Plant Cell Rep (2004) 23:32–38DOI 10.1007/s00299-004-0776-0

G E N E T I C T R A N S F O R M A T I O N A N D H Y B R I D I Z A T I O N

M. Cordero de Mesa · N. Santiago-Dom�nech ·F. Pliego-Alfaro · M. A. Quesada · J. A. Mercado

TheCaMV 35S promoter is highly active on floral organsand pollen of transgenic strawberry plants

Received: 28 October 2003 / Revised: 31 January 2004 / Accepted: 2 February 2004 / Published online: 27 March 2004� Springer-Verlag 2004

Abstract We have evaluated the expression of thereporter b-glucuronidase (GUS) gene driven by thecauliflower mosaic virus 35S (CaMV 35S) promoter inflowers and pollen from 14 independent transgenicstrawberry lines. Of the 14 lines evaluated, 13 (92.8%)showed GUS activity—as estimated by the histochemicalGUS assay—in some floral organs, with expression beingmost common in the flower stem, sepals, petals, ovaryand stigma. Ten of these thirteen transgenic lines (77%)showed GUS activity in pollen, although the percentagesof positive pollen per flower varied greatly among thedifferent lines. A study of the GUS expression duringpollen maturation showed that the (CaMV 35S) promotershowed low expression in pollen from flower buds beforeanthesis but was activated in mature pollen followinganther dehiscence. The percentages of pollen grains thatshowed GUS activity ranged from 2.1% to 46.3%. Thesepercentages were similar or even higher when maturepollen was stored dry at room temperature for 2 weeks.After 5 weeks of storage, the percentages of GUS-positivepollen decreased in two of the six lines analysed but re-mained at similar values in the other four lines. GUSactivity was also measured in protein extracts of maturepollen by means of the fluorometric GUS assay, with thevalues obtained ranging from 3.8 mmol MU mg pro-tein�1 h�1 to 0.26 mmol MU mg protein�1 h�1. Contrary tothe generally held view that the CaMV 35S promoter isvirtually silent in pollen, we conclude that it is highlyexpressed in transgenic strawberry pollen.

Keywords CaMV 35S promoter · Fragaria � ananassa ·GUS activity · Pollen · Transgenic plant release

Abbreviations CaMV 35S: Cauliflower mosaic viruspromoter · GUS: b-Glucuronidase (EC 3.2.1.31) ·MU: 4-Methyl umbelliferone · nos: Nopaline synthasepromoter · nptII: Neomycin phosphotransferase ·X-Gluc: 5-Bromo-4-chloro-3-indolyl-b-d-glucuronic acid

Introduction

The environmental spread of transgenes and their prod-ucts through pollen dispersal is one of the main stumblingblocks to the commercial release of transgenic plants. Inaddition to gene flow through cross-pollination with wildrelative species, pollen can be a vehicle to the spread oftransgenic proteins that are either expressed or depositedon the grain. The dispersal of transgene products viapollen poses potential risks, such as ecological effects onnon-target beneficial insects in the case of insecticidalproteins (Poppy 2000), the inhalation of airborne aller-gens or the introduction of allergenic proteins into thefood chain by bee contamination of honey (Eady et al.1995; Rogers and Parkes 1995). The likelihood of trans-genic protein dispersal via pollen will initially depend onthe activity of the promoter driving the transgene in themale gametophyte. In this respect, information concern-ing the activity of promoters commonly used to drivegene expression in transgenic plants, like the cauliflowermosaic virus promoter (CaMV 35S) or nopaline synthasepromoter (nos), is scarce and limited to a few species.

