BACKGROUND
transgenic plants have become an essential component in ecological research, allowing the precise study of gene functions under field conditions  <cit> .
despite progress in the development of more efficient transformation techniques, the unpredictable and stochastic occurrence of transgene silencing and epigenetic alternations after the tissue culture step remain unsolved problems for most plant species  <cit> .
basically two forms of gene silencing have been described, transcriptional gene silencing , in which gene expression is directly blocked, and posttranscriptional gene silencing  in which mrna is degraded  <cit> .
ptgs has been exploited as a very powerful tool for reverse genetic studies and is revolutionizing plant ecology, particularly for non-model plants, where the introduction of “silencing-constructs” in self-compatible inverted repeat  or antisense  orientations enables the targeted silencing of endogenous genes in trans <cit> .
unfortunately, this process can be undermined by unwanted tgs, if the promoter of the transgene is de novo methylated, thereby diminishing the expression of the silencing-construct  <cit> .
de novo dna methylation can be highly sequence-specific for a transgene, as a result of the process called rna-directed dna methylation   <cit> .
however, the pattern of establishment and prerequisites for the methylation process remain elusive  <cit> .
characteristic symptoms of unwanted transgene silencing are spatially variegated or transient gene expression levels, patterns which have been observed in several different plant taxa including nicotiana tabacum <cit> , petunia hybrida <cit> , arabidopsis thaliana <cit> , gentiana triflora x g. scabra <cit>  and even in some transgenic woody plants such as grapevine   <cit>  and birch trees   <cit> .
the wild tobacco  is an annual plant, native to the great basin desert in the western united states and is used as a model organism to study traits important for survival under real world conditions, in particular the role of jasmonic acid  in plant defense against herbivores  <cit> .
n. attenuata has been frequently transformed with many different sense-expression, inverted repeat  or antisense  silencing-constructs to manipulate different layers of plant defense for field studies of gene function  <cit> .
a stably transformed plant is only useful for ecological experiments if the transgene-altered phenotype remains stable over the entire period of plant development.
in the glasshouse the life cycle of n. attenuata takes about 70–80 days until the plant produces seeds and develops from a vegetative rosette-stage, through stalk elongation, into the generative flowering phase.
over the course of development the plant reconfigures its defense strategy from largely inducible to constitutive deployment of various jasmonate-mediated chemical defenses  <cit> .
transgenerational phenotypic stability is also essential if different lines are to be crossed to combine traits so that parental phenotypes can be faithfully transmitted in a hemizygous state to the subsequent hybrid generations.
the n. attenuata line ir-acx <dig> was created to suppress a particular step in the ja biosynthesis pathway due to the silencing of the endogenous acetyl-coa-transferase  <dig> , but as recently shown the ability to suppress ja accumulation was lost when t <dig> generation plants were used during a field experiment  <cit> .
similar findings of leaky or lost phenotypes in n. attenuata lines have been reported in other studies  <cit>  highlighting the importance of the early detection of “unstable” plant lines.
the methylated form of cytosine was discovered more than 60 years ago  <cit> , but despite the very high amounts found in wheat seedlings, it was long considered only as a “minor base” in plant genomes  <cit> .
its importance in epigenetic gene regulation is increasingly being recognized, but the overall process remains poorly understood  <cit> .
if a genomic sequence functions as a promoter, de novo methylation can lead to transcriptional silencing of the downstream gene  <cit> .
cytosine methylation plays an important role in many cellular processes such as tissue-specific gene expression, embryogenesis or genomic imprinting  <cit> .
nevertheless, its generally accepted main function in plants is in the control of “invasive elements” such as transposons or viral sequences  <cit> .
in contrast to mammals, plants not only methylate cytosines in cg dinucleotides, but also in all other possible sequence contexts at chg and chh positions   <cit> .
the symmetric dna methylation patterns at cg and chg sites can be sustained during semiconservative dna replication and are therefore somatically and even meiotically stable  <cit> .
a methylation at the chh position is called asymmetric, because it has no mirror position on the complementary dna strand and hence will be lost during the dna replication process.
for maintenance during mitosis, an asymmetric site needs therefore a constant signal as a permanent de novo methylation trigger  <cit> .
although most aspects of epigenetic inheritance are understood, somatic cells are considered to be relative static and the principles of methylation establishment in vegetative grown plants remain unclear  <cit> .
the aim of this study was to illuminate the timing of the transgene inactivation process and to summarize our strategy for an optimized selection of n. attenuata plants with desired, stable phenotypes in a set of antimicrobial peptide expressing lines.
since a combination of tgs and ptgs effects can lead to a progressive shutdown of transgene expression  <cit> , we were interested in finding early indicators and methods to avoid or even predict unwanted transgene silencing in the wild tobacco n. attenuata.
RESULTS
non-mendelian segregation of the resistance marker as the first indicator of transgene silencing
to be able to work with n. attenuata lines that constitutively express antimicrobial peptides under a 35s promoter, we created eleven different transformation vectors containing eleven different antimicrobial peptide genes .
from each construct more than  <dig> independent transformed plant lines were created and in total the segregation data of  <dig> plant lines were observed over three generations of inbreeding.
for a high probability in selecting stable expressing plant lines, we used the optimized screening protocol described in gase et al.
