BACKGROUND
plant biotechnology has great potential to speed up crop improvement and meet the needs of a growing world population.
a wide variety of commercial crops with important agronomic traits have already been produced successfully.
similar methods have also made it possible to use plants as bioreactors for economic production of pharmaceuticals.
although various plant expression systems have been established as bioreactors, the wider use of plant-derived pharmaceuticals has been limited by low yields due to the dozens of important parameters critically reviewed by egelkrout et al.
<cit> .
various strategies to maximize transgene expression have been investigated to meet the demands of fundamental research or industrial applications.
employing a strong promoter for transcription is the first method of choice.
the most widely used constitutive promoter for transgene expression in plants is the 35s promoter of cauliflower mosaic virus   <cit> .
the use of tandem repeats of the fragment between positions - <dig> and - <dig>  enhances the level of promoter activity up to  <dig> fold  <cit> .
the rice act <dig> or maize ubi <dig> promoters are constitutive promoters from plants that can deliver high expression of foreign genes in mononcots  <cit> .
other plant constitutive promoters have also been isolated  <cit> , but few of them have been extensively used.
in addition to these promoters from natural highly expressed genes, recombinant promoters designed by combining different cis-regulatory elements and core promoters can be useful alternatives  <cit> .
for example, the pemu promoter, which coupled the anaerobic responsive elements from the maize adh <dig> gene and ocs-elements from the octopine synthase gene with a truncated maize adh <dig> promoter, allowed high expression in cereal cells  <cit> .
a g-box motif  tetramer fused to a camv -90/35s basal promoter conferred stable, strong expression in both dicot and monocot plants  <cit> .
in another study, insertion of an amplification promoting sequence  upstream of an expression cassette was found to increase the transcription of the adjacent heterologous genes by  <dig>  to  <dig>  fold in tomato and tobacco, respectively  <cit> .
in addition to these efforts to promote transcription, codon optimization, proper signal localization peptide and a translation initiation sequence  are also used to achieve enhanced protein translation and stability  <cit> .
another source of constitutive regulatory elements is cryptic gene-regulatory elements.
the plant cryptic promoter tcup conferred strong constitutive expression when fused to a reporter gene in both tobacco and arabidopsis <cit> , and was also highly active in a wide range of dicot plants including alfalfa, canola, cauliflower, tomato and pea as well as in conifers  <cit> .
the strong constitutive feasibilities of the tcup promoter made it possible to drive the expression of a selectable marker gene for recovery of transformed plants through both organogenesis and embryogenesis  <cit> , and proved to be a useful alternative to camv 35s as it does not interact with tested promoters  <cit> .
although the expression of tcup is reported to be minimal in monocot plants such as wheat, barley, maize and oat  <cit> , we considered that it would be interesting to study the possible expression and application of tcup in rice.
higher levels of gene expression may also be achieved through increasing the number of copies of a transgene.
this may be achieved by selfing heterozygous lines  <cit>  or crossing homozygous lines obtained from independent transformation events  <cit>  and also by using constructs with multiple plant transcription units under the control of the same or different promoters as cited by egelkrout et al.
<cit> .
however, these methods are rather time-consuming and inconvenient.
in this report, we demonstrate that a fragment  derived from tcup was active in a tissue specific manner in rice transgenic plants and behaved as a weak promoter, driving a selectable marker gene for rice transformation.
interestingly, such selection favored the recovery of transgenic plants with higher expression levels of a foreign gene controlled by a constitutive promoter, an effect ascribed to the additive result of multiple t-dna insertions.
the effect of a weak promoter driving a selectable marker on the elevation of transformation copy number and transgene expression was also confirmed by using the nopaline synthase  promoter.
thus our findings suggest a new and simple way to improve transgene expression in rice irrespective of the promoters used to control them.
RESULTS
tissue specific expression of the tcup <dig> promoter in rice
as in a previous study, the - <dig> tcup truncation had similar activity to the full length tcup promoter as shown by the 5′ deletion assay in arabidopsis <cit> .
here we synthesized a fragment named tcup <dig> from - <dig> to + <dig> relative to the transcription start site of the natural tcup promoter .
the tcup <dig> promoter was the same as entcup <dig>  <cit>  except that the upstream translational initiation codon in the leader sequence was retained and a kozak consensus sequence  <cit>  including the atg codon was introduced after + <dig>
to determine the tcup <dig> promoter activity in rice and its ability to drive selectable marker gene expression for rice transformation, the tcup <dig> promoter was subcloned to a t-vector and digested for fusion respectively to the hpt selectable marker gene or gus reporter gene in the same binary vector .
this vector was also designed to test whether the hpt expression controlled by tcup <dig> can confer resistance to the usual hygromycin selection in rice.
after 21-days selection on 50 mg/l hygromycin, calli were randomly selected for histochemical gus assay.
as shown in figure 2bc, the gus gene was expressed on the primary callus showing that these cells were living after the hygromycin selection.
after a second round of selection, hygromycin resistant calli were chosen for regeneration.