TheCaMV 35S promoter is widely used in transfor-mation experiments due to its high activity in most tissuesof a broad range of species. Based on its wide expression,CaMV 35S is generally considered to be a constitutivepromoter, although physiological and/or environmentalfactors, such as developmental stage (Pret’ov� et al.2001), photoperiod and temperature (Schnurr and Guerra2000), can modulate its expression. Despite its wide-spread use in biotechnological plant improvement pro-grams, our knowledge of CaMV 35S activity on repro-ductive tissues and, particularly, in pollen grains is scantand confusing. To date transient transformation experi-

Communicated by L. Pe�a

M. C. de Mesa · N. Santiago-Dom�nech · F. Pliego-Alfaro ·M. A. Quesada · J. A. Mercado ())Departamento de Biolog�a Vegetal,Universidad de M�laga,29071 M�laga, Spaine-mail: mercado@uma.esTel.: +34-952-137522Fax: +34-952-13-1944

ments of pollen with the GUS reporter gene under thecontrol of CaMV 35S have yielded no conclusive results.Nishihara et al. (1993) detected GUS activity in pollen offive species transformed by particle bombardment. Sim-ilarly, Twell et al. (1989) found low but consistent levelsof GUS activity in bombarded pollen of tobacco andtomato. In contrast, GUS expression was not observed ineither bombarded pollen of Nicotiana glutinosa and Lil-ium longiflorum (Van der Leede-Plegt et al. 1992) or inelectroporated maize microspores (Fennell and Haupt-mann 1992). Reports on CaMV 35S expression in pollenfrom stably transformed plants are also inconclusive; forexample, no promoter expression has been detected inmature pollen of tobacco, tomato (Van der Leede-Plegt etal. 1992), petunia (Mascarenhas and Hamilton 1992) andArabidopsis (Wilkinson et al. 1997). However, Twell etal. (1989) and Wilkinson et al. (1997) reported low levelsof GUS activity driven by CaMV 35S in tobacco pollen.In any case, the expression of this promoter is several foldlower than the expression of such pollen-specific pro-moters as lat52 from tomato (Twell et al. 1989).

Similar to these Solanaceae crops, strawberry plantsare also included in several biotechnological breedingprogrammes (Faedi et al. 2002). During the 1990s, reli-able protocols for the genetic transformation of straw-berry were established for several commercial cultivars(Barcel� et al. 1998; James et al. 1990; Mathews et al.1995). Recently, these have been successfully employedto modify selected traits like insect resistance (Watt et al.1999), herbicide tolerance (Morgan et al. 2002) and fruitsoftening (Jim�nez-Bermffldez et al. 2002). While most ofthese transformations used the CaMV 35S promoter tocontrol transgene expression, there is no informationavailable on the expression of this promoter in strawberryreproductive tissues and pollen. The aim of the investi-gation reported here was to evaluate the activity of theCaMV 35S promoter in the different organs of thestrawberry flower and in the pollen grains.

Materials and methods

Plant material

Transgenic Fragaria � ananassa Duch. plants, cv. Chandler, hadbeen obtained previously by the Agrobacterium-biolistic combinedmethod developed for this species (Cordero de Mesa et al. 2000).These plants were transformed with the pGUSINT plasmid (Van-canneyt et al. 1990) containing the nptII gene for kanamycin re-sistance and the uidA reporter gene (coding for GUS) under thecontrol of a single (CaMV 35S) promoter. Primary transgenics weregrown in a greenhouse and propagated vegetatively via runners(cultivated strawberry is not reproduced by sexual crosses). Plantsused in this work correspond to the second and third vegetativegenerations and were evaluated in consecutive years. Fourteen in-dependent transgenic lines, eight copies per line, as well as non-transformed plants were used. The plants were grown in a green-house under natural temperature and photoperiod, and flower andpollen samples were obtained at the middle of the fruiting season(April-May).

Histochemical detection of GUS activity in flowers and pollen

Expression of the GUS gene in whole strawberry flowers and iso-lated pollen samples was detected histochemically following theprocedure of Jefferson et al. (1987). Samples were vacuum infil-trated for 5 min in the staining solution (1 mM X-gluc in 50 mMphosphate buffer pH 7) and incubated for 24 h at 37C. The flowerswere then washed in 95% ethanol to extract the chlorophylls.Flowers were collected at anthesis, and pollen samples were ob-tained by crushing isolated anthers in a small volume of GUSstaining solution. Six flowers per line from different plants wereanalysed in two consecutive years.