<cit> .
this includes the use of flow cytometry , diagnostic pcrs , qrt-pcr  and southern blotting .
the segregation analysis of the resistant marker provides not only information about zygosity but can additionally reveal independent segregating loci and the occurrence of unwanted transgene silencing very early in the screening process.
a regenerated t <dig> plant should ideally harbor only one t-dna copy in a single locus, which is usually inherited as a simple, dominant mendelian trait.
according to mendel’s law of independent assortment  <cit> , offspring derived from self-pollination would show an expected 3: <dig> ratio, with 25% of the seedlings sensitive to hygromycin.
from our segregation data of  <dig> independently transformed n. attenuata lines most of the seedlings showed hygromycin sensitivity in the calculated range .
we considered all seedlings with 10–50% sensitivity as being offspring from a hemizygous mother plant and selected only these for further inbreeding .
epigenetic mechanisms could lead to deviations from mendelian segregation ratios and all seedlings with unusual high numbers of sensitivity  were therefore excluded from further screening  <cit> .
in the second generation , we usually seek seedlings with 0% sensitivity, indicating that they originated from a homozygous plant .
as a selection criterion, all sibling plants of the same line should not deviate from any of the expected ratios and show also 0% or 10–50% sensitivity.
the occurrence of a single plant with non-mendelian segregation  would lead to an exclusion of the entire transgenic line.
in the t <dig> generation, the progenies from the homozygous plants were again tested for stability and any newly occurring segregation led to the exclusion of the line .
the majority of seedlings of the more than  <dig> analyzed plants segregated within the expected ranges, nevertheless  <dig> of  <dig> lines  were excluded in the t <dig> stage,  <dig> of  <dig>  in the t <dig> stage and  <dig> of  <dig>  in the t <dig> stage, due to non-mendelian segregation patterns.
altogether 43% of the antimicrobial peptide expressing n. attenuata lines were excluded for this reason.
the t <dig> seedlings from three independent lines  indicated nearly a complete loss of resistance, with sensitivity rates comparable to wild-type seedlings.
because of this drastic and uniform switch within only one plant generation, these three lines provided the opportunity to further investigate the otherwise unpredictable occurrence of gene silencing.
variability in transgene expression precedes loss of resistance
to select appropriate transgenic lines with high levels of transgene expression, we routinely analyze homozygous t <dig> plants by qrt-pcr during the screening process.
as an example, we show the transgene expression profiles for three antimicrobial peptides  in  <dig> independently transformed n. attenuata lines .
several plant lines showed the desired high and uniform levels of gene expression, whereas others showed low or variable gene expression levels among independently transformed lines expressing the same constructs .
the offspring of these plants was tested on hygromycin containing media, to confirm enduring resistance within the t <dig> seedlings .
of particular interest were lines ice  <dig> , pna  <dig>  and pna  <dig> , because they nearly completely lost hygromycin resistance in the t <dig> generation and before this, showed even variable expression of the antimicrobial peptide genes in the t <dig> .
to analyze how much a complete loss of resistance correlates with the downregulation of the neighboring transgene, we compared expression levels in both generations  from lines pna  <dig> , pna  <dig>  and ice  <dig> .
to ensure similar growth conditions, the germination was performed on hygromycin-free media and lines showing stable transgene expression  were included as positive controls.
the expression analysis again revealed a very high plant-to-plant variability in the t <dig> stage , but also a very strong down-regulation of gene expression in the t <dig> generation.
this was consistent with the observed loss of hygromycin resistance .
comparing the t <dig> and t <dig> stage, plants of line ice  <dig> .
<dig> showed a  <dig> fold , lines pna  <dig> .
<dig> a  <dig> fold  and lines pna  <dig> .
<dig> a  <dig> fold  reduced transgene expression, respectively .
compared to the stable expressing control lines , the results of the transgene silencing were much more apparent and line ice  <dig> .
<dig> showed  <dig> fold , line pna  <dig> .
<dig>  <dig> fold  and pna  <dig> .
<dig>  <dig> fold  lower gene expression levels, respectively .
in summary, transgenic lines that indicated a loss of hygromycin resistance had an at least  <dig> fold lower transgene expression, compared to a stable lines expressing the same constructs.
multiple t-dna insertions in silenced lines
several reports describe a correlation between the incidence of unwanted gene silencing and high transgene copy number, making the selection of single copy t-dna insertions by southern blotting a very important part of the screening process  <cit> .
the southern blot analysis of the silencing affected lines ice  <dig> .
<dig>  pna  <dig> .
<dig> and pna  <dig> .