<dig> gus positive transgenic lines were recovered and most of them showed a similar expression pattern.
a representative line  shows that gus activity is mainly observed in the root tip, leaf and anthers, but is absent in lateral roots, the seed coat and ovary.
this indicates that the tcup <dig> promoter is expressed in a tissue specific manner in rice, unlike the constitutive expression previously observed in tobacco  <cit> , but that it can still be successfully used for selectable marker gene expression and recovery of transgenic rice plants.
the tcup <dig> promoter is a weak but functional promoter in rice as compared with camv 35s
we further compared the relative strength of the tcup <dig> and camv 35s promoters in terms of their effectiveness in selectable marker gene expression and consequent tolerance to the selective agent.
the double enhancer camv 35s promoter  <cit>  derived from pbi <dig>  <cit>  was inserted in front of the hpt expression cassette.
another sgfp expression cassette driven by the maize ubi- <dig> promoter  <cit>  was introduced within the t-dna as a visual marker for successful transformation .
the tcup <dig> promoter was constructed using the same strategy as camv 35s.
the two vectors were used to transform rice calli derived from mature seeds of cultivar nipponbare.
the percentage of calli with cells expressing gfp was determined at the end of the first round selection  from two independent transformations with each vector .
almost all calli  infected with the camv 35s-hpt-gfp vector had gfp-expressing sectors which reduced to  <dig> % and  <dig> % when infected with tcup1-hpt-gfp.
this result suggested that the transformation capacity conferred by the tcup <dig> promoter mediated selection was lower than that of camv 35s.
although a large proportion of the calli were transformed by the hpt-gfp vector controlled by tcup <dig>  the growth of these calli was slow, as shown in figure 3b, whereas calli transformed using camv 35s grew vigorously during both the first  and second  rounds of selection.
the weights of calli were measured at the beginning of s <dig> and s <dig>  as shown in figure 3d, there were no statistical differences at the start of s <dig> between the camv 35s and tcup <dig> controlled vectors in two replicated experiments .
following s <dig>  there were approximately  <dig>  to  <dig>  fold increases of weight in calli selected with the camv 35s vector but only  <dig> to  <dig>  fold increases with the tcup <dig> vector.
the apparently low tolerance to hpt selection indicated that the tcup <dig> promoter drives relatively weak gene expression in rice.
the tcup <dig> promoter driving low expression of the selective marker gene nevertheless provides enhanced exogenous gene expression
the strength of the tcup <dig> promoter in regulating gene transcription was further analyzed.
after the second round of selection, proliferated hygromycin resistant calli from both camv 35s and tcup <dig> treatments were collected for rna extraction and cdna synthesis.
the relative expression levels of hpt and gfp were analyzed by quantitative real-time rt-pcr .
as expected, the hpt mrna level in resistant calli controlled by tcup <dig> was much lower than in those controlled by camv 35s , correlating with their respective growth during selection.
conversely however, the relative gfp expression in resistant calli selected by the tcup <dig> vector was found to be 4- <dig> times higher than in those selected by camv 35s from two independent experiments .
similarly, the green fluorescence emitted from the resistant calli was stronger in the tcup <dig> than in the camv 35s treatment .
the difference in the gfp transcripts between the camv 35s and tcup <dig> vectors was also confirmed in the regenerated plantlets  .
a population of  <dig> independent lines from each treatment was randomly chosen for analyzing gfp transcripts in leaves.
the range of gfp mrna levels  in lines selected using tcup <dig> was higher than in those using camv 35s.
differences in -Δct between the two highest gfp expressing lines containing each vector was about  <dig> , and half the lines in the population selected by the tcup <dig> vector had gfp expression levels similar to or higher than the highest one selected by camv 35s.
it seemed therefore that selection with a weakly expressed marker gene contributed to higher expression of the exogenous gene.
to investigate this correlation, we replace the tcup <dig> promoter with the nopaline synthase  promoter of agrobacterium tumefaciens <cit>  for selectable marker expression.