To analyse the expression of the CaMV 35S promoter duringpollen maturation and storage, we collected pollen at differentdevelopmental stages: flower buds before anthesis, flowers at an-thesis without visible dehiscent pollen and flowers with dehiscentpollen. In the first two cases, pollen was released by crushingisolated anthers in GUS staining solution. Dehiscent pollen fromeach individual flower was collected by gently shaking the flowersand subsequently fractionated into three samples. One of thesesamples was immediately assayed for GUS activity, and the otherswere stored in crystal vials at 25C. After 2 weeks and 5 weeks ofstorage, the pollen was stained for GUS activity. Four to six in-dependent samples for each line, obtained from different plants,were analysed. A minimum of 100 pollen grains per sample wasrecorded, and the percentage of pollen grains showing GUS activitywas estimated from all of the grains recorded, both viable and non-viable grains.

Fluorometric assay for GUS activity

GUS enzyme activity was measured in the pollen grains by thefluorometric assay, as described by Jefferson et al. (1987). Samplesof dehiscent pollen from six to ten flowers, collected by shaking theflowers on a petri dish, were homogenized in 2 ml of extractionbuffer [50 mM phosphate buffer, pH 7, 10 mM EDTA, 0.1% (v/v)Triton-X 100, 10 mM b-mercaptoethanol]. Aliquots of the extracts(100 ml) were added to 1 ml of assay buffer (extraction buffersupplemented with 1 mM MU), prewarmed and incubated at 37C.After 0, 5 and 20 min of incubation, 100-ml samples were removedand placed in 1.9 ml stop buffer (200 mM sodium carbonate).Fluorescence was measured using a DyNA Quant 200 fluorometer(Hoefer Pharmacia Biotech). Protein concentrations in the extractswere determined by the Bradford assay (Bradford 1976), and GUSactivity was expressed as micromole MU per hour per milligramprotein. Three independent extractions were performed for eachline.

Southern analysis

Genomic DNA was extracted from young strawberry leaves usingthe DNeasy plant mini kit (Qiagen, Valencia, Calif.). Previous tothis, the plant material had been washed several times with washingbuffer to eliminate DNA contaminants (Mercado et al. 1999). Tenmicrograms of DNA was digested overnight with EcoRI in thepresence of 1 mM spermidine, electrophoresed in a 0.8% agarosegel and then transferred to Hybond N+ membranes. A 900-bp PCRfragment containing the nos-nptII gene was used as probe. Thisprobe was labelled with digoxigenin using the DIG High PrimeDNA labelling and detection starter kit (Roche, Indianapolis, Ind.).The filter was prehybridised at 42C in DIG Easy Hyb buffer andhybridised overnight in the same buffer containing the probe. Thefilter was then washed and the probe detected following the man-ufacturer’s instructions.

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Results

GUS activity driven by the CaMV 35S promoterin strawberry flowers

Fourteen transgenic lines transformed with the pGUSINTplasmid were used in this investigation. The transgenicnature of these plants was previously assessed by PCRanalysis and confirmed in several lines based on kana-mycin segregation of seeds (Cordero de Mesa et al. 2000).Despite the presence of large amounts of polyphenols andpolymeric carbohydrates in strawberry tissue, which madeit difficult to extract pure DNA suitable for molecularanalysis, we successfully obtained purified DNA bycombining a previous protocol (Mercado et al. 1999) witha commercial DNA extraction kit. The presence of T-DNA was confirmed in these samples by Southern hy-bridisation with a nos-nptII probe (Fig. 1).

GUS activity was absent in all of the tissues from wild-type flowers; that is, under the staining conditions em-ployed no blue coloration was observed. However, 13 ofthe 14 independent transgenic lines containing the pGU-SINT plasmid (92.8%) showed GUS activity in theirfloral organs. An example of GUS staining in strawberryflowers is shown in Fig. 2. The transgenic plants wereanalysed during two consecutive years, and very minor

differences in GUS activity were observed between thetwo cycles of vegetative propagation studied. No statis-tically significant differences were found using an anal-ysis of frequencies at P=0.05. Not all of the transgeniclines displayed the same pattern of GUS expression(Fig. 3). GUS activity was generally found in the flowerstem, sepals, petals, ovary, stigma and pollen; this patternwas exhibited by 8 of the 13 lines (61.5%) that showedGUS staining in some floral organs. The presence of GUSactivity in the anther and filament was detected in 50% ofthe transgenic lines. In contrast, GUS staining in the style