<dig> indicated in the xbai digest no evidence for abnormalities, but the digest with ecorv indicated two t-dna insertions for all three lines .
unusually, the second t-dna fragment showed nearly the same size in all three independently transformed lines.
since the fragment size resembles the size of the entire transgenic cassette from left to right border  this indicates the integration of two t-dna copies adjacent to each other, which could be responsible for the observed transgene silencing in these lines.
however, multiple t-dna copies at two independent loci can be also identified much earlier in the screening process by their unusual segregation rate .
in our dataset, only a very small portion of lines  showed a segregation rate around  <dig> % in the t <dig> stage and were considered as harboring transgenes at two independent loci .
this enables an early exclusion of these lines from the further screening process.
the southern blot indicated for most of the analyzed lines only single t-dna insertions, including the stable control lines  .
sensitive seedlings showed increased nos promoter methylation
unwanted or unintended transgene silencing was commonly associated with an increase in methylation within the promoter region of the transgene  <cit> .
since we found evidence for epigenetic gene silencing , we analyzed promoter methylation levels in the transgenic cassette by bisulfite sequencing.
seedlings from line ice  <dig>  showed a transitional loss of hygromycin resistance and we separated hygromycin sensitive  and hygromycin resistant  seedlings to compare nos promoter methylation levels within a 294 bp fragment .
among these isogenic seedlings, the resistant phenotypes were consistent with the methylation levels and sensitive seedlings had increased methylation levels, particular in the chg and chh sites .
interestingly, the ctg at the 84th position  was entirely methylation free in resistant seedlings, but to 100% methylated in sensitive seedlings .
since this site is located directly downstream of a ccaat box  <cit>  it appears to be particularly important for the transcription process.
hypermethylation of the 35s promoter
for the methylation analysis of the 35s promoter, individual reverse primers were designed for the two different expression cassettes  which allowed amplification of nearly the entire 35s promoter sequence .
within the 346 bp fragment a total of  <dig> cg,  <dig> chg and  <dig> chh sites were found as potential targets for methylation.
to allow the direct comparison of promoter methylation differences in  t <dig> and  t <dig> seedlings, all seeds were germinated on hygromycin-free media.
the analysis of line pna  <dig>  indicated in the t <dig> stage seedlings methylation levels of  <dig> %  cg and  <dig> %  chg methylation, respectively .
in comparison, the methylation rates of t <dig> stage seedlings  were more than  <dig> fold increased with  <dig> %  cg and  <dig> %  chg methylation.
at the asymmetric sites, the chh methylations levels were  <dig> fold increased from  <dig> %  in t <dig> to  <dig> %  in t <dig> seedlings.
the clearly increased levels of 35s promoter methylation were consistent with the observed loss of gene expression in this generation .
de novo cytosine methylation is only acquired during vegetative growth
to trace methylation changes of the 35s promoter at different times during plant growth, we sequentially sampled leaf material  <dig>   <dig> and 60 days post germination  .
the three silencing affected lines  showed in both generations  much higher 35s promoter methylation rates compared to the control lines  .
the line pna  <dig> .
<dig> indicated the lowest methylation levels and showed throughout the sampling period over both generations mean rates of only  <dig> %  in cg,  <dig> %  in chg and  <dig> %  in chh methylation .
these extremely low methylation levels indicate a complete dna conversion during the bisulfite treatment and therefore a negligible false positive signal due to incomplete conversion.
the second control was investigated until the t <dig> generation  and showed in all three generations consistent low rates of 35s promoter methylation .
these two stable expressing lines indicated no tendency for an increase in 35s promoter methylation after a generational change or during vegetative growth .
in contrast, the unstable lines ice  <dig> , pna  <dig>  and pna  <dig>  all showed increasing levels of 35s promoter methylation during growth .
as a consequence the methylation levels deviated strongly between seedlings and flowering plants within the same generation.
for instance, the chg methylation levels of line pna  <dig>  indicated only  <dig> %  in seedlings but  <dig> %  in flowering plants.
this resembles an absolute methylation increase during plant development of more than 77% within only 45 days.
the most rapid cytosine methylation increase was observed between seedlings and rosette-stage plants, where the cg and chg levels changed within 15 days with a velocity of more than 3% per day .
although the ice  <dig>  line started initially with higher methylation levels in seedling stage, it followed a similar trend and all three independent lines showed a similarly dramatic increase in methylation over time .
during the growth of t <dig> plants, the promoter methylation levels increased only slightly and reached a plateau-like level at around 90% for cg and chg sites and ca.
30% for chh sites.
surprisingly, at the generational transition low differences could be observed between the t <dig> and t <dig> plants.
the mean methylation levels of the t <dig> seedlings were highly similar to the levels found in the flowering t <dig> plants .
even the comparison of the individual frequencies at the individual positions indicated no methylation resetting or enhancement across the generational change .
it should be explicitly mentioned that the t <dig> generation seeds were not collected from exactly the same plants used here as t <dig> generation.
both seed generations had been collected beforehand and both generations were grown simultaneously adjacent to each other in the glasshouse.
regardless, the intensity of the methylation increase was highly reproducible and the patterns from both generations matched perfectly .
among all analyzed clones certain asymmetric positions were only methylated at very low frequencies.
in particular, the cytosines at the 14th and 160th position showed, for instance, 0% methylation in both generations .