the nos promoter is constitutively expressed in all tissues examined in tobacco  <cit> , but is much weaker than the camv 35s full promoter in petunia  <cit> .
there are similar differences in rice as shown in figures 3b and 3d; the growth of calli infected with the nos-hpt-gfp vector was slower than that with the vector controlled by camv 35s in two experimental repeats, although the transformation rates were comparable between these two promoters .
furthermore, the transcriptional level of hpt in resistant calli selected by nos was significant lower than in those selected by camv 35s , while both the transcript levels and fluorescence intensity of gfp were greater  as had occurred with tcup <dig>  interestingly, the range of gfp expression levels in transgenic plants regenerated from resistant calli selected by nos was found to be even higher than in those selected by tcup <dig> .
these data confirm that higher exogenous gene expression in transgenic rice can be reliably obtained by using weak promoters to express the selectable marker gene.
elevated gene expression via weak promoter selection correlates with increased t-dna copy numbers
previous work comparing different promoters driving selectable markers suggested their possible effects on the copy numbers of the transgene  <cit> .
we therefore investigated whether the higher expression levels of the exogenous gene after using weak promoters for selection was caused by increased t-dna copy numbers.
the copy numbers of integrated t-dna in transgenic plants produced with each of the three vectors was estimated by southern blot analysis.
lines with either the highest  or lowest  gfp expression were chosen from each transgenic population based on the results of qrt-pcr .
the lines with high gfp expression from populations transformed using nos or tcup <dig> were found to have more hybridizing bands and stronger band signals  than the high gfp expressing lines transformed with the vector controlled by camv 35s , which varied from  <dig> to  <dig> bands.
most of the lines with low gfp expression from populations transformed with the vectors controlled by nos or tcup <dig> were also found to have multiple hybridizing bands , but all of the low gfp expressing lines transformed with camv 35s contained only a single band .
based on the main hybridizing bands, there were about 2 t-dna copies averagely integrated in lines transformed with the camv 35s vector, but at least  <dig> integrated t-dna copies in lines transformed with the nos and tcup <dig> vectors.
these results suggest that transgene expression is increased by the accumulation of multiple t-dnas under selection with the attenuated marker gene.
the gfp fluorescence in roots of t <dig> progeny from several lines with high levels of gfp expression and multiple inserted copies were characterized .
most lines were stable in gfp expression and similar in expression to their primary lines, except that line  <dig>  selected by tcup <dig> controlled vector had lower gfp fluorescence than line  <dig> .
the higher gfp expressing lines with multiple copies selected by nos and tcup <dig> promoters retained brighter gfp fluorescence in their progeny than those selected by 35s .
discussion
the tcup <dig> promoter is a functional cis element for rice transformation
a recent study on the tobacco cryptic constitutive promoter  in arabidopsis had shown its great ability to eliminate nonspecific interactions between transgene promoters  <cit> .
this attractive property gave rise to the idea of applying the tcup expression system in rice.
it was reported that the tcup sequence could direct transgene expression in both dicots and monocots, but the expression level of the reporter gene seemed lower in monocot cell suspensions than in dicot leaf tissue as measured by the numbers of gus-positive spots  <cit> .
although enhanced tcup derivatives had been used to drive the neomycin phosphotransferase ii  gene and selection for kanamycin resistant transformants in tobacco, cauliflower, alfalfa and arabidopsis <cit> , the performance of these elements in rice was not known.
we therefore examined its expression style and strength in rice and using it for selection of transformants.
the two experiments were done together by fusing the tcup <dig> fragment to both the hpt selectable marker and the gus reporter.
finally, a total of  <dig> gus positive lines were obtained and most of them showed similar staining patterns and inheritance to their progenies .
thus it was clearly demonstrated that the tcup <dig> fragment tested in this study, which comprised all three regions essential for tcup activity in dicots  <cit> , was also active as a cis-regulatory element in rice, although the expression was limited to certain tissues and organs.
the gus expression in transformed calli suggested that tcup <dig> could mediate successful rice transformation.
this was confirmed subsequently, although the selection process was prolonged because of the slow growth of resistant calli.
growth remained slow even if the upstream out-of-frame atg was eliminated by mutation , despite the report that this modification enhanced native tcup activity  <dig> to  <dig> fold  <cit> .
these results indicated that, as in other monocots, the tcup cis-regulatory element was active but less effective in rice.
the difference in behavior may be due to differences in transcription mechanisms between dicots and monocots, such as different transcription factors  <cit> .
