Antisense-mediated suppression of transgene expression targeted

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117049_349_326_1481.pdf Journal of Experimental Botany, Vol. 49, No. 326, pp. 1481±1490, September 1998 Antisense-mediated suppression of transgene expression targeted specifically to pollen

Joy E. Wilkinson1, Keith Lindsey2and David Twell3

Department of Biology, University of Leicester, University Road, Leicester LE17RH, UK

Received 3 July 1997; Accepted 23 April 1998

Abstractrestrictions on the use of genes from unrelated plants and

even from animals and micro-organisms. However, theA potential problem in the field release of transgeniccommercial release of transgenic plants raises concernsplants is the spread of foreign gene products viaabout the impact on the wider environment, especiallypollen. Therefore, the use of the tomato pollen-specificthe potential for the uncontrolled spread of the transgenelat52gene promoter was investigated as a means ofand its product. Dispersal of transgenes through cross-targeting antisense RNA to pollen without affectingpollination with wild and related species has been widelytransgene expression elsewhere in the plant. A trans-

explored (Crawleyet al., 1993; ScheZeret al., 1993;genic tobacco line, T115, which showed GUS expres-ScheZer and Dale, 1994; Timmonset al., 1995). However,sion in pollen, leaves and roots was retransformedpotential problems associated with the release of trans-with a construct containing the pollen-specificlat52gene products via pollen itself have not been thoroughlypromoter driving the GUS encodinguid Agene in anti-investigated.sense orientation. From 24 independent transformantsOf particular concern is the release of plants containingobtained, 19 showed a significant reduction in pollengenes such as those which encode insecticidal proteinsGUS activity. Of these lines, four showed a reprodu-like theBacillus thuringiensis(Bt) toxin (Perlaket al.,cible antisense effect in pollen in the next generation,1990; Wunnet al., 1996) and trypsin inhibitor proteinwhile it was shown in one line that GUS activity in(Boulter, 1989) which may aVect the ecology of localleaves and roots was also unaffected. To ascertainpollen-eating insects. Also under consideration for releasethe effectiveness of the antisense strategy to down-are genes encoding ribosome inactivating proteinsregulate very high levels of pollen expression, a(Logemannet al., 1992), seed storage proteins of thelat52-gusantisense construct was introduced intoglutenin class (Shewryet al., 1995) and human serumtobacco lines containinglat52-gus, which had pollenalbumin (Sijmonset al

., 1990) which may have theGUS activity of up to 250 times greater than in linecapacity to become air-borne allergens should theyT115. Results showed that 30 out of 34 independentbecome ectopically localized in pollen (Fox, 1994). Therelines exhibited a significant antisense effect in pollen,is also the potential for these foreign genes and theirconfirming the effectiveness of pollen-targeted anti-products to enter the human food chain via bee contam-sense startegy to reduce undesirable expression inination of honey. Indeed, it has been shown by Eadypollen independent of expression level in pollen.et al. (1995) that the reporter gene,uid A, remained

transcriptionally active and its enzyme product retained Key words: Antisense,b-glucuronidase,lat52promoter,

high levels of activity for several weeks when expressedpollen, transgenic plant release.in pollen stored in honey. Pollen therefore represents a

route whereby transgene products can aVect the wider

Introduction

environment and potentially may contaminate the human food chain. The use of transgenic plants in agriculture is attractive in

that, unlike conventional plant breeding, there are few It has been shown previously that the promoters1Present address: Plant Sciences Unit, Unilever Research, Colworth House, Sharnbrook, Beds. MK44 1LQ, UK

2Present address: Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK.

3To whom correspondence should be addressed. Fax:+44 116 2522791. E-mail: twe@le.ac.uk

© Oxford University Press 1998Downloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

1482Wilkinsonet al.