we grouped the asymmetric chh sites into low, medium and high methylated positions and found that the overall methylation preference was nucleotide-specific with higher probability at certain positions  compared to others  .
these findings were similar to the site-specific preferences of asymmetric positions found in a genome-wide analysis of the epigenome in arabidopsis  <cit> .
the epigenetic status of the transgene was equally inherited by parental lines
since we commonly combine phenotypes of transgenic plants by crossing, we wanted to determine whether the heredity of a silenced transgene might be parent-of-origin-specific.
we performed reciprocal crosses between wild-type and transgenic lines and tested the hemizygous offspring for hygromycin resistance.
the crosses with the silencing affected lines  all showed high levels of hygromycin sensitivity, independent of the direction of the cross revealing equal inheritance of the silenced allele through both female and male gametes .
the crossings with the stable expressing control lines  always retained their hygromycin resistance.
although crossings had, in certain cases, the potential to reduce silencing  <cit> , we did not observe a reduction compared to plants produced from self-pollinations.
equivalent transgene inactivation in ir-lines
unwanted transgene inactivation is not restricted to sense expression lines and has been reported frequently for inverted repeat  constructs, which can also lose their in trans silencing ability  <cit> .
in the process of producing several hundred ir-lines for the targeted silencing of endogenous n. attenuata genes involved in plant defense against herbivores, we have observed several incidents of resistance marker loss in several ir-lines over the past decade.
most recently, this was observed in the t <dig> generation of the ir-acx <dig> line, which normally shows a reduced ability to accumulate jasmonic acid  after wounding due to the in trans silencing of the endogenous acx <dig> gene  <cit> .
consistently with our previous observations, the t <dig> seedlings of ir-acx <dig> also lost the ability to grow on hygromycin containing media .
to test the general applicability of a cell-culture induced transgene reactivation we included this ir-line as a candidate for the secondary regeneration process.
restored gene expression after secondary regeneration
most methods used to arrest the progress of transgene silencing include the use of cytidine analogs or viral methylation inhibitors, but these substances can cause severe growth and developmental abnormalities  <cit> .
the cell culture step of the plant transformation procedure itself has been shown to be a significant source of methylation changes  <cit>  and we evaluated whether the addition of a secondary cell culture step could interfere with the somatic acquirement of de novo methylation for the recovery of phenotypes in transformed but epigenetically silenced n. attenuata lines.
explant cultures were created from hypocotyls of transgenic homozygous t <dig> seedlings of lines pna  <dig> , ice  <dig>  and ir-acx <dig> and were called “secondary regenerants”.
the offspring of the secondary regenerants showed a large variability in hygromycin resistance.
most strikingly 41% of the regenerated plants produced offspring with full resistance to hygromycin and only 24% showed a resistance loss as seen after conventional propagation of these lines .
to test if these phenotypically “recovered” plants also were restored in the expression of the transgene, we isolated rna from rosette-stage plants.
the gene expression analysis indicated much higher gene expression levels  after the secondary regeneration, compared to conventionally propagated plants of the same line .
most of the regenerated lines now showed gene expression levels very similar to those of the stable expressing lines .
the transgene activity of the ir-acx <dig> line was not tested by gene expression analysis, but instead we determined the ability to suppress ja accumulation after simulated herbivory, as it would be performed during an experiment.
all offspring from the tested ir-acx <dig> regenerants showed suppressed ja accumulation, compared to wild-type plants .
this indicated an actively expressed ir-construct and a recovered in trans silencing ability of the endogenous acx <dig> gene after secondary regeneration.
to explore the durability of this recovery, we germinated the subsequent generation  on hygromycin containing media.
here the progression of marker gene silencing returned with the characteristic highly variable plant-to-plant pattern of hygromycin sensitivity .
lines with low or variable gene expression levels had the highest probability of losing the resistance in the subsequent generation indicating a negative correlation between strength of transgene expression and the subsequent loss of the resistance marker.
finally, at least one line from each of the pna and ice regenerants , but seven of the ir-acx <dig> regenerants  showed enduring resistance up to the t <dig> generation.
discussion
erratic occurrence of unwanted transgene silencing
this study summarizes our experience in the overall occurrence of transgene silencing during the screening of n. attenuata plants and provides guidance in identifying and avoiding unstable plant lines.
erratic occurrence and variegated phenotypes are commonly reported phenomena of transgene silencing and have been shown in many different plant species  <cit> .
this was recently illustrated for n. benthamiana plants, transformed with a 35s:gfp construct  <cit> .
these plants showed erratic and non-uniform gfp expression phenotypes, which differed strongly among isogenic sibling plants, but also among tissues from the same plant.
if no visual marker is used, as in our case, the accurate selection based on the resistance marker turns out to be extremely important.
here, the miscellaneous inactivation pattern could be found in the intermediate resistance stages of seedlings or so called “gradual silencing”  <cit> .
we frequently found intermediate resistant seedlings together with a non-mendelian distribution .
we hypothesize that the gene silencing starts in the 35s promoter and then gradually spreads into the nos promoter of the resistance marker, as discussed in mishiba et al.