the two effects of multiple t-dna copy number on transgene expression
it was previously reported that an average of  <dig>  copies of t-dna were integrated in rice plants of nipponbare when using the double camv 35s promoter in pcambia <dig> for hpt expression  <cit> , which is comparable to our results using the double camv 35s promoter derived from pbi <dig>  higher transgene copy numbers are always obtained by the biolistic method, but have never been reported in transgenic rice plants produced via agrobacterium-mediated methods, which is usually considered to have lower  transgene copy number integration  <cit> .
in our experiments, lines with extremely high numbers of t-dna inserts  were frequently obtained when using nos or tcup <dig> promoters for selectable marker gene expression.
in fact, these numbers could be underestimates as some of the strong hybridization signals may represent two or more inserts with a similar size migrating together as one band.
our data suggest that these multi-copy events can be screened out conditionally, for example here by attenuating hpt gene expression.
the inadequate expression of the selectable marker under the threshold of hyg pressure  may lead to preferential survival of high copy number cells with active hpt expression, as apparently occurred in experiments to transform tall fescue by the peg mediated method under elevated selection pressure from increased hyg concentration   <cit> .
these data indicate that the selection pressure set by both the marker gene expression level and the concentration of the corresponding selective agent is the dominant factor that influences t-dna copy number in the resultant transgenic plants.
therefore, even if the 35s promoter is used for selectable marker gene expression, transformants with high copy number of t-dna would be obtained when a very highly selective agent was applied.
the correlation between copy number and gene expression is complicated because multicopy t-dna insertions are associated either with transgene silencing or with additive transgene expression depending on the nature of the inserts  <cit> .
the negative effects of multicopy events have been extensively studied and are often discussed in relation to transformation related biotechnologies  <cit> .
generally, plants with a single copy of the transgene are preferred as they are genetically and phenotypically more stable than those with multiple copies  <cit> .
in this study, we found that the transgene expression and copy number were positively correlated: high gfp expressers always had multiple t-dna copies but the low gfp expressers had fewer; however some individuals with multiple copies had low gfp expression .
it therefore appears that multiple copies of t-dna inserts tend to contribute to exogenous transgene expression more than to suppression or inactivation in our case, although the expression/suppression of multiple t-dna copies varied between different studies, most likely as a result of different promoters/t-dna insertion elements being studied, or different transformation methods used  <cit> .
however, there was no linear relationship between copy number and transgene expression, perhaps because expression was also suppressed by some t-dna inserts.
the use of weak promoters to drive selection for effective gene expression in plants
in the study described here, we compared the effects of three different promoters in driving selectable markers for rice transformation.
the constitutive camv 35s promoter  yielded fast growth of callus sectors on the selection medium with 50 mg/l hygromycin, but much slower growth of calli was observed when using the tcup <dig> promoter and an intermediate rate using the nos promoter.
the growth performance of the transformed calli directly suggested to us the relative strengths of these three promoters, but it was not accurate enough to determine their absolute activities, because the overall resistance depended on the number of initial transformed cells, and where and how the t-dna were inserted.
we therefore tried to dissect promoter activities by comparing their transcribed hpt mrna levels in transgenic lines with single copy insertion.
it was an unexpected finding that the populations produced using the nos and tcup <dig> vectors were prone to have multiple insertions.
this suggested to us that these promoters were too weak to produce enough resistant protein for minimal selection unless transcriptionally active insertions were accumulated.
even so, the hpt transcripts were still far lower than those produced by camv 35s , similar to the result from resistant calli .
on the other hand, the increased copy number also elevated the constitutive expression of the gfp gene.
although it is difficult to select the homozygous line in the progeny derived from a transformant with high copy number for the purposes of genetic study, exogenous transgene expression level is still relatively stable in the next generations as we observed .
this finding provides a new strategy to select for high transgene expressers which is urgently needed for many purposes, such as the economic production of pharmaceuticals using plants as bioreactor expression systems and the generation of genetically modified plants with improved agronomic traits, especially for those vegetatively propagated plant such as potato.
it will also help in the analysis of cis-regulatory elements when using reporter genes, particularly for those promoters with weak activities.
remarkably, the tcup <dig> promoter would be a good choice for specific promoter analysis because it may also have low interaction capacity in rice.
CONCLUSIONS
here we report the weak activity of tcup derived promoter  in transgenic rice and the application of this weak promoter in driving selection for rice transformation.
the expression of a foreign gene  was increased both in resistant calli and regenerated plantlets, and this was caused by the large numbers of t-dna inserts.
the use of a second weak promoter  confirmed these findings.
this research work suggests an effective way to maximize transgene expression and its potential applications, such as specific marker line creation and vegetative plant breeding.
our findings also suggest that care should be taken in using a weak promoter for selection if a simple insertion pattern is desired.