T115 showed it to contain T-DNA at a single locus. The commonly in use in generating transgenic lines, namely lat52-gusline contained theuid Agene under the control of the the CaMV 35S (Odellet al., 1985) andnos(Anet al., tomatolat52promoter (Twellet al., 1990) and was shown by

1988) promoters show great variability in pollen expres-

genetic analysis to contain the construct at a single locus. Both sion between species and also between independent trans- the T115 andlat52-guslines used were homozygous for the transgenes. All tobacco plants were grown under extended genic lines (Wilkinsonet al., 1997). In this instance it daylength conditions in the greenhouse consisting of 18 h light was possible to isolate lines in which pollen expression followed by 6 h dark. could not be detected. It is envisaged, therefore, that transgene dispersal through pollen may be prevented by

Bacterial strains and plasmids

isolating transgenic lines in which no transgene expression Theuid Agene was cloned in an antisense orientation in the or product is detectable in pollen, but which has the vector pMOG22 (obtained from MOGEN) by using PCR required expression pattern elsewhere in the plant. ampli®cation to obtain the gene with required restriction enzyme sites. The PCR reaction mix contained 10 ng pLAT52±7 However, it is likely that there will be instances in which as DNA template (harbouring the full-lengthuid Agene), a transgenic plant line containing the desired trait is also

280 ng of each primer (SUG 1 5∞-CAACGAGCTCATG-

linked with expression in pollen. TTACGTCCTGTAGAAAC-3∞;SUG25∞-CAACAGATCTC- A possible means of eliminating any spread of products ATTGTTTGCCTCCCTGCTG-3∞), and was carried out using through pollen would be to de¯ower all plants, but this

2 units of Taq polymerase (Promega) in the supplied Mg2+-

containing reaction buVer. Ampli®cation was carried out in a would be diYcult to implement on a large scale. Antisense Techne PHC±3 thermal cycler using the following conditions:

RNA technology, and its sister technology, co-

initial denaturation for 4 min at 95°C; then 25 cycles of primer suppression (Greenet al., 1986; Jorgensen, 1990, 1993), annealing and extension by Taq polymerase for 2 min at 72°C, is routinely used to down-regulate the activity of speci®c denaturation for 1 min at 95°C; and a ®nal extension step for genes and could be applied in a ®eld situation. The success

8 min at 72°C. PCR products were separated in a 1.5% (w5v)

agarose gel (SeaKem) and stained with ethidium bromide. A of antisense in down-regulating speci®c genes, combined band of the expected size (1.8 kb) was obtained which was cut with the use of a promoter that could target the produc- from the gel and DNA isolated using Geneclean (BIO 101 tion of antisense transcripts to a particular organ or tissue Inc.). The incorporation ofSstI andBglII restriction enzyme type prompted an investigation into the possibility of sites into the PCR primers enabled the product to be cloned using a pollen-speci®c promoter to target antisense into pBluescript (Stratagene). Thelat52promoter and nos 3∞ terminator fragments were ligated into the vector to make speci®cally to pollen. pLAT52-sug. A 2.75 kb fragment containing thelat52promoter,

This paper shows how antisense-mediated down-

antisenseuid Aand 3∞nos terminator sequences was removed regulation ofuid Aexpression can be targeted speci®cally from pLAT52-sug using the restriction enzymesSalI andEco to pollen without aVecting transgene expression elsewhere RI and cloned into theXhoI andEcoRI sites of pMOG22 to in the plant. The eVectiveness of the antisense strategy create pMOG52-sug (Fig. 1). pMOG22 and pMOG52-sug were directly transformed intoAgrobacterium tumefaciensLBA4404 was also shown in tobacco lines containing very high (Oomset al., 1982) using the technique of HoÈfgen and levels of GUS activity in pollen.

Willmitzer (1988).

Plant transformation

Materials and methods

Tobacco leaf discs of tobacco lines T115 andlat52-guswerePlant materialobtained from cultured shoots and were transformed essentially

as described by Horschet al. (1985) with selection on mediumThe tobacco line T115, containing theuid Agene under the

contol of the-90 bp CaMV 35S minimal promoter (Topping containing 100 mg l-1kanamycin sulphate and 20 mg l-1

hygromycin B (Calbiochem). Agrobacteria were eliminatedet al., 1991) was obtained from Dr J Topping (Department of

Biological Sciences, University of Durham). Genetic analysis of by addition of 200 mg l-1cefotaxime (Claforan, Roussel).

Fig. 1.Diagram showing pMOG52-sug construct.Downloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

Suppression of transgene expression1483

Segregation analysis for kanaymcin and hygromycin resistance was used to determine T-DNA locus number. This was determined by plating out 100±130 seeds, surface-sterilized with

5% (v/v) sodium hypochlorite solution for 20 min, on half-

strength Murashige and Skoog (1962) medium supplemented with 10 g l-1sucrose, 100 mg l-1kanamycin sulphate and

20 mg l-1hygromycin B, respectively. The proportion of

resistant seedlings was counted at 21 d post germination.