<cit> .
here the advantage of a head-to-head orientation of both promoters becomes clear, as it places them in close vicinity and a loss of the resistance marker would provide an accurate harbinger of the forthcoming silencing within the expression cassette.
the following three indicators were our major criteria for the early detection of plant lines affected by unwanted gene silencing:  unusual segregation rates with >50% of sensitive seedlings,  intermediate phenotypes of seedlings with unclear levels of resistance and  large differences in gene expression among isogenic plants.
we suggest from our experience that testing the subsequent generations for resistance would be the easiest way to ensure stable transgene expression in n. attenuata.
it is generally advisable to keep the number of generations as small as possible in transgenic plants, since with each new generation the probability of silencing increases.
these selection criteria are independent of the mechanism responsible for the transgene silencing process, whether it be by tgs or ptgs  <cit> .
as long as the selected plant lines show uniform levels of gene expression and mendelian pattern of inheritance for the resistance marker, they could be considered as “stable” and used for further experiments .
sense transgene silencing in nicotiana attenuata
the intensity of transgene silencing can vary greatly among different plant species.
in transgenic gentian plants  the 35s enhancer sequence showed progressive methylation, independently of copy number and position of insertion  <cit> .
all tested gentian lines showed strong de novo methylation, whereas the same construct was methylated with much lower rates in n. tabacum <cit> .
even among closely related nicotiana species the spontaneous silencing of a transgene was associated with higher methylation levels in n. benthamiana than in n. tabacum <cit> .
similar observations were made with unstable transgene expression in n. plumbaginifolia <cit> .
these reports are consistent with the hypothesis of a more rigorous gene silencing machinery in wild diploid plant species, than in the cultivated tetraploid crop.
gene silencing by dna methylation is a natural defense mechanism against viruses, transposons and other form of “invasive elements”  <cit> .
plants have a more complex and sophisticated gene silencing apparatus than animals do and make use of cytosine methylation at multiple sites in combination with histone modifications and harbor a vast variety of small rnas  <cit> .
plants even have a signal transmission pathway for small rnas, which can act as mobile signals to direct rddm systemically  <cit> .
the very active systemic spreading of the silencing signal through the phloem was first observed in the solanaceous plants, tobacco and tomato  <cit>  and later demonstrated also for arabidopsis  <cit> .
from our initial  <dig> independent sense expression n. attenuata lines we omitted 43% after three generations due to indications of gene silencing.
n. attenuata has a highly sophisticated suite of defenses against herbivores  <cit>  and it might be, that this plant also has an active methylation apparatus to protect its genome against genetic manipulations  <cit> , which michael wassenegger once aptly called a “gene silencing-based resistance against transgene overexpression”  <cit> .
factors influencing transgene silencing – the gene dosage effect
factors which have often been shown to increase the probability of transgene silencing are the transgene copy number and the strength of expression  <cit> .
in addition, t-dna rearrangements, read-through transcripts or improperly terminated or non-polyadenylated mrna are also associated with transgene silencing  <cit> .
certainly position effects and integration into heterochromatin have been frequently reported in association with local gene silencing  <cit> , but integrations into euchromatin can be similarly silenced and more recent studies suggest that overall, the insertion position plays only a minor role  <cit> .
strong viral promoters, such as the 35s cauliflower mosaic virus promoter, were thought to produce “aberrant rna” after exceeding a certain threshold of expression  <cit> .
the progressive methylation of the 35s promoter and the observed downregulation of the transgene in the lines ice  <dig> , pna  <dig>  and pna  <dig>  might be mainly induced by the presence of two t-dna copies in close proximity to each other .
these complex insertions at a single locus can trigger transgene silencing as shown in earlier studies  <cit> .
any form of repeated t-dna arrangement appears to increase the overall silencing probability  <cit> .
but despite the intensity, the methylation increase occurred relatively late in these lines and the loss of the resistance marker was not revealed until the t <dig> generation .
we hypothesize that the expression of the two t-dna copies remains below a threshold level when plants are hemizygous.
once homozygous in the t <dig>  these thresholds are exceeded and the sum of the four t-dna copies likely initiate the silencing process.
this scenario would explain why also all hemizygous t <dig> sibling plants of these lines were inconspicuous and showed no abnormal segregation.
the attempt to reactivate a silenced transgene by crossing with wild-type plants, to create hemizygous offspring with reduced t-dna copy number, was partly successful in arabidopsis and petunia  <cit> .
in our case the sensitivity in crossings did not differ from self-pollination, probably because the methylation levels had already accumulated past the silencing threshold in flowering t <dig> plants .
similar as reported for n. tabacum hybrids, we found no evidence of a specifically maternal or paternal contribution to the inactivation process  <cit> .
further monitoring of the crosses could be still interesting if after ongoing propagation, demethylation might occur, as has been seen in other backcrosses with wild-type plants  <cit> .
successive increase of de novo methylation during development
usually, epigenetic modifications were considered to be stable in somatic cells and during normal plant development  <cit> .
most substantial epigenetic changes have been reported during gamete formation and embryogenesis in plants  <cit> .