Fig. 1 Southern blot analysis of DNA extracted from selectedtransgenic lines (lanes 8, 12, 11, 15). The membrane was hybrid-ised with a digoxigenin-labelled probe containing the chimeric nos-nptII gene. P pGUSINT plasmid digested with EcoRI, C DNA fromcontrol, non-transformed plants

Fig. 2A–F Histochemicalstaining for GUS activity drivenby the CaMV 35S promoter instrawberry flowers and pollen.A, C Control non-transformedflower showing the absence ofstaining. B Transgenic flowerfrom line GUS7 showing GUSactivity in most of its tissues. DTransgenic flower from lineGUS1 showing GUS activity infloral pistils. E Mature pollenfrom control non-transformedplants showing the absence ofGUS activity. F Mature trans-genic pollen from line GUS12stained for GUS activity. Inboth E and F, pollen was ob-tained by gently shaking dehis-cent anthers. Bars (E, F):100 mm

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and receptacle was only observed in two (15.4%) and one(7.7%) line, respectively. It is noteworthy that 10 of the13 transgenic lines (77%) showed GUS activity in thepollen grains.

CaMV 35S promoter expression in strawberry pollen

Initially our study was focused at the time of flower an-thesis. Pollen grains from both the control plants and thedifferent transgenic lines were collected by crushing theanthers of recently opened flowers and then stained withX-gluc to detect GUS activity. These samples contained amixture of pollen from dehiscent and non-dehiscent an-thers. No blue staining was observed in any of the pollensamples obtained from wild-type strawberry plants. In thetransgenic plants, the percentages of stained pollen grainsranged from 0.4% in the GUS1 line to 25.6% in the GUS8line (Fig. 4). In five of the ten pollen-positive lines (50%),stained pollen was observed in all of the flowers assayed.These lines (GUS2, -8, -10, -11 and -12) together withGUS15 that also showed a high percentage of GUS-stained pollen were selected for further analysis.

As a second step, we evaluated the activity of theCaMV 35S promoter during transgenic strawberry pollenmaturation and after 2 weeks and 5 weeks of storage(Table 1). In the six selected lines analysed, the per-centages of stained grains were low when pollen wasobtained from the anthers of closed flower buds or re-cently opened flowers with non-dehiscent anthers (with-out visible pollen on the anther surface). In contrast, thepercentages of positive grains increased significantlyduring the later stages of pollen development. In maturepollen grains, which we obtained by gently shakingflowers with dehiscent anthers, the percentages of stainedgrains ranged from 2.1% to 46.3%, corresponding tolines 15 and 12, respectively. After 2 weeks of storageat room temperature, pollen grains displayed similar(GUS10 and -11) or higher (GUS2, -8, -12 and -15)percentages of GUS staining than freshly assayed dehis-cent pollen. The highest percentage of stained pollen,75%, was observed in the GUS12 transgenic line. After5 weeks of storage, the pattern of expression was variable,depending on the transgenic line: the percentage of pol-len grains showing GUS activity slightly increased inGUS2, remained at similar values in GUS8, -10 and -11and decreased significantly in GUS12 and, specially, inGUS15, where no stained pollen was detected in any ofthe sample analysed. This trend was observed in twodifferent growing seasons with plants belonging to two

Fig. 3 Percentages of independent transgenic strawberry lines thatdisplayed GUS activity driven by the CaMV 35S promoter in thedifferent floral organs and in pollen grains. Percentages were cal-culated from the 13 lines that showed GUS activity in some floralorgans. Empty bars correspond to plants from the second round ofvegetative propagation, diagonally stripped bars correspond toplants belonging to the third cycle of propagation

Fig. 4 Percentages of pollen grains that showed GUS activitydriven by the CaMV 35S promoter in the different transgenicstrawberry lines. Pollen was obtained by crushing the anthers ofrecently opened flowers. A minimum of 100 pollen grains fromeach flower was analysed. Values are means € standard deviationof six flowers from different plants per line