Histochemical localization of GUS activity

Tobacco pollen was stained for GUS activity in microtitre wells, and whole seedlings in 1.5 ml microcentrifuge tubes, for up to

16 h at room temperature (17±22°C) in 1 mM 5-bromo-4

chloro-3-indolylb-d-glucuronic acid (X-glu, Biosynth) accord- ing to the method of JeVersonet al. (1987). The buVer was modi®ed by the use of histochemical staining buVer comprising

100 mM sodium phosphate (pH 7.0), 10 mM EDTA, and 0.1%

(v/v) Triton X-100 (Stomp, 1990). It was determined that, under these conditions, GUS staining in T115 pollen remained localized without spreading to neighbouring grains, whereas pollen from lines oflat52-gustobacco required the addition of

5 mM potassium ferricyanide to localize staining (Lojda, 1970;

Fig. 2.GUS activity in leaves (a) and roots (b) of line T115. Time

Mascarenhas and Hamilton, 1992).

period refers to number of days post-germination on MS plates.

Standard deviations are marked by vertical lines.

Isolation and staining for GUS activity in microspores Following measurement of bud length, one anther was removed and placed into a 1.5 ml microfuge tube containing 0.5 ml

Fig. 3.Activation of T115 andlat52promoters during pollen development. T115 bud length of (a) 20 mm (b) 22 mm and (c) 25 mm.Lat52bud

length of (d) 19 mm, (e) 22 mm and (f ) 25 mm. Scale bars: 40mm in all sections.Downloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

1484Wilkinsonet al.

0.3 M mannitol. The anther was gently squashed against the

sion was the tomatolat52gene promoter which has been side of the tube until a white cloud of microspores was released. shown to have high activity speci®c to pollen in tomato, The liquid was transferred to a fresh tube using a pipette tip, tobacco andArabidopsis(Twellet al., 1990). The timing centrifuged at 2000 rpm for 2 min in a microfuge and the of activation of GUS expression in pollen of the line supernatant removed. The microspores were washed once in

0.3 M mannitol and then resuspended in histochemical staining

T115 was compared to the tobacco linelat52-gusby

buVer containing X-glu before being left to stain in a ¯at- staining microspores and pollen at varying stages of bottomed microtitre plate. development, as determined from the bud length (Twell et al., 1993). Results indicated that in both instances

Fluorimetric assays for GUS activity

GUS activity was ®rst detectable in spores from Crude protein extracts were made from tissues according to

15±17 mm buds, though staining of a greater proportion

JeVersonet al. (1987), and GUS activities determined using of the spores was observed in 17 mm buds for the

4-methyl-umbelliferone glucuronide (MUG, Sigma) as sub-

lat52-gusline. Expression was then observed to increase strate. The ¯uorescent product 4-methylumbelliferone (4-MU)

rapidly to 100% stained spores in 25 mm buds in bothwas assayed using a Perkin Elmer LS±50 Luminescence

Spectrometer, using an excitation wavelength of 365 nm and an lines (Fig. 3). Pollen GUS expression in the line T115 emission wavelength of 455 nm. Protein concentrations in was therefore activated temporally in a similar manner extracts were determined using the Bradford reagent (Bio- to thelat52-gustransgenic plants (Twellet al., 1990). Rad), according to manufacturer's instructions. Speci®c GUS enzyme activities were expressed as pmol 4-MU produced min-1

Effect of antisense on pollen expression

of reaction mg-1protein.

The mean GUS activty in mature pollen from 10

plants of the homozygous T115 line was 1831 pmol

Results

4-MU min-1mg-1(±505 sd). Since thelat52promoter

is gametophytically expressed in the vegetative cell of theAnalysis of transgenic line T115pollen grain (Twellet al., 1990; Twell, 1992), integrationUse of tagging techniques in which plants are transformedat a single locus would aVect only 50% of the pollen inwith theuid Agene under the control of the-90 bpthe ®rst (T1) generation. Therefore, the expectation wasCaMV 35S minimal promoter (Lindseyet al., 1993),that lines transformed with a single active antisenseenabled identi®cation of a transgenic tobacco line, T115,gene would be expected to show a reduction in pollenwhich possessed GUS activity in leaves, roots and pollen