progressive demethylation events that could be observed in endosperm tissue were interpreted as a way to reinforce transposon methylation in the embryo  <cit> .
since transgene silencing has been often described as a sudden switch of the phenotype between plant generations, a similar mechanism might have been responsible for enhancing transgene methylation during the reproductive phase.
our observation of a high variability in rosette-stage plants  lead to the hypothesis that epigenetic changes might start already early during vegetative growth and increase with different velocities amongst individual plants.
other studies suggested a somatic inactivation as well, pointing to evidence of diminishing expression of a reporter gene during development  <cit> .
however, in these studies, methylation levels were not analyzed in different stages of plant development.
our methylation kinetic showed a strong somatic increase during growth, but nearly no changes between the generations, resembling a continuous inheritance of the methylation status to the offspring .
the recent model of a methylation reinforcement during the reproductive stage, as seen for transposons  <cit> , seems to be not applicable to the de novo methylation of transgenes.
successive analysis of methylation changes have largely been restricted to tissue cultures or micropropagated plants  <cit> .
in a long-term callus cultures of pearl millet , a gradual decrease in gus activity could be associated with increased methylation levels, 18 month after transformation  <cit> .
in potato, a successive increase of gene silencing could be shown during a 5 year period of vegetative propagation  <cit> .
in contrast, we found within only 15 days of normal plant development an absolute increase of 50% in total cg methylation.
developmental methylation increases reported in flax and arabidopsis were only observed after treatment with dna demethylating agents and therefore more a remethylation to the former status  <cit> .
likewise, the demethylated genome of arabidopsis ddm <dig> mutants showed remethylation after complementation with the wild-type allele  <cit> .
however, it required multiple generations to reach approximately wild-type levels and methylation changes in different plant stages were not compared.
a plant stage dependent transgene expression is particular problematic if certain phenotypes  can be only observed in late developmental stages.
for ecological field experiments in which plant fitness measurements play a central role, it is important to ensure transgene functionality over the entire plant life during a field season.
indeed, the strong transgene silencing effects we saw in our lines can be the result of an orchestrated combination of different transcriptional and posttranscriptional effects, which together contribute to the downregulation of the transgene.
since gene expression levels might not be comparable among different  plant stages, the survey of the cytosine methylation levels was the more appropriate method to visualize changes during development.
comparable analysis of the timing of gene silencing in chicken cells indicated that histone hypoacetylation and transcriptional shutdown occurs even before the promoter shows hypermethylation  <cit> .
inhibition of transgene silencing
cytidine analogs and methytransferase blockers are commonly used treatments to prevent gene silencing in cell cultures.
these chemicals can inhibit the transgene methylation process and have been successfully applied in plant, as well as in animal cell cultures  <cit> .
however, a treatment of cell cultures differs substantially from that of an intact organism.
the stable co-expression of silencing inhibitors in n. benthamiana and n. tabacum plants resulted in much higher transgene expression levels, but both plant species suffered from abnormal growth and altered leaf morphologies, which would invalidate their use in ecological experiments  <cit> .
although plants are surprisingly able to tolerate even mutations in genes of the dna methylation pathway , the knockdown of the expression of these genes leads to the accumulation of developmental abnormalities  <cit> .
the gene silencing machinery is an important part of the gene regulatory mechanism and their disturbance has global negative effects on development  <cit> .
to date, there is no nuanced method available of selectively recovering only a single silenced transgene without causing substantial collateral damage to genome-wide methylation patterns.
reactivation of transgene expression through cell culture to rescue phenotypes for ecological research
the cell culture step of the plant transformation process is a common source of unintended side effects  <cit> .
the somaclonal variations that result from the de- and re-differentiation steps of cell culturing can be of genetic or epigenetic origin.
since dna methylation patterns were highly variable among regenerated plants, an altered dna methylation machinery during cell culture conditions had been suggested  <cit> .
most studies in different plant species found a genome-wide trend towards hypomethylation after a tissue culture step with even the possibility of restoring the activity of a former deactivated transgene  <cit> .
recently, an epigenome analyses in rice  revealed the details of the genome-wide loss of dna methylation after regeneration  <cit> .
we demonstrated for transgenic n. attenuata plants, that a secondary callus regeneration step could be used to recover transgene expression in the offspring of the regenerated plants.
in this way, the desired gene expression levels could be achieved, even from plants with progressively silenced constructs .
however, the transgene was re-silenced within most of the regenerants after two generations, highlighting the temporary character of the recovery.
regardless, the onset of gene silencing was successfully deferred for one generation with plants that produce many seeds, which provides a long-term source of material for further experiments.
similar attempts in gentian plants failed and the gene suppression persisted, probably because already silenced leaf tissue was used for the secondary regeneration  <cit> .
therefore we used hypocotyl tissue of t <dig> seedlings, which were still resistant and indicated a relative low methylation rate .
we hypothesize, that a cell culture induced transgene recovery mainly functions by interfering with the somatic de novo methylation process, rather than actively demethylating a transgene.
the offspring of the regenerants were phenotypically normal, making this method suitable for ecological research.