Table 1 GUS staining in pol-lena from different transgenicstrawberry lines

GUS2 GUS8 GUS10 GUS11 GUS12 GUS15

Flower buds 1.6€0.9b 0.1€0.1 0.2€0.2 0.5€0.7 1.7€1.4 0.1€0.1Non-dehiscent anthers 1.4€0.8 0.4€0.1 0.1€0.2 0.1€0.1 0.4€0.5 0Dehiscent anthers 24.4€6.8 20.0€9.9 8.1€2.4 8.8€11.7 46.3€18.9 2.1€1.4Pollen stored 2 weeks 31.2€11.5 37.1€25.4 9.4€3.7 4.8€5.7 75.1€39.2 13.0€6.9Pollen stored 5 weeks 45.0€17.2 37.9€34.2 12.4€4.3 5.2€5.5 19.5€12.7 0a Pollen samples were collected from flower buds before anthesis, from flowers at anthesis just beforeanther dehiscence and from flowers with dehiscent anthers. GUS activity was also assayed in pollenafter 2 weeks and 5 weeks of storage.b Values are the mean € standard deviation of four or six independent flowers. A minimum of 100pollen grains per flower and stage was recorded.

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cycles of vegetative propagation. Segregation of GUSactivity in strawberry pollen did not follow simple Men-delian ratios, probably due to the complex nature of theoctoploid strawberry genome. Therefore, it was not pos-sible to establish a correlation between GUS expression inthe pollen grains and the number of T-DNA copies.

The presence of GUS activity in mature pollen wasfurther verified by measuring GUS activity quantitativelyby the fluorometric assay. No activity was detected in theprotein extracts from control pollen. In contrast, trans-genic lines showed significant levels of GUS activity,ranging from 3.8 mmol MU mg protein�1 h�1 in GUS8 to0.26 mmol MU mg protein�1 h�1 in GUS12 and GUS15(Fig. 5). These values fall within the range of those ob-tained in leaf petioles or fruit protein extracts from thesame transgenic lines.

Discussion

The purpose of the present investigation was to evaluatethe activity of the CaMV 35S promoter in reproductivetissue of strawberry plants and, especially, in pollengrains. With respect to the first-mentioned, GUS activitydriven by the CaMV 35S promoter in floral tissue wasdetected in 13 of the 14 independent transgenic linesanalysed. The sole line that did not show any blue col-oration in its flowers also showed a negative reactionwhen its vegetative tissue was analysed for GUS activity.This positive correspondence between expression in veg-etative and reproductive tissues occurred in all of theplants analysed in the present study. Consequently, weconsider the CaMV 35S promoter to be constitutivelyactive in strawberry plants. Contrary to this result, intransgenic Arabidopsis plants, Wilkinson et al. (1997)found that only 57% of the lines analysed displayed GUSactivity in flowers driven by CaMV 35S, although all linesexhibited GUS activity in the leaves. The most common

pattern of GUS expression observed in strawberry wasalso different from the one reported in Arabidopsis. Bothpatterns consist of GUS expression in the flower stem andsepals and low expression in the style. However, whilemore than 80% of the transgenic strawberry lines showedGUS activity in petals, stigma and pollen, in Arabidopsisflowers expression in these tissues was either restricted toa few lines or was absent, as in the case of pollen. Asconcluded by Wilkinson et al. (1997), it is clear that theactivity of the CaMV 35S promoter varies greatly betweendifferent plant species and that its expression cannot bepredicted with certainty. Therefore, more analysis of ex-pression in different species would be required to obtain aclearer picture.