(Toppinget al., 1991). This line was chosen as a candidate to attempt targeting of antisense-uid Ato pollen since the eVect on GUS activity levels in leaves and roots could be determined to see if they were also aVected. This line was characterized in detail to obtain data for GUS activity in roots and leaves and also to determine when GUS activity became detectable during pollen development This enabled standard measurements to be taken once the line was retransformed with the antisense construct. To eliminate developmental eVects on GUS expression in roots and leaves, GUS assays were performed on the ®rst leaf and on roots of seedlings taken at intervals between 21 and 28 d post-germination on MS plates, as described in Materials and methods. Activity in seedling leaves increased during this time period from 59.82 to

285.6 pmol 4-MU min-1mg-1protein, but stabilized

at around 270±280 pmol 4-MU min-1mg-1protein at

26±28 d (Fig. 2a). A diVerent temporal pattern was

observed with seedling root tissue, in which GUS activity initially decreased from 18770 to 10522 pmol

4-MU min-1mg-1protein between days 21±23, but

was found to stabilize at around 12060±10500 pmol

4-MU min-1mg-1protein from 23±28 d (Fig. 2b). In

Fig. 4.GUS activity in pollen of T115 lines transformed with pMOG52-sug (a) and pMOG22 (b). The thin solid line (Ð) lines transformed with the antisense GUS construct, all indicates mean GUS activity found in the T115 line, the broken lines seedling leaf and root assays were therefore carried out (A) and (б) represent the standard deviation above and below the on tissue at 26±28 d post-germination. mean, respectively. The thick solid line (Ð) represents half the mean pollen GUS activity level in T115.

The promoter of choice to drive antisenseuid Aexpres-Downloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

Suppression of transgene expression1485

GUS activity from approximately 1800 to 900 pmol analysis of the progeny of 13 of the lines, which had

4-MU min-1mg-1protein. initially showed the greatest reduction in pollen GUS

A total of 24 independent T115 lines transformed with activity (A1±A11, A17, and A20) indicated that four of the antisense construct pMOG52-sug were obtained, and these lines (A3, A5, A11, and A20) continued to show a

20 lines transformed with the control vector pMOG22.

reproducible down-regulation of GUS activity (Fig. 5). Analysis of pollen GUS activity in lines containing For example, in line A5, examination of 10 siblings in the pMOG52-sug showed that 19 out of 24 lines (79%) had T2 generation revealed the presence of two populations a signi®cant reduction in pollen GUS activity to at least with GUS activity in pollen of approximately 300 half the level found in the parental T115 line (Fig. 4a). and 700 pmol 4-MU min-1mg-1protein, respectively Analysis of lines containing pMOG22 indicated that the (Fig. 5b). Segregation analysis con®rmed that line A5

process of transformation and regeneration did not causecontained the antisense construct present at a single

a decrease in GUS activity in pollen (Fig. 4b). A total oflocus and that the two populations corresponded to

two of the control lines, B2 and B12, appeared to showhomozygous and hemizygous lines (data not shown).

a marked reduction in pollen GUS expression, but stain-Histochemical staining provided further evidence thatuid

ing of pollen from these lines showed that 100% of theAexpression was down regulated (Fig. 6a±e). pollen stained, though the intensity of staining was not

as high as in some other lines. These lines were thereforeEffect of antisense on leaf and root expressionnot aVected by the control vector, but were at the extreme

Results obtained from the measurement of GUS expres- end of variation in GUS expression present in this popula- sion in the leaves and roots of antisense lines are shown tion. Mean GUS activity for the 20 lines was 1862 pmol in Fig. 7a and b. An examination of the lines which

4-MU min-1mg-1protein, which was similar to the

showed a positive antisense eVect in both the T1 and T2mean ®gure of 1831 pmol 4-MU min-1mg-1protein

generations in pollen (lines A3, A5, A11, and A20) calculated for the T115 line.

T115bantisense GUS lines were selfed and further indicated that root expression was decreased substantially

Fig. 5.An examination of GUS activity in siblings of the T2 generation of antisense containing lines A3 (a), A5 (b), A11 (c), and A20 (d). The

solid line (Ð) represents half the average GUS activity seen in the T115 parental line.Downloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

1486Wilkinsonet al.

Fig. 6.Histochemical staining of pollen and seedlings to show antisense eVect targeted to pollen. (a) Pollen of parental line T115; (b) pollen from

a heterozygous antisense containing line A5.4; (c) pollen from a homozygous antisense containing line A5.3; (d) pollen from line A20.1; (e) pollen

from line A11.3; (f ) seedling of T115; (g) seedling of control non-antisense containing line B3; and (h) seedling of antisense containing line A5.