CONCLUSIONS
there is considerable interest in the creation of transformed plant lines with stable and heritable phenotypes, but the dynamics of epigenetic mechanisms during plant development can lead to gradual changes within a single generation and “transgene half-life” could compromise long-term experiments.
overall, the regulation of cytosine methylation in vegetative tissue seems be more dynamic than previously thought.
unlike in animals in which the germline is sequestered, plants develop germ cells directly from somatic cells relative late in their life cycle.
any vegetative acquired change of the genome could therefore be potentially submitted to the offspring, giving plants the potential to flexibly adapt to a rapid changing environment  <cit> .
apparently epigenetic processes can play a much greater role in driving plant evolution than previously thought  <cit> .

 transgenic plants have become an essential component in ecological research, allowing the precise study of gene functions under field conditions. despite progress in the development of more efficient transformation techniques, the unpredictable and stochastic occurrence of transgene silencing and epigenetic alternations after the tissue culture step remain unsolved problems for most plant species. basically two forms of gene silencing have been described, transcriptional gene silencing, in which gene expression is directly blocked, and posttranscriptional gene silencing in which mrna is degraded. ptgs has been exploited as a very powerful tool for reverse genetic studies and is revolutionizing plant ecology, particularly for non-model plants, where the introduction of “silencing-constructs” in self-compatible inverted repeat or antisense orientations enables the targeted silencing of endogenous genes in trans. unfortunately, this process can be undermined by unwanted tgs, if the promoter of the transgene is de novo methylated, thereby diminishing the expression of the silencing-construct. de novo dna methylation can be highly sequence-specific for a transgene, as a result of the process called rna-directed dna methylation. however, the pattern of establishment and prerequisites for the methylation process remain elusive. characteristic symptoms of unwanted transgene silencing are spatially variegated or transient gene expression levels, patterns which have been observed in several different plant taxa including nicotiana tabacum, petunia hybrida, arabidopsis thaliana, gentiana triflora x g. scabra and even in some transgenic woody plants such as grapevine and birch trees. the wild tobacco is an annual plant, native to the great basin desert in the western united states and is used as a model organism to study traits important for survival under real world conditions, in particular the role of jasmonic acid in plant defense against herbivores. n. attenuata has been frequently transformed with many different sense-expression, inverted repeat or antisense silencing-constructs to manipulate different layers of plant defense for field studies of gene function. a stably transformed plant is only useful for ecological experiments if the transgene-altered phenotype remains stable over the entire period of plant development. in the glasshouse the life cycle of n. attenuata takes about 70–80 days until the plant produces seeds and develops from a vegetative rosette-stage, through stalk elongation, into the generative flowering phase. over the course of development the plant reconfigures its defense strategy from largely inducible to constitutive deployment of various jasmonate-mediated chemical defenses. transgenerational phenotypic stability is also essential if different lines are to be crossed to combine traits so that parental phenotypes can be faithfully transmitted in a hemizygous state to the subsequent hybrid generations. the n. attenuata line ir-acx was created to suppress a particular step in the ja biosynthesis pathway due to the silencing of the endogenous acetyl-coa-transferase, but as recently shown the ability to suppress ja accumulation was lost when t generation plants were used during a field experiment. similar findings of leaky or lost phenotypes in n. attenuata lines have been reported in other studies highlighting the importance of the early detection of “unstable” plant lines. the methylated form of cytosine was discovered more than 60 years ago, but despite the very high amounts found in wheat seedlings, it was long considered only as a “minor base” in plant genomes. its importance in epigenetic gene regulation is increasingly being recognized, but the overall process remains poorly understood. if a genomic sequence functions as a promoter, de novo methylation can lead to transcriptional silencing of the downstream gene. cytosine methylation plays an important role in many cellular processes such as tissue-specific gene expression, embryogenesis or genomic imprinting. nevertheless, its generally accepted main function in plants is in the control of “invasive elements” such as transposons or viral sequences. in contrast to mammals, plants not only methylate cytosines in cg dinucleotides, but also in all other possible sequence contexts at chg and chh positions. the symmetric dna methylation patterns at cg and chg sites can be sustained during semiconservative dna replication and are therefore somatically and even meiotically stable. a methylation at the chh position is called asymmetric, because it has no mirror position on the complementary dna strand and hence will be lost during the dna replication process. for maintenance during mitosis, an asymmetric site needs therefore a constant signal as a permanent de novo methylation trigger. although most aspects of epigenetic inheritance are understood, somatic cells are considered to be relative static and the principles of methylation establishment in vegetative grown plants remain unclear. the aim of this study was to illuminate the timing of the transgene inactivation process and to summarize our strategy for an optimized selection of n. attenuata plants with desired, stable phenotypes in a set of antimicrobial peptide expressing lines. since a combination of tgs and ptgs effects can lead to a progressive shutdown of transgene expression, we were interested in finding early indicators and methods to avoid or even predict unwanted transgene silencing in the wild tobacco n. attenuata.  