA into a single plant cell.
2) Integration of the DNA into the plant cell genome.
3) Conversion of the transformed cell into a whole plant.

11/9/2019 Creating a better future 2

 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)

11/9/2019 Creating a better future 3

 Agrobacterium-mediated
Transformation

11/9/2019 Creating a better future 4

 Biology of the Agrobacterium-
plant interaction

The only known natural example

of inter-kingdom DNA transfer

11/9/2019 Creating a better future 5

11/9/2019 Creating a better future 6
11/9/2019 Creating a better future 7
The genus Agrobacterium has a wide host
range:

 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.

11/9/2019 Creating a better future 8

11/9/2019 Creating a better future 9
11/9/2019 Creating a better future 10
 Ti Plasmid

11/9/2019 Creating a better future 11

 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.

11/9/2019 Creating a better future 12

 Why is Agrobacterium used for producing
transgenic plants?

 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.

11/9/2019 Creating a better future 13

 Important genes encoded by Ti plasmid

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
Opines and agrocinopines are NUTRIENTS for A.tumefacies.
They can not be used by other bacterial species
It provides unique niche for A.tumefaciens
11/9/2019 Creating a better future 14
Agrobacterium-induced plant tumors contain
high concentrations of :

 Plant hormones (auxin, cytokinin)

 Opines (octopine, nopaline)

11/9/2019 Creating a better future 15

11/9/2019 Creating a better future 16
11/9/2019 Creating a better future 17
11/9/2019 Creating a better future 18
 Plant signals

 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.

11/9/2019 Creating a better future 19

 Structure of the T-DNA

 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.

11/9/2019 Creating a better future 20

  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 T-
DNA with DNA of interest, making
Agrobacterium a vector for genetic
transformation of plants.

11/9/2019 Creating a better future 21

 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’ --P35S…Bt…Tnos--//--P35S…nptII…Tnos---3’

11/9/2019 Creating a better future 22

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

11/9/2019 Creating a better future 23

Termination sequence: DNA sequence signaling end of
the gene during transcription.

TNOS: Nopaline synthase gene from Agrobacterium

tumefaciens

11/9/2019 Creating a better future 24

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

11/9/2019 Creating a better future 25

 Selectable marker genes commonly used in cereal
transformation

11/9/2019 Creating a better future 26

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
11/9/2019 Creating a better future 27
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)

11/9/2019 Creating a better future 28

 GFP: So many ways to be green

11/9/2019 Creating a better future 29

11/9/2019 Creating a better future 30
Plasmid map

XBE B BX EH RBR
LBR XB E B H XBE BX H
ampR ColE1 ori 35S
nos nptII ssu 35S gusA-intron
bar nos 35S 35S crtI
ter ter (monocot) ter

XhoI

XhoI
XhoI
XhoI

lox
lox
ColE1 ori spc/sptr RK2

pBECKS2000.4

E XH XBE BX EH RBR
LBR XBX BE BX H B
ampR ColE1 ori
nos
nos nptII hph 35S gusA-intron 35S 35S crtI 35
nos

ter (monocot) S

XhoI
XhoI
ter

XhoI
XhoI

lox
lox
ColE1 ori spc/sptr RK2

pBECKS2000.6

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

11/9/2019 Creating a better future 31
 Production of T-strand
 Every induced
Agrobacterium cell
produces one T-strand.
 VirD1 and VirD2 are
involved in the initial T-
strand processing, acting
as site-and strand-specific
endonucleases.

11/9/2019 Creating a better future 32

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

11/9/2019 Creating a better future 33

 Intercellular transport
 Transport of the T-
complex 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.

11/9/2019 Creating a better future 34

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

11/9/2019 Creating a better future 35

 T-DNA integration is not highly
sequence-specific
 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.

11/9/2019 Creating a better future 36

 RICE TRANSFORMATION PROTOCOL

1) Vector Construction
2) Bacterial Culture and maintenance
3) Agrobacterium infection
4) 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

11/9/2019 Creating a better future 37

Biotechnologist of the day
Maud Hinchee
 UC-Davis (BS & PhD)
 Univ Wash (MS)
 At Monsanto for nearly 20
years—developed “…methods to
specifically target our genetic
engineering tool, Agrobacterium,
to the right cells at the right
time.”
 Produced Roundup Ready
Soybean
 Now Chief Technology Officer--
ArborGen
Agroinfiltration-transient method of expressing
transgenes
Agroinfiltration—forcing Agrobacterium with
transgenes into leaves
Agroinfiltration--tobacco
 Stable transformation using
Agrobacterium
 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
 Arabidopsis floral dip

: www.plantmethods.com/content/2/1/16/figure/F1
 Most plants still need tissue culture for
transformation and regeneration

http://wwww.cirad.fr/presentation/programmes/biotrop/resultats/images/agrobac.gif
 Key steps for traditional Agrobacterium-
mediated transformation

 Infection (cocultivation) and DNA transfer—

Agrobacterium strain and acetosyringone
 Kill off unwanted Agrobacterium after gene
transfer
 Selection methods to prevent escapes
 Plant regeneration
 Advantages of Agrobacterium-mediated
transformation
• 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
 Disadvantages of Agrobacterium-mediated
transformation
• 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)