With respect to the GUS activity driven by theCaM V35S promoter in pollen, we found that 77% of thetransgenic lines analysed displayed GUS activity in pollengrains, with expression being highly variable among thedifferent lines. These results are in contrast to previousresults that showed theCaMV 35S promoter to be virtuallyinactive in pollen and, consequently, to have a low po-tential to cause harmful effects through pollen expression(Rogers and Parkes 1995; Wilkinson et al. 1997). In bothtransient transformation experiments and in stably trans-formed plants, a lack of CaMV 35S promoter activity oronly low levels of activity have been reported in thepollen grains of several plant species, such as tobacco,tomato, petunia and Arabidopsis (Mascarenhas andHamilton 1992; Twell et al. 1989; Van der Leede-Plegt etal. 1992; Wilkinson et al. 1997). However, similar to ourobservations, the CaMV 35S promoter appears to be ac-tive in pollen from different pine species (Fernando et al.2000). Furthermore, although a single CaMV 35S pro-moter is ineffective in inducing GUS expression in to-bacco pollen, Conner et al. (1999) detected GUS activityin pollen from transgenic tobacco lines containing adouble CaMV 35S promoter. It seems clear that while theactivity of the CaMV 35S promoter in pollen grains maybe dependent on the species studied, some of the dis-crepancies among the results of the various investigationsmay also be a result of the developmental stage of thepollen used for analysis. Our results show that the activityof the CaMV 35S promoter in strawberry pollen dependsmainly on the maturation stage of the grains. Thus, itsexpression is low in immature grains, prior to anther de-hiscence, but it is activated in fully mature pollen grains,at least during the first 2 weeks after anther dehiscence.The level of the transgene product, namely GUS activity,clearly increased from the stage of anther dehiscence tothe period 2 weeks later in five of the six lines analysed.This increment in activity was probably due to an acti-vation of the CaMV 35S promoter and not to an accu-mulation of GUS protein, since the latter has been shownto have a half-life of 50 h in mesophyll protoplasts (Jef-ferson et al. 1987). Similarly, the unexpected high ex-pression of the promoter from the potato Lhca3.St.1 gene,which encodes a light-harvesting protein, in transgenictobacco pollen was only observed in fully mature pollenbut it remained active in senesced dried anthers (Conner

Fig. 5 GUS activity in protein extracts of mature pollen fromtransgenic plants measured by the fluorometric assay. Pollen wascollected by shaking flowers with dehiscent anthers onto a petridish. Values are the Mean € standard deviation of three indepen-dent extractions

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et al. 1999). It is noteworthy that the CaMV 35S promoterremained active in strawberry pollen even after 5 weeksof pollen storage under dry conditions. The activity of thetransgene promoters after a long time of pollen storage isnot a novel result. Eady et al. (1995) found high levels ofGUS activity driven by the pollen-specific promoter lat52in tobacco pollen stored up to 6 weeks in honey. Mas-carenhas (1990) separated pollen genes into two classes—one group comprises genes that are expressed soon aftermeiosis and are undetectable in mature pollen, and asecond group includes genes that are first detected aroundthe time of microspore mitosis and that continue to ac-cumulate as the pollen grains mature. The expressionpattern of CaMV 35S detected in strawberry pollen mayindicate that this promoter shares some cis-regulatorysequences with promoters from genes of this latter group.However, although several putative regulatory motifshave been identified, the molecular basis of the specificityof pollen gene expression is still unknown (McCormick1993). More probably, due to the complex structure of theCaMV 35S promoter, which contains two major enhancerdomains and at least five subdomains that synergisticallyconfer developmental and tissue-specific expression(Benfey et al. 1989, 1990), it may be able to interact witha wide range of native gene enhancer sequences expressedin mature pollen and/or during pollen germination.

Gametophytic segregation of GUS activity in straw-berry did not follow the classic Mendelian segregationratios as expected in diploid species. Fragaria � ananassais an octoploid hybrid plant that arose as an interspecificcross between F. virginiana and F. chiloensis. It is as-sumed that the octoploid strawberry has mixed poly-ploidy—disomic and polysomic (Senanayake and Bring-hurst 1967). The inheritance pattern in this species istherefore complex, which makes it difficult to establishthe correlation between GUS expression in pollen and T-DNA copy number.

In conclusion, our analysis of the CaMV 35S promoterin pollen from different transgenic strawberry linesshowed a consistent expression pattern—the CaMV 35Spromoter was either silent or had very little activity inimmature pollen, but it was active in later stages, espe-cially following anther dehiscence. These results indicatethat, contrary to the general view, the CaMV 35S pro-moter is active in pollen and, therefore, is a factor to betaken into consideration in the biosafety assessment oftransgenic plants carrying this promoter.

Acknowledgements This work was supported by the Ministerio deCiencia y Tecnolog�a, Spain and EU FEDER funds (grant nos.IFD97/0843C0504 and AGL 2001-1897).

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