 plant biotechnology has great potential to speed up crop improvement and meet the needs of a growing world population. a wide variety of commercial crops with important agronomic traits have already been produced successfully. similar methods have also made it possible to use plants as bioreactors for economic production of pharmaceuticals. although various plant expression systems have been established as bioreactors, the wider use of plant-derived pharmaceuticals has been limited by low yields due to the dozens of important parameters critically reviewed by egelkrout et al.. various strategies to maximize transgene expression have been investigated to meet the demands of fundamental research or industrial applications. employing a strong promoter for transcription is the first method of choice. the most widely used constitutive promoter for transgene expression in plants is the 35s promoter of cauliflower mosaic virus. the use of tandem repeats of the fragment between positions - and - enhances the level of promoter activity up to fold. the rice act or maize ubi promoters are constitutive promoters from plants that can deliver high expression of foreign genes in mononcots. other plant constitutive promoters have also been isolated, but few of them have been extensively used. in addition to these promoters from natural highly expressed genes, recombinant promoters designed by combining different cis-regulatory elements and core promoters can be useful alternatives. for example, the pemu promoter, which coupled the anaerobic responsive elements from the maize adh gene and ocs-elements from the octopine synthase gene with a truncated maize adh promoter, allowed high expression in cereal cells. a g-box motif tetramer fused to a camv -90/35s basal promoter conferred stable, strong expression in both dicot and monocot plants. in another study, insertion of an amplification promoting sequence upstream of an expression cassette was found to increase the transcription of the adjacent heterologous genes by to fold in tomato and tobacco, respectively. in addition to these efforts to promote transcription, codon optimization, proper signal localization peptide and a translation initiation sequence are also used to achieve enhanced protein translation and stability. another source of constitutive regulatory elements is cryptic gene-regulatory elements. the plant cryptic promoter tcup conferred strong constitutive expression when fused to a reporter gene in both tobacco and arabidopsis, and was also highly active in a wide range of dicot plants including alfalfa, canola, cauliflower, tomato and pea as well as in conifers. the strong constitutive feasibilities of the tcup promoter made it possible to drive the expression of a selectable marker gene for recovery of transformed plants through both organogenesis and embryogenesis, and proved to be a useful alternative to camv 35s as it does not interact with tested promoters. although the expression of tcup is reported to be minimal in monocot plants such as wheat, barley, maize and oat, we considered that it would be interesting to study the possible expression and application of tcup in rice. higher levels of gene expression may also be achieved through increasing the number of copies of a transgene. this may be achieved by selfing heterozygous lines or crossing homozygous lines obtained from independent transformation events and also by using constructs with multiple plant transcription units under the control of the same or different promoters as cited by egelkrout et al.. however, these methods are rather time-consuming and inconvenient. in this report, we demonstrate that a fragment derived from tcup was active in a tissue specific manner in rice transgenic plants and behaved as a weak promoter, driving a selectable marker gene for rice transformation. interestingly, such selection favored the recovery of transgenic plants with higher expression levels of a foreign gene controlled by a constitutive promoter, an effect ascribed to the additive result of multiple t-dna insertions. the effect of a weak promoter driving a selectable marker on the elevation of transformation copy number and transgene expression was also confirmed by using the nopaline synthase promoter. thus our findings suggest a new and simple way to improve transgene expression in rice irrespective of the promoters used to control them.  tissue specific expression of the tcup promoter in rice as in a previous study, the - tcup truncation had similar activity to the full length tcup promoter as shown by the 5′ deletion assay in arabidopsis. here we synthesized a fragment named tcup from - to + relative to the transcription start site of the natural tcup promoter. the tcup promoter was the same as entcup except that the upstream translational initiation codon in the leader sequence was retained and a kozak consensus sequence including the atg codon was introduced after + to determine the tcup promoter activity in rice and its ability to drive selectable marker gene expression for rice transformation, the tcup promoter was subcloned to a t-vector and digested for fusion respectively to the hpt selectable marker gene or gus reporter gene in the same binary vector. this vector was also designed to test whether the hpt expression controlled by tcup can confer resistance to the usual hygromycin selection in rice. after 21-days selection on 50 mg/l hygromycin, calli were randomly selected for histochemical gus assay. as shown in figure 2bc, the gus gene was expressed on the primary callus showing that these cells were living after the hygromycin selection. after a second round of selection, hygromycin resistant calli were chosen for regeneration. gus positive transgenic lines were recovered and most of them showed a similar expression pattern. a representative line shows that gus activity is mainly observed in the root tip, leaf and anthers, but is