Scale bars: 33mm (a, b, c), 65mm (d, e), and 3.5 mm (f, g, h). in three of these lines (A3, A11 and A20) whereas a plasmid pMOG52-sug was transformed intolat52-gus tobacco lines. While line T115 had a mean pollen GUS signi®cant decrease in leaf expression was present only in expression of 1831 pmol 4-MU min-1mg-1protein,line A11. The line A5, though appearing to have lower the mean for thelat52-guslines was 445 528 pmolleaf and root GUS activity than the mean, did retain

4-MU min-1mg-1protein, almost 250-fold higher. Aexpression within the range measured for the whole

total of 35lat52-guslines containing the antisense con-population. This line therefore appears to have antisense

struct were generated and 24 lines were generated trans-successfully targeted to pollen without substantially

formed with the pMOG22 control vector. A total of 30aVecting leaf and pollen expression. Histochemical stain-

out of 34 of the antisense-containing lines showed aing of 8-d-old seedlings also indicated that root expressionsigni®cant reduction in pollen GUS expression to approxi-was not aVected (Fig. 6f±h). Results for the control linesmately half that in the parental line, as expected ifB2 to B4 are also shown in Fig. 7 and indicate that againantisense is functioning to reduce expression (Fig. 8a).the process of transformation and regeneration did notThough some of the control lines also showed reducedaVect leaf and root expression.GUS activity levels (Fig. 8b), histochemical staining

showed that in all cases 100% of the pollen stained blue,Assessing the effectiveness of antisensewhereas in the antisense-containing lines 14 out of 18

To determine whether very high levels of GUS expression lines analysed showed populations in which approxi-

mately half of the pollen did not stain (Fig. 8c).could be down-regulated by pollen-targeted antisense, theDownloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

Suppression of transgene expression1487

more speci®c patterns of activity have also been used to target antisense transcripts to particular tissues. For example, leaf-speci®c inhibition ofuid Agene expression driven by the CaMV 35S promoter was achieved in tobacco by using a light-regulated promoter obtained fromArabidopsis(Cannonet al., 1990); and seed-speci®c inhibition of CaMV 35S drivenuid Agene expression was achieved in tobacco by using a seed storage protein promoter from soybean (Fujiwaraet al., 1992). The availability of a promoter with high activity speci®c to pollen made it possible to target antisense transcripts to pollen. The ability to target antisense to gametophytic tissue has implications in plant breeding as well as in preventing the possible spread of transgenes through pollen. Analysis of T115 tobacco lines retransformed with the antisense construct, pMOG52-sug, indicated that 79% of the 24 independent transformants showed a reduction in pollen GUS activity to approximately half that seen in the T115 parental line (Fig. 4a). A maximum 50% reduc- tion in pollen GUS activity was expected for lines con- taining the antisense construct at a single locus in the ®rst (T1) generation due to segregation at meiosis. Study of the subsequent T2 generation of 13 of the lines which had the greatest reduction in GUS activity showed that four of these lines continued to show a reduction in Fig. 7.GUS activity in leaves (a) and roots (b) of 26±28-d-old seedlings pollen GUS activity (Fig. 5). This indicated that of lines transformed with pMOG52-sug. The solid line (Ð) indicates inheritance of the initial antisense phenotype was un- mean GUS activity found in the T115 line, the broken lines (A) and (б) represent the standard deviation above and below the mean, stable in some instances. It has been noted that certain respectively. Vertical lines indicate the standard deviation for the total clones exhibit high rates of instability when selfed of three samples taken for each independent transgenic line. (Cherdshewasartet al., 1993) and several workers have shown thattransgene inactivation may be due to promoter methylation (for example, see Kilbyet al., 1992; Meyer,

Discussion

1995). The four lines which continued to display an

antisense eVect produced siblings which indicated theThe ability to target gene expression to speci®c tissues is

a useful tool in the ®eld of genetic engineering. In this presence of both homozygous and hemizygous popula-

tions. GUS activity in homozygous lines was reducedpaper, it has been shown that by the use of a pollen-

speci®c promoter and antisense RNA technology, speci®c to a minimum of approximately 300 pmol 4-MU

min-1mg-1protein, a reduction to 17% of originaldown-regulation of a gene in pollen can be achieved

without aVecting its expression elsewhere in the plant. activity in the T115 line. This residual activity may be

due to antisense not functioning at 100% eYciency, or toAntisense technology has been used very successfully

to reduce the expression of various genes in several plant a mismatch in the activity of the promoters: for example,

if the T115 promoter was activated slightly before thespecies. For example, the reduction of polygalacturonase

gene expression in tomato (Smithet al., 1988); thelat52promoter in developing pollen, any GUS protein

synthesized fromuid Atranscripts would remain stableinhibition of chalcone synthase in petunia (van der Krol

et al., 1988); the reduction of patatin in potato tubers and enabling detection of residual activity.

