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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]

123

Plant Cell Tiss Organ Cult (2010) 102:245250

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

246 Plant Cell Tiss Organ Cult (2010) 102:245250

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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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2010 Yasmin Rosepetals