2010 yasmin rosepetals
TRANSCRIPT
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O R I G I N A L P A P E R
Transient gene expression in rose petals via Agrobacteriuminfiltration
Aneela Yasmin Thomas Debener
Received: 15 December 2009 / Accepted: 1 March 2010 / Published online: 18 March 2010
Springer Science+Business Media B.V. 2010
Abstract The study of gene function in roses is hampered
by the low efficiency of transformation systems and thelong time span needed for the generation of transgenic
plants. For some functional analyses, the transient expres-
sion of genes would be an efficient alternative. Based on
current protocols for the transient expression of genes via
the infiltration of Agrobacterium into plant tissues, we
developed a transient expression system for rose petals. We
used b-glucuronidase (GUS) as a marker gene to optimize
several parameters with effects on GUS expression. The
efficiency of expression was found to be dependent on the
rose genotype, flower age, position of petals within a
flower, Agrobacterium strain and temperature of co-culti-
vation. The highest GUS expression was recorded in petals
of the middle whirls of half-bloomed flowers from cultivars
of Pariser Charme and Marvel.
Keywords Rosa Rose petals
Agrobacterium mediated transient expression
Abbreviations
T-DNA Transfer deoxyribonucleic acid
RNAi RNA interference
GUS b-glucuronidase
OD Optical density
Introduction
Stable transformation is an important tool for the functional
analyses of genes by genetic complementation through
overexpression or by gene silencing. However, the gener-
ation of stable transformants readily available for func-
tional analyses is a lengthy process. The production of
stable transgenic plants requires at least 34 months for
Arabidopsis (Zhang et al. 2006), 23 months for Nicotiana
species (Clemente 2006) and 912 months for Rosa (Dohm
et al. 2001; Marchant et al. 1998). Alternatives include
transient assays by either particle bombardment or Agro-
bacterium-mediated transformation. For particular target
traits, transient assays are less time-consuming, less labo-
rious and therefore more cost-effective (Wroblewski et al.
2005). In transient assays, it is possible to measure gene
expression within a very short time, independent of the
regeneration of a transformed cell (Kapila et al. 1997). For
stable Agrobacterium-mediated transformation, the T-DNA
has to be integrated into the host genome, whereas in
transient assays, non-integrated copies of T-DNA present
in the nucleus of the host can also be expressed (Kapila
et al. 1997). Therefore, genes could be expressed up to
1000 fold higher than in stable transformants (Janssen and
Gardner 1989). Transient assays have been successfully
utilized for genetic complementation (Zottini et al. 2008;
Van der Hoorn et al. 2000), RNAi experiments (Schob
et al. 1997), the assessment of resistance genes (Santos-
Rosa et al. 2008; Schweizer et al. 1999), protein trafficking
(Batoko et al. 2000) and recombinant protein production
(Sheludko et al. 2007).
Roses are among the most economically important
ornamental crops, and therefore several protocols for
regeneration and transformation have been published
(Dohm et al. 2001; Marchant et al. 1998). However,
A. Yasmin T. Debener (&)
Institute of Plant Genetics, Department of Molecular Breeding,
Leibniz University of Hannover, Herrenhauser Str. 2, 30419
Hannover, Germany
e-mail: [email protected]
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DOI 10.1007/s11240-010-9728-2
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protocols for stable transformation suffer from low trans-
formation efficiencies and a lengthy regeneration process.
In this study, we report the establishment and optimi-
zation of a transient gene expression system based on the
infiltration of Agrobacterium into rose petals. The assay
was optimized by evaluating the effects of rose cultivars,
bacterial strains and different physical and chemical factors
on the expression ofb-glucuronidase.
Materials and methods
Plant material
Three tetraploid rose cultivars, Pariser Charme, Heck-
enzauber and Marvel, as well as the tetraploid experi-
mental hybrid 91/100-5 and the diploid hybrid 88/124-46
were used in the present study. An inbred line of Nicotiana
benthamiana was obtained from E. Maiss at the Institute
for Plant Protection, Leibniz University, Hannover, Ger-many. All rose genotypes are part of the genotype collec-
tion of the Institute for Plant Genetics, Leibniz University
of Hannover, and the plant material was maintained in
greenhouses under semi-controlled conditions.
Agrobacterium strains and genetic constructs
The Agrobacterium strains used in this study were
GV3101::pMP90, C58C1, EHA105 (Hellens et al. 2000)
harboring the construct 35S:GUS-intron in pBINPLUS
(Van Engelen et al. 1995) and WT 80.1 (Universitat
Hannover) harboring the construct 35S:GUS-intron in
pBIN19 (Bevans 1984).
Agrobacteria were grown in YEP liquid and on an agar
(15 g/l) medium supplemented with Kanamycin (50 mg/l)
and Rifampicin (10 mg/l) according to Wroblewski et al.