absent in lateral roots, the seed coat and ovary. this indicates that the tcup promoter is expressed in a tissue specific manner in rice, unlike the constitutive expression previously observed in tobacco, but that it can still be successfully used for selectable marker gene expression and recovery of transgenic rice plants. the tcup promoter is a weak but functional promoter in rice as compared with camv 35s we further compared the relative strength of the tcup and camv 35s promoters in terms of their effectiveness in selectable marker gene expression and consequent tolerance to the selective agent. the double enhancer camv 35s promoter derived from pbi was inserted in front of the hpt expression cassette. another sgfp expression cassette driven by the maize ubi- promoter was introduced within the t-dna as a visual marker for successful transformation. the tcup promoter was constructed using the same strategy as camv 35s. the two vectors were used to transform rice calli derived from mature seeds of cultivar nipponbare. the percentage of calli with cells expressing gfp was determined at the end of the first round selection from two independent transformations with each vector. almost all calli infected with the camv 35s-hpt-gfp vector had gfp-expressing sectors which reduced to % and % when infected with tcup1-hpt-gfp. this result suggested that the transformation capacity conferred by the tcup promoter mediated selection was lower than that of camv 35s. although a large proportion of the calli were transformed by the hpt-gfp vector controlled by tcup the growth of these calli was slow, as shown in figure 3b, whereas calli transformed using camv 35s grew vigorously during both the first and second rounds of selection. the weights of calli were measured at the beginning of s and s as shown in figure 3d, there were no statistical differences at the start of s between the camv 35s and tcup controlled vectors in two replicated experiments. following s there were approximately to fold increases of weight in calli selected with the camv 35s vector but only to fold increases with the tcup vector. the apparently low tolerance to hpt selection indicated that the tcup promoter drives relatively weak gene expression in rice. the tcup promoter driving low expression of the selective marker gene nevertheless provides enhanced exogenous gene expression the strength of the tcup promoter in regulating gene transcription was further analyzed. after the second round of selection, proliferated hygromycin resistant calli from both camv 35s and tcup treatments were collected for rna extraction and cdna synthesis. the relative expression levels of hpt and gfp were analyzed by quantitative real-time rt-pcr. as expected, the hpt mrna level in resistant calli controlled by tcup was much lower than in those controlled by camv 35s, correlating with their respective growth during selection. conversely however, the relative gfp expression in resistant calli selected by the tcup vector was found to be 4- times higher than in those selected by camv 35s from two independent experiments. similarly, the green fluorescence emitted from the resistant calli was stronger in the tcup than in the camv 35s treatment. the difference in the gfp transcripts between the camv 35s and tcup vectors was also confirmed in the regenerated plantlets. a population of independent lines from each treatment was randomly chosen for analyzing gfp transcripts in leaves. the range of gfp mrna levels in lines selected using tcup was higher than in those using camv 35s. differences in -Δct between the two highest gfp expressing lines containing each vector was about, and half the lines in the population selected by the tcup vector had gfp expression levels similar to or higher than the highest one selected by camv 35s. it seemed therefore that selection with a weakly expressed marker gene contributed to higher expression of the exogenous gene. to investigate this correlation, we replace the tcup promoter with the nopaline synthase promoter of agrobacterium tumefaciens for selectable marker expression. the nos promoter is constitutively expressed in all tissues examined in tobacco, but is much weaker than the camv 35s full promoter in petunia. there are similar differences in rice as shown in figures 3b and 3d; the growth of calli infected with the nos-hpt-gfp vector was slower than that with the vector controlled by camv 35s in two experimental repeats, although the transformation rates were comparable between these two promoters. furthermore, the transcriptional level of hpt in resistant calli selected by nos was significant lower than in those selected by camv 35s, while both the transcript levels and fluorescence intensity of gfp were greater as had occurred with tcup interestingly, the range of gfp expression levels in transgenic plants regenerated from resistant calli selected by nos was found to be even higher than in those selected by tcup. these data confirm that higher exogenous gene expression in transgenic rice can be reliably obtained by using weak promoters to express the selectable marker gene. elevated gene expression via weak promoter selection correlates with increased t-dna copy numbers previous work comparing different promoters driving selectable markers suggested their possible effects on the copy numbers of the transgene. we therefore investigated whether the higher expression levels of the exogenous gene after using weak promoters for selection was caused by increased t-dna copy numbers. the copy numbers of integrated t-dna in transgenic plants produced with each of the three vectors was estimated by southern blot analysis. lines with either the highest or lowest gfp expression were chosen from each transgenic population based on the results of qrt-pcr. the lines with high gfp expression from populations transformed using nos or tcup were found to have more hybridizing bands and stronger band signals than the high gfp expressing lines transformed with the vector controlled by camv 35s, which varied from to bands. most of the lines with low gfp expression from populations transformed with the vectors controlled by nos or tcup were also found to have multiple hybridizing bands, but all of the low gfp expressing lines transformed with camv 35s contained only a single band. based on the main hybridizing bands, there were about 2 t-dna copies averagely integrated in lines transformed with the camv 35s vector, but at least integrated t-dna copies in lines transformed with the nos and tcup vectors. these results suggest that transgene expression is increased by the accumulation of multiple t-dnas under selection with the attenuated marker gene. the gfp fluorescence in roots of t progeny from several lines with high levels of gfp expression and multiple inserted copies were characterized. most lines were stable in gfp expression and similar in expression to their primary lines, except that line selected by tcup controlled vector had lower gfp fluorescence than line. the higher gfp expressing lines with multiple copies selected by nos and tcup promoters retained brighter gfp fluorescence in their progeny than those selected by 35s. discussion the tcup promoter is a functional cis element for rice transformation a recent study on the tobacco cryptic constitutive promoter in arabidopsis had shown its great ability to eliminate nonspecific interactions between transgene promoters. this attractive property gave rise to the idea of applying the tcup expression system in rice. it was reported that the tcup sequence could direct transgene expression in both dicots and monocots, but the expression level of the reporter gene seemed lower in monocot cell suspensions than in dicot leaf tissue as measured by the numbers of gus-positive spots. although enhanced tcup derivatives had been used to drive the neomycin phosphotransferase ii gene and selection for kanamycin resistant transformants in tobacco, cauliflower, alfalfa and arabidopsis, the performance of these elements in rice was not known. we therefore examined its expression style and strength in rice and using it for selection of transformants. the two experiments were done together by fusing the tcup fragment to both the hpt selectable marker and the gus reporter. finally, a total of gus positive lines were obtained and most of them showed similar staining patterns and inheritance to their progenies. thus it was clearly demonstrated that the tcup fragment tested in this study, which comprised all three regions essential for tcup activity in dicots, was also active as a cis-regulatory element in rice, although the expression was limited to certain tissues and organs. the gus expression in transformed calli suggested that tcup could mediate successful rice transformation. this was confirmed subsequently, although the selection process was prolonged because of the slow growth of resistant calli. growth remained slow even if the upstream out-of-frame atg was eliminated by mutation, despite the report that this modification enhanced native tcup activity to fold. these results indicated that, as in other monocots, the tcup cis-regulatory element was active but less effective in rice. the difference in behavior may be due to differences in transcription mechanisms between dicots and monocots, such as different transcription factors. the two effects of multiple t-dna copy number on transgene expression it was previously reported that an average of copies of t-dna were integrated in rice plants of nipponbare when using the double camv 35s promoter in pcambia for hpt expression, which is comparable to our results using the double camv 35s promoter derived from pbi higher transgene copy numbers are always obtained by the biolistic method, but have never been reported in transgenic rice plants produced via agrobacterium-mediated methods, which is usually considered to have lower transgene copy number integration. in our experiments, lines with extremely high numbers of t-dna inserts were frequently obtained when using nos or tcup promoters for selectable marker gene expression. in fact, these numbers could be underestimates as some of the strong hybridization signals may represent two or more inserts with a similar size migrating together as one band. our data suggest that these multi-copy events can be screened out conditionally, for example here by attenuating hpt gene expression. the inadequate expression of the selectable marker under the threshold of hyg pressure may lead to preferential survival of high copy number cells with active hpt expression, as apparently occurred in experiments to transform tall fescue by the peg mediated method under elevated selection pressure from increased hyg concentration. these data indicate that the selection pressure set by both the marker gene expression level and the concentration of the corresponding selective agent is the dominant factor that influences t-dna copy number in the resultant transgenic plants. therefore, even if the 35s promoter is used for selectable marker gene expression, transformants with high copy number of t-dna would be obtained when a very highly selective agent was applied. the correlation between copy number and gene expression is complicated because multicopy t-dna insertions are associated either with transgene silencing or with additive transgene expression depending on the nature of the inserts. the negative effects of multicopy events have been extensively studied and are often discussed in relation to transformation related biotechnologies. generally, plants with a single copy of the transgene are preferred as they are genetically and phenotypically more stable than those with multiple copies. in this study, we found that the transgene expression and copy number were positively correlated: high gfp expressers always had multiple t-dna copies but the low gfp expressers had fewer; however some individuals with multiple copies had low gfp expression. it therefore appears that multiple copies of t-dna inserts tend to contribute to exogenous transgene expression more than to suppression or inactivation in our case, although the expression/suppression of multiple t-dna copies varied between different studies, most likely as a result of different promoters/t-dna insertion elements being studied, or different transformation methods used. however, there was no linear relationship between copy number and transgene expression, perhaps because expression was also suppressed by some t-dna inserts. the use of weak promoters to drive selection for effective gene expression in plants in the study described here, we compared the effects of three different promoters in driving selectable markers for rice transformation. the constitutive camv 35s promoter yielded fast growth of callus sectors on the selection medium with 50 mg/l hygromycin, but much slower growth of calli was observed when using the tcup promoter and an intermediate rate using the nos promoter. the growth performance of the transformed calli directly suggested to us the relative strengths of these three promoters, but it was not accurate enough to determine their absolute activities, because the overall resistance depended on the number of initial transformed cells, and where and how the t-dna were inserted. we therefore tried to dissect promoter activities by comparing their transcribed hpt mrna levels in transgenic lines with single copy insertion. it was an unexpected finding that the populations produced using the nos and tcup vectors were prone to have multiple insertions. this suggested to us that these promoters were too weak to produce enough resistant protein for minimal selection unless transcriptionally active insertions were accumulated. even so, the hpt transcripts were still far lower than those produced by camv 35s, similar to the result from resistant calli. on the other hand, the increased copy number also elevated the constitutive expression of the gfp gene. although it is difficult to select the homozygous line in the progeny derived from a transformant with high copy number for the purposes of genetic study, exogenous transgene expression level is still relatively stable in the next generations as we observed. this finding provides a new strategy to select for high transgene expressers which is urgently needed for many purposes, such as the economic production of pharmaceuticals using plants as bioreactor expression systems and the generation of genetically modified plants with improved agronomic traits, especially for those vegetatively propagated plant such as potato. it will also help in the analysis of cis-regulatory elements when using reporter genes, particularly for those promoters with weak activities. remarkably, the tcup promoter would be a good choice for specific promoter analysis because it may also have low interaction capacity in rice.  here we report the weak activity of tcup derived promoter in transgenic rice and the application of this weak promoter in driving selection for rice transformation. the expression of a foreign gene was increased both in resistant calli and regenerated plantlets, and this was caused by the large numbers of t-dna inserts. the use of a second weak promoter confirmed these findings. this research work suggests an effective way to maximize transgene expression and its potential applications, such as specific marker line creation and vegetative plant breeding. our findings also suggest that care should be taken in using a weak promoter for selection if a simple insertion pattern is desired. 
in this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. the correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase. the use of weak promoter derived selectable markers leads to a high t-dna copy number and then greatly increases the expression of the foreign gene. techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. the method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice. however, in the resistant calli and regenerated transgenic plants selected by the use of tcup the constitutive expression of green fluorescent protein was dramatically increased as a result of the additive effect of multiple t-dna insertions. the tcup promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the gus reporter, but was much weaker than the camv 35s promoter in driving a selectable marker for growth of resistant calli. tcup a fragment derived from the tobacco cryptic promoter, was tested for its activity in rice by fusion to both a β-glucuronidase reporter and a hygromycin phosphotransferase selectable marker.
 techniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. the conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent.  in this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tcup a fragment derived from the tobacco cryptic promoter, was tested for its activity in rice by fusion to both a β-glucuronidase reporter and a hygromycin phosphotransferase selectable marker. the tcup promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the gus reporter, but was much weaker than the camv 35s promoter in driving a selectable marker for growth of resistant calli. however, in the resistant calli and regenerated transgenic plants selected by the use of tcup the constitutive expression of green fluorescent protein was dramatically increased as a result of the additive effect of multiple t-dna insertions. the correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase.  the use of weak promoter derived selectable markers leads to a high t-dna copy number and then greatly increases the expression of the foreign gene. the method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice. tcuppromoter activityselectable markertransgene expressioncopy numberrice 