Control populations of T115 transformed with a `non-(HoÈfgen and Willmitzer, 1992); and the reduction of

amylose content in grain starch by the expression of antisense' vector, pMOG22, showed no decrease in pollen

GUS activity (Fig. 4b), indicating that it was the presenceantisensewaxygene mRNA in rice (Shimadaet al., 1993).

The most common promoter used to drive the produc- of the antisense construct which resulted in decreased

pollen GUS activity and not the process of transformationtion of antisense transcripts is the CaMV 35S promoter

(Bourque, 1995). This promoter is active in a wide range and regeneration. The control populations also showed

no reduction in GUS activity in leaves and roots.of plant tissues (JeVersonet al., 1987; Odellet al., 1985);

and its use in antisense constructs generally leads to a However, three of the four antisense containing lines

which continued to show an antisense eVect in the T2non-speci®c reduction in gene expression. Promoters withDownloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

1488Wilkinsonet al.

Fig. 8.GUS activity in pollen of lat52-gus lines transformed with pMOG52-gus (a) and pMOG22 (b). The thin solid line (Ð) indicates mean GUS

activity found in the lat52-gus line, the broken lines (A) and (б) represent the standard deviation above and below the mean, respectively.

The thick solid line (Ð) represnts half the mean pollen GUS activity level in lat52-gus plants. Histochemical staining of pollen to illustrate the

antisense eVect is shown in (c), with pollen from a pMOG22 transformed control, D10 (i), and pollen from lines C16 (ii) and C18 (iii) transformed

with pMOG52-sug. Scale bars: 65mm(c).

generation (lines A3, A11 and A20) also appeared to taken into account in any targeting experiment and,

consequently, lines may have to be screened in order toshow a reduction in GUS activity in roots, while line A11

also showed a reduction in leaf expression (Fig. 7). Line obtain one with the desired characteristics.

The majority of the models currently postulated on theA5, however, showed the expected reduction in pollen

GUS activity, but no reduction in leaf or root tissue, possible mechanism by which antisense causes its eVects

rely on the formation of an RNA:RNA hybrid betweenindicating that the antisense eVect had been successfully

targeted to pollen. the sense and antisense constructs (for example, see

Templeet al., 1993). It was therefore expected that useThe reduction of root and leaf expression in some of

the antisense lines is not entirely unexpected. Tagging of the highly activelat52promoter driving antisense

transcript production would be eVective in a line such asexperiments in which constructs containing a functioning

uid Agene with either no promoter or a minimal promoter T115 where pollen GUS activity was low, since the

antisense transcripts would be expected to be in vastare introduced into tobacco, have shown that up to 75%

of transgenic lines had GUS activity in roots while 22% excess and form hybrids with most or all available sense

transcripts. However, thelat52promoter was also eVect-showed some leaf activity (Lindseyet al., 1993; Topping

et al., 1991). It is therefore possible that some of the ive when used to target antisense transcripts in transgenic

tobacco lines containing thelat52promoter drivinguidantisense constructs have inserted downstream of root or

leaf elements which activate antisense transcript produc-Ain the sense orientation.

EVectiveness of the antisense did not, in this instance,tion in roots and/or leaves. This eVect would have to beDownloaded from https://academic.oup.com/jxb/article/49/326/1481/532143 by guest on 24 July 2023

Suppression of transgene expression1489

rely on the over-abundance of antisense transcripts relat-

Acknowledgements

ive to sense transcripts. The use of the same promoter The ®nancial support of the Ministry of Agriculture, Fisheries combination, such as thelat52against thelat52, may and Food is gratefully acknowledged. This work was carried result in eVective antisense down-regulation since both out under MAFF Plant Health Licence number 1277/337/13. promoters will be regulated temporally and spatially in the same way. Both sense and antisense transcripts would

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