non-mendelian segregation of the resistance marker as the first indicator of transgene silencing to be able to work with n. attenuata lines that constitutively express antimicrobial peptides under a 35s promoter, we created eleven different transformation vectors containing eleven different antimicrobial peptide genes. from each construct more than independent transformed plant lines were created and in total the segregation data of plant lines were observed over three generations of inbreeding. for a high probability in selecting stable expressing plant lines, we used the optimized screening protocol described in gase et al.. this includes the use of flow cytometry, diagnostic pcrs, qrt-pcr and southern blotting. the segregation analysis of the resistant marker provides not only information about zygosity but can additionally reveal independent segregating loci and the occurrence of unwanted transgene silencing very early in the screening process. a regenerated t plant should ideally harbor only one t-dna copy in a single locus, which is usually inherited as a simple, dominant mendelian trait. according to mendel’s law of independent assortment, offspring derived from self-pollination would show an expected 3: ratio, with 25% of the seedlings sensitive to hygromycin. from our segregation data of independently transformed n. attenuata lines most of the seedlings showed hygromycin sensitivity in the calculated range. we considered all seedlings with 10–50% sensitivity as being offspring from a hemizygous mother plant and selected only these for further inbreeding. epigenetic mechanisms could lead to deviations from mendelian segregation ratios and all seedlings with unusual high numbers of sensitivity were therefore excluded from further screening. in the second generation, we usually seek seedlings with 0% sensitivity, indicating that they originated from a homozygous plant. as a selection criterion, all sibling plants of the same line should not deviate from any of the expected ratios and show also 0% or 10–50% sensitivity. the occurrence of a single plant with non-mendelian segregation would lead to an exclusion of the entire transgenic line. in the t generation, the progenies from the homozygous plants were again tested for stability and any newly occurring segregation led to the exclusion of the line. the majority of seedlings of the more than analyzed plants segregated within the expected ranges, nevertheless of lines were excluded in the t stage, of in the t stage and of in the t stage, due to non-mendelian segregation patterns. altogether 43% of the antimicrobial peptide expressing n. attenuata lines were excluded for this reason. the t seedlings from three independent lines indicated nearly a complete loss of resistance, with sensitivity rates comparable to wild-type seedlings. because of this drastic and uniform switch within only one plant generation, these three lines provided the opportunity to further investigate the otherwise unpredictable occurrence of gene silencing. variability in transgene expression precedes loss of resistance to select appropriate transgenic lines with high levels of transgene expression, we routinely analyze homozygous t plants by qrt-pcr during the screening process. as an example, we show the transgene expression profiles for three antimicrobial peptides in independently transformed n. attenuata lines. several plant lines showed the desired high and uniform levels of gene expression, whereas others showed low or variable gene expression levels among independently transformed lines expressing the same constructs. the offspring of these plants was tested on hygromycin containing media, to confirm enduring resistance within the t seedlings. of particular interest were lines ice, pna and pna, because they nearly completely lost hygromycin resistance in the t generation and before this, showed even variable expression of the antimicrobial peptide genes in the t. to analyze how much a complete loss of resistance correlates with the downregulation of the neighboring transgene, we compared expression levels in both generations from lines pna, pna and ice. to ensure similar growth conditions, the germination was performed on hygromycin-free media and lines showing stable transgene expression were included as positive controls. the expression analysis again revealed a very high plant-to-plant variability in the t stage, but also a very strong down-regulation of gene expression in the t generation. this was consistent with the observed loss of hygromycin resistance. comparing the t and t stage, plants of line ice. showed a fold, lines pna. a fold and lines pna. a fold reduced transgene expression, respectively. compared to the stable expressing control lines, the results of the transgene silencing were much more apparent and line ice. showed fold, line pna. fold and pna. fold lower gene expression levels, respectively. in summary, transgenic lines that indicated a loss of hygromycin resistance had an at least fold lower transgene expression, compared to a stable lines expressing the same constructs. multiple t-dna insertions in silenced lines several reports describe a correlation between the incidence of unwanted gene silencing and high transgene copy number, making the selection of single copy t-dna insertions by southern blotting a very important part of the screening process. the southern blot analysis of the silencing affected lines ice. pna. and pna. indicated in the xbai digest no evidence for abnormalities, but the digest with ecorv indicated two t-dna insertions for all three lines. unusually, the second t-dna fragment showed nearly the same size in all three independently transformed lines. since the fragment size resembles the size of the entire transgenic cassette from left to right border this indicates the integration of two t-dna copies adjacent to each other, which could be responsible for the observed transgene silencing in these lines. however, multiple t-dna copies at two independent loci can be also identified much earlier in the screening process by their unusual segregation rate. in our dataset, only a very small portion of lines showed a segregation rate around % in the t stage and were considered as harboring transgenes at two independent loci. this enables an early exclusion of these lines from the further screening process. the southern blot indicated for most of the analyzed lines only single t-dna insertions, including the stable control lines. sensitive seedlings showed increased nos promoter methylation unwanted or unintended transgene silencing was commonly associated with an increase in methylation within the promoter region of the transgene. since we found evidence for epigenetic gene silencