Sci.Int.

(Lahore),25(2),287-290,2013

ISSN 1013-5316; CODEN: SINTE 8

287

AGROBACTERIUM-MEDIATED STABLE TRANSFORMATION OF

ARABIDOPSIS THALIANA PLANTS USING -GLUCURONIDASE
(GUS) GENE
Aneela Yasmin1, Akhtar Ali Jalbani2 , Shahla Baloch1
1

Department of Biotechnology, Faculty of Crop Production

Sindh Agriculture University, Tandojam, Pakistan
2
Information Technology Center,
Sindh Agriculture University, Tandojam, Pakistan

ABSTRACT: This study is carried to optimize Arabidopsis transformation protocol using -glucuronidase
(GUS) gene as a reporter gene to analyse gene expression in heterologous system. The method we used to
facilitate our studies is Agrobacterium mediated floral dip method with minor modifications. Floral dip
method is the most popular protocol for the production of stable transformants of Arabidopsis thaliana.
Agrobacterium has the ability to transfer foreign DNA into intact plant cells. In floral dip method, the
developing inflorescences of Arabidopsis were dipped in the suspension of overnight grown Agrobacterium
carrying the genes of interest for about 10 seconds. This dipping of inflorescences targeted the female
gametes resulting in transformed seeds. The transformed seeds were then plated in vitro on a selective
medium and screened for primary transformants (T0). T0 transgenic lines were then planted to get T1
followed by sowing for T2 and T3 respectively. Using this method T2*- transgenic plants, homozygous for
transgene, were obtained in 10.5 months.
Keywords: Arabidopsis thaliana, Floral dip method, GUS reporter gene
1. INTRODUCTION
Generating stable transformants is an important tool to
validate the functionality of genes. Usually, the
transformation of plant cells or tissues is carried out by
delivering foreign genes into plant cells (Herrera-Estrella et
al., 2005)[1]. Agrobacterium tumefaciens and/or biolistic are
used to deliver new genes in the cells which are then
regenerated and verified to get final transgenic plants ready
for functional analysis. It is a lengthy process that requires
skilled manpower and sophisticated laboratory facilities to
conduct in vitro regeneration step. Although this approach is
time consuming this is widely adopted method to produce
transgenic plants in most of the crops. Tissue culture
imposes stress to plant tissues during dedifferentiation and
differentiation processes ultimately resulting in somaclonal
variation and DNA modification (Labra et al. 2004)[2]. Thus
plant transformation method without using in vitro
conditions would reduce the time generating plant
transgenics. In addition to improving transformation
methodology it is also important to identify a heterologous
system that could be transformed easy and in short time to
study foreign gene expression and functions independent of
the regulatory elements present in the source of the gene
under investigation. Arabidopsis, a model plant, is such a
heterologous system. Its genome is completely sequenced
and needs 3-4 month for the production of stable transgenic
plants (Zhang et al. 2006)[3]. For the first time Feldmann
and Marks (1987)[4] demonstrated the production of
transgenics without in vitro step. They co-cultivated
germinating seeds of Arabidopsis thaliana and
Agrobacterium tumefaciens with genes of interest in a
binary expression vector and obtained transgenic plantlets
with low efficiency. Later on Bechtold et al. (1993)[5]
conducted a breakthrough study to transform uprooted
flowering plants of Arabidopsis by vacuum infiltration in
agro-suspension. The uprooted plants were then replanted;
seed were harvested and screened for transgenics. This

method improved the transformation efficiency dramatically.

Later on this transformation procedure was improved
considerably (Clough and Bent, 1998)[6]. Major
modifications include omission of using uprooted plants and
vacuum infiltration. Agrobacterium is natural genetic
engineer; it has the ability to transfer foreign DNA into
intact plant cells. When the developing flowers of
Arabidopsis are dipped in the suspension of Agrobacterium
carrying the genes of interest the female gametes are
transformed and form transgenic seeds (Desfeux et al.,
2000)[7]. Now a day the most simplified method of
Arabidopsis transformation is floral dip method.
Here we report the optimization of Agrobacterium mediated
stable transformation of Arabidopsis using -glucuronidase
(GUS; Jefferson et al. 1987)[8] reporter gene for utilizing
Arabidopsis as heterologous system to study resistance
genes of roses in future. Rose is a woody plant that is
difficult to regenerate in vitro and to apply molecular
biological techniques due polyploidy (Yasmin, 2010;
2011)[9,10].
2. MATERIALS AND METHOD
a) Plant Material
Arabidopsis thaliana [Columbia-0 (wild)] plants were used
in present study. Seeds were grown in a clay pot using
standard methods. 10 Germinated seedlings were then sown
in pots (4 in.x4 in.) and pots were covered by a nylon sheet
fixed by rubber band to avoid soil leakage during dipping in
bacterial suspension. Plants then grown in a growth chamber
under short days for one month. Later the growth conditions
were changed to long-day to induce inflorescence. Pots must
be watered properly to keep plants healthy plants as the
efficiency of transformation depends on the health of plants.
b) Bacteria and Constructs
In this study Agrobacterium strain GV3101::pMP90
carrying construct 35S:GUS-intron in pBINPLUS (Hellens
et al. 2000[11]; Van Engelen et al. 1995)[12] was used. YEP

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liquid and/ or solid (agar= 15g/L) media was used to

grow bacteria .Media was supplemented with 50mg/L of
Kanamycin, 10mg/L of Rifampicin and 25mg/L Gentamycin
for the selection of bacteria and pMP90, virulence vector.
c) Floral Dip Method and Seed Harvesting
An overnight liquid culture (500ml; 280C for 20-24 hours)
of Agrobacterium was started a day before the main
experiment of transformation (Yasmin and Debener, 2010).
The bacteria were pelleted in a centrifuge at room
temperature and 4500 rpm for 15-20 min. Pelleted bacteria
were washed in sterile water and re-suspended in sterile
solution of 3% sucrose (100-200ml). The prepared bacterial
suspension was supplemented with 0.02% of tween-20. The
pots of plants were inverted to dip aerial parts of plants in
the bacterial suspension for 10 second followed by 3-5
seconds of draining bacterial suspension from dipped plants.
Dipped plants were then placed in a box fitted with a cover
to maintain high humidity for one day. Next day plants were
taken out from the box and shifted to growth chamber to
grow and set seed for about a month. As the siliques turned
brown watering was stopped and drying plants wrapped
using wax paper and seeds were collected and stored at room
temperature until further processing.
d) Screening Primary Transformants
Standard MS media plates were prepared containing
carbenicillin (100 mg L1) and kanamycin (50mg L-1) for
selection of primary transformants. Heavily contaminated
seeds were sterilized by gas treatment. Small samples of
seeds were put into Eppendorf tubes and were placed in a
thoroughly cleaned desiccator. A beaker containing 200 ml
of 5.3% sodium hypochloride solution was placed in the
desiccator and 2ml of HCl was added to the hypochloride
solution; desiccator was closed immediately and evacuated
briefly with pump. The samples were left overnight for
sterilization and taken out next morning in a fume hood or
under clean bench for plating. The sterilized seeds were
suspended in sterile 0.05% agarose (about 30-40 ml seed per
ml agarose) and the suspension was spread onto selection
plates. Plates were dried under laminar flow hood and seeds
were vernalized by placing them at 40C for 3 days. Then
plates were moved to tissue culture growth room in long-day
conditions. After 12 days, the transformants were easily
distinguished as seedlings with green cotyledons, true leaves
and roots whereas non-transformed seedlings even if
germinated their cotyledons were turned brown within few
days (figure 1). The true transgenics plants obtained during
primary screening were designed as T0 and transplanted
again in soil to get T1 followed by seed collection and
sowing to get T2, T2* respectively as explained in Table 1
to get homozygous lines.
e) Histochemical Assay
The seedlings of Arabidopsis were collected and
histochemical assay was performed (Jefferson et al.
1987)[8]. The samples were submerged in staining solution,
vacuumed to facilitate infiltration of staining solution and
incubated overnight at 37 C. Stained samples were treated
with 70% ethanol to bleach chlorophyll. GUS expression
levels were visually monitored (figure 2).

Sci.Int.(Lahore),25(2),286-290,2013

Figure 1: In vitro selection of transgenics on kanamycin

containing MS media.

Figure 2: Histochemical analysis of seedlings of transgenic

Arabidopsis carrying GUS gene.

3. RESULTS AND DISCUSSION

Here we report a modified version of floral dip method
based on Zhang et al. (2006).[3] Kanamycin resistance and
-glucuronidase (GUS) genes were used as reporter genes to
stream line a protocol for the generation of transformed
homozygous lines of Arabidopsis to study heterologous gene
expression in this plant. It took about 10.5 months to get
transgenic homozygous lines (Table 1). Agrobacterium
mediated floral dip method eliminates initial callus induction
and plant regeneration ultimately reducing time, labor and
use of expensive equipment (Zhang et al. 2006).[3]
Although we found that the transformation efficiency of
floral dip method is not high, an Arabidopsis plant produces
thousands seeds by self-pollination in a single
transformation experiment resulting in many independent
transgenic lines within short time.
Zhang et al. (2006)[3] used 5% sucrose solution
supplemented with 0.02% (vol./vol.) of a surfactant, Silwet
L-77 in contrast we get the identical results by using 3%
sucrose solution supplemented with the same concentration
of tween-20. In agreement to Zhang et al. (2006)[3] the
higher concentrations of surfactant found toxic to plants.
However, in contrast to them when we used 5% and/or
higher concentration of sugar solution, it promoted fungal
growth at the plant base during first 24 hours after dip in
high humid conditions. We found 3% sucrose solution
optimal to get the similar results. We replaced the usual
bleach and water based sterilization of seeds by gaseous
sterilization as describe in methods section in detail. This
method of sterilization is less tedious and simple.
We applied the above described parameters and performed
transformation experiment in three replicates. GUS intron
gene was used as a reporter gene. In each experiment 10
plants were transformed and on average 14 transgenic lines
were recovered at the end of primary screening. Five out of
those 14 lines were selected randomly to generate T1,
followed by T2, and T2* respectively. In our view the
success of this procedure mainly depends on the health and

Sci.Int.(Lahore),25(2),287-290,2013

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Table 1: The generation of Arabidopsis stable homozygous lines

Time
required
4-months

3-months

Practical steps
carried out
Floral parts of
plants were dipped
in agro-suspension
followed by seed
collection and in
vitro selection of
transformants.
Transformants
were rescued and
transferred to
climate chamber
from in vitro
culture. Seeds were
collected after
selfing, separately
from each
transformant and
designated as
single line.

Generation
No.

Resultant
genotype

T0

All Gg
Figure 3: PCR-based confirmation of Agrobacterium using nptII specific primers. Single colonies of Agrobacterium were
selected and checked by Npt-II primers in standard PCR
conditions (product size 600bp at 600C).

Arabidopsis plants can be utilized for large-scale

transformation projects such as enhancer-trapped library
construction or generation of T-DNA tagged mutant lines
T1

Cross: Gg
x Gg
Result: 1:3
(GG: Gg:
Gg: gg)

From each line 30

plants were selfed
and seed were
collected.

T2

GG x GG =
all GG
Gg x Gg =
1:3
gg x gg =
all rr

Seeds of all 30
plants of each
transformant line
were screened in
vitro and
homozygous lines
were selected.

T2*

All GG

3-months

15-days

1 kb

physiological state of the plant. There are many reports that

favor our observation (Gelvin 2003[13; Wroblewski et al.
2005[14]; Zhang et al. 2006)[3]. Healthy plants with
immature flower clusters are ideal for floral dip
transformations. In addition to this it is important to confirm
the presence of the gene that has to be transferred in the
plant. This could be done by PCR with specific primers. In
our case we used npt II primers to confirm the presence of
our construct (figure 3).
It is conclude that the floral dipping procedure is quite
simple to perform and can produce many transgenic lines
just after one experiments as in our case we found 14
transgenic lines in one experiment. Arabidopsis plants are
easy to grow and maintain in a climate chamber. Using floral
dip method it is quite simple to introduce specific gene
constructs such as resistance genes and generate
homogyzous lines ready for functional analysis. Further,.

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Sci.Int.(Lahore),25(2),286-290,2013