(2005). In addition to these antibiotics, Gentamycin (25 mg/
l) was added to GV3101 cultures for the selection of pMP90.
For some variations of the induction of virulence genes
in Agrobacterium, bacterial suspensions were supple-
mented with 0, 100 and 200 lM of acetosyringone and a
non-ionic surfactant Breakthru (Evonic Industries, Joh
et al. 2005) at final concentrations of 0, 10, 100 and
1,000 ppm (v/v).
Petal infiltrations and incubation conditions
Flowers of all rose cultivars were harvested and placed in
translucent plastic containers on wet paper towels. One day
before infiltration, an overnight liquid culture of Agro-
bacterium was started according to Wroblewski et al.
(2005) from single colonies of bacteria freshly grown on
agar plates. The following day, bacteria were collected by
centrifugation at 22C and 4,500 rpm for 15 min. The
pellet was washed once with sterile distilled water and
resuspended in sterile distilled water at OD600: 0.40.5
(Wroblewski et al. 2005). The bacterial suspension was
infiltrated from a hole punctured at the base of the petal
using a 1-ml needleless syringe (Schob et al. 1997;
Wroblewski et al. 2005). Alternatively, bacterial suspen-
sions were infiltrated via vacuum infiltration. For this,petals were submerged in the bacterial suspensions in
Falcon plastic tubes and placed in a desiccator. Infiltration
was then performed at 200 mbar for 5 min. The infiltrated
petals were kept on a wet tissue paper, in a rectangular
transparent box, with a cover, in a temperature-controlled
incubator and in the dark until they were assayed for GUS
expression.
Histochemical assay
The histochemical assay was performed according to Jef-
ferson et al. (1987). On average, 30 petals per treatment inseven replicated experiments were evaluated. Vacuum was
used to facilitate the infiltration of the staining solution into
the petals and tobacco leaves. Samples were incubated in
staining solution overnight at 37C, and chlorophyll was
removed by fixation in 70% ethanol.
Data analysis
b-glucuronidase (GUS) expression levels were visually
rated on a scale from 0 to 3, indicating from no expression
(score 0) to very high expression (score 3; Fig. 1). N.
benthamiana was used as a positive control in all experi-
ments. The effect of different parameters on GUS expres-
sion was evaluated using the Chi Square test of
independence and the KruskalWallis and Wilcoxon exact
tests as implemented in the R-software (R Development
Core Team 2009).
Results
The first infiltration experiments with the Agrobacterium
strain GV3101 harboring pBINPLUS::GUS-Intron in petals
of the variety Pariser Charme resulted in various levels of
GUS expression (Fig. 1). Although the visual score for
GUS expression exceeded the Nicotiana control in some
replications, significant variability was observed between
individual petals. Two different infiltration methods were
tested for their feasibility and effectiveness. Infiltration
with l-ml syringes without needles led to quick and com-
plete infiltrations of the whole petals. Although vacuum
infiltration led to a complete and even infiltration of the
petals, this treatment soaked the delicate petals and led to
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premature senescence. These observations were consistent
among all rose genotypes tested here. Therefore, in all
subsequent experiments, the petals were infiltrated with a
syringe.
A number of variables were tested, including the host
genotype, Agrobacterium strain, flower age, petal position
within a flower, additives to the bacterial growth media,
bacterial density, temperature during co-cultivation and co-cultivation time.
Influence of the host genotype
Five different rose genotypes were evaluated for their
compatibility to agro-infection by monitoring GUS
expression. The host genotype had a highly significant
effect on the level of GUS expression (KruskalWallis Test
P = 2.2 e-16). The two rose varieties of Pariser Charme
(Fig. 1) and Marvel displayed very high GUS expression
levels, whereas genotypes 91/100-5, 88/124-46 and
Heckenzauber seemed to be resistant to agro-infection,
showing little or no GUS expression. Pariser Charme
displayed the highest intensity of GUS expression, with the
levels in some petals showing even stronger expression
than the leaves of N. benthamiana (Fig. 2).
Influence of the Agrobacterium strain
Four Agrobacterium strains (GV3101, EHAI05, C58C1
and 80.1), each harboring GUS-Int, were evaluated for
their ability to infect different rose genotypes (Fig. 3).
Strain 80.1 is a wild-type Agrobacterium that has been
isolated from roses and pre-characterized as leading to
significant levels of GUS expression in previous experi-
ments (data not shown). Almost no effect of the type of
bacterial strain could be observed on the GUS expression.
The only significant differences occurred after the inocu-
lation of the genotypes of Marvel (KruskalWallisP = 3.7 e-7) and 88/124-46 (KruskalWallis P = 0.0023).
Both Marvel and 88/124-46 strain A80.1 produced sig-
nificantly weaker GUS signals as compared to all of the
other strains (P values between 0.00023 and 8.16e-7 for
Marvel and between 0.0002 and 0.028 for 88/124-46). In
all of the other combinations, no significant differences
could be detected. Because strain GV3101 gave the highest
average expression level and as it had been used in several
published studies for agroinfiltration, it was selected for
further studies. As a host genotype, Pariser Charme was
selected to optimize different physical and biological fac-
tors that could influence the transient expression of a for-
eign gene in this system.
Effects of flower age and petal position
The GUS expression levels of petals from buds of Pariser
Charme before opening (stage 1), after the flowers had just
opened (stage 2) and with fully opened flowers (stage 3), as
well as petals from the outer whirl from the middle of the
flower and the inner whirl of petals, were compared. The
Fig. 1 Pattern of scoring for the histochemical GUS assay in rose petals. Scores are indicated below the pictures of three different staining
intensities
Fig. 2 Pariser Charme petals
infiltrated by the Agrobacterium
suspension at OD600 = 0.5
harboring a GUS-Intron
construct: a before GUS
staining; b after the
histochemical GUS assay
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highest level of expression was found in stage 2 flowers
(mean value of expression = 2.13) as compared to stage 1
(mean value of expression = 0.69, P = 2.0e-5) and stage
3 (mean value of expression = 1.47, P = 0.016) flowers.
Within the stage 2 flowers, petals from the middle of the
flowers displayed the highest GUS expression as compared
to the outer and inner whirl petals (P values between
0.0003 and 0.0029). However, the variation between petals
of the same flower stage and the same whirl was very high,
with standard deviations between 0.64 and 0.95.
Effect of acetosyringone and additives
The effect of acetosyringone on GUS expression was tested
for the strain GV3101 on all host genotypes and was found
to be non-significant (KruskalWallis P = 0.326). In
addition, the surfactant Breakthrough was used to promote
an even distribution of bacterial suspensions in petals. No
significant differences in GUS expression were noted. At
higher concentrations (100 and 1,000 ppm), it promoted
early senescence in petals, and the highest concentration
was even lethal to petals and led to necrosis within 24 h.
Effect of bacterial density
To determine the optimal concentration of bacteria for
GUS expression, the bacterial suspensions were adjusted to
OD600 levels of 0.1, 0.3, 0.5 0.8, 1.0, 1.5, 2.0, 3.0 and 4.
GUS expression was observed only for bacterial densities
between OD600 0.5 and 4.0 (Fig. 4). Among these densi-
ties, no significant differences could be detected. The
optimal OD was found to be 0.5 in Pariser Charme and
Marvel. In contrast to this, even the highest densities did
not lead to GUS signals in the remaining rose genotypes
(data not shown).
Effect of incubation temperature
b-glucuronidase expression in infiltrated rose petals was
recorded at four different temperatures, 19, 22, 25 and
28C. The effect of the temperature during the co-culti-vation was found to be significant (KruskalWallis
P = 2.2e-16). Temperatures of 19 and 25C revealed sig-
nificantly lower GUS expression levels as compared to
22C (Fig. 4). At 28C, GUS expression levels were very
low and were almost non-detectable.
Fig. 3 Effect of host genotype and Agrobacterium strain on the
expression of GUS in rose petals. Indicated on the vertical axis are the
mean values for the GUS scores, from 0 to 2.5, as shown in Fig. 1
Fig. 4 Mean values of GUS scores for the effect of bacterial density
(a), the effect of cultivation temperature (b) and the effect of time of
co-cultivation (c). The y-axis indicates the mean values of the GUS
scores; the x-axes indicate the different treatments within each factor.
Different letters above each column indicate significant differences of
the mean values at P\ 0.05
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Effect of co-cultivation time
The time of co-cultivation had a significant effect on the
level of GUS expression (Fig. 4, KruskalWallis P = 7.3
e-14). GUS expression was detectable from the second day
after infiltration. However, significant levels of GUS
expression occurred only after day three. The highest
intensity of GUS expression was detected between daysthree and seven, after which expression decreased signifi-
cantly (Fig. 4).
Discussion
The transient expression of genes is an indispensable
analytical tool for studying gene function in plants. The
infiltration of Agrobacterium suspensions into plant organs
(agroinfiltration) is a fast and highly efficient method that
does not require expensive equipment. Therefore, several
protocols for various plant species have been publishedover the last 13 years in which factors that influence the
infection process, and therefore the expression efficiency,
were investigated (Kapila et al. 1997; Wroblewski et al.
2005; Santos-Rosa et al. 2008; Zottini et al. 2008). Among
the most important factors identified thus far are the
genotype of the host plant, the Agrobacterium strain, the
pre-culture of the host plant and of the Agrobacteria and
the temperatures at which the co-cultivation of Agrobac-
terium and the host are conducted (Wroblewski et al. 2005;
Joh et al. 2005; Zottini et al. 2008).
Here, we report the optimization of a transient gene
expression assay in rose petals based on the infiltration of
Agrobacterium suspensions.
The agroinfiltration was optimized, and the data reveal
that agroinfiltration is dependent on the host genotypes,
flower age, petal position, bacterial density and tempera-
ture. Several factors influencing transient gene expression
after Agrobacterium infiltration have been reported before
(Dillen et al. 1997; Kapila et al. 1997; Kim et al. 2009;
Santos-Rosa et al. 2008; Wroblewski et al. 2005). It has
been reported that the genetic background of the host sig-
nificantly influences the efficiency of transient expression
in lettuce, Arabidopsis and grapevine (Wroblewski et al.
2005; Santos-Rosa et al. 2008; Zottini et al. 2008). Here,
we also observed that two rose genotypes (Pariser Char-
me and Marvel) had a significantly higher susceptibility
to Agrobacterium infections as compared to three other
genotypes (Heckenzauber, 91/100-5 and 88/124-46). This
result was confirmed in a preliminary study among 30 cut
roses, among which only one genotype showed significant
GUS expression levels (data not shown). To date, we can
make no assumptions on the number and the nature of the
genetic factors influencing the efficiency of agroinfections.
The analysis of segregating progeny from defined crosses
between susceptible and resistant genotypes would be a
strategy to address this question.
During our studies, considerable variation was observed
in the expression of GUS in flowers of different ages and
within a flower from petal to petal. Similar levels of vari-
ations in expression are reported within single plants, in
plants of different ages, or even in tissues of differentdevelopmental stages of single plants of Arabidopsis,
Nicotiana, pepper, cotton, tomato and lettuce (Wroblewski
et al. 2005; Joh et al. 2005). The middle petals of stage 2
rose flowers were found to be optimal for the transient
expression studies carried out here. As both Marvel and
Pariser Charm are multi-petalled genotypes with an aver-
age number of more than 50 petals per flower, each flower
will yield more than 10 highly susceptible petals for tran-
sient expression experiments.
In some of the published reports, the use of different
infiltration media (McIntosh et al. 2004) and the addition of
acetosyringone (Kapila et al. 1997) and surfactants (Johet al. 2005) significantly improved the expression of for-
eign genes. Acetosyringone is known for its ability to
induce the virulence genes of Agrobacterium necessary to
transfer T-DNA (McCullen and Binns 2006). In the present
study, neither the addition of acetosyringone nor the
addition of surfactants improved the transient expression in
rose petals. This is in agreement with Wroblewski et al.
(2005), who investigated these factors in Arabidopsis and
lettuce. Temperature is also considered to be a determinant
for Agrobacterium-mediated gene transfer in plants (Dillen
et al. 1997). Rose petals were incubated at 19, 22, 25 and
28C for 4 days after infiltration. The highest GUS
expression was observed at 22C. This result suggests that
the regulation of T-DNA transfer through vir genes is
temperature dependent, as previously demonstrated by
Dillen et al. (1997).
In contrast to temperature, the density ofAgrobacterium
suspensions had no significant effect over a broad range of
OD values from 0.5 to 4.0. Only densities less than 0.5 did
not lead to visible GUS expression. This is in contrast to
the results from several other studies conducted by Santos-
Rosa et al. (2008) and Kim et al. (2009), who found at least
weak expression down to densities of OD 0.1. One expla-
nation for this could be due to physiological differences
between petals and leaves, although this remains highly
speculative unless comparative experiments have been
conducted on leaves. It is interesting to report that the
tested bacterial densities did not reveal any kind of necrosis
or withering in the rose petals, as were observed in tobacco
and tomato by Wroblewski et al. (2005). Another differ-
ence to published reports lies in the time from which GUS
expression is visible. Whereas previous studies reported at
least weak GUS expression from 1 day after the infiltration
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of agrobacteria (Kim et al. 2009), we detected GUS
expression at significant levels only from day three on and
weak signals at only day two after infiltration. The reasons
for this difference are as elusive as those for the lack of
expression at low densities.
Because we only screened a small number of the
available genetic variants of both the host plant and the
Agrobacterium, there is also a great potential to furtheroptimize the system by including additional rose and
Agrobacterium genotypes.
The data presented here demonstrate the utility of rose
petals as a suitable system for carrying out transient
expression studies. Transient gene expression in rose petals
now allows the characterization of both petal-specific
genes and constitutively expressed rose genes in a short
time. We are in the process of evaluating this system for its
suitability to functionally characterize rose resistance
genes.
Acknowledgments The first author is thankful to Deutscher Aka-demischer Austausch Dienst (DAAD), Higher Education Commission
of Pakistan (HEC) and Sindh Agriculture University, Tandojam for
the award of scholarship and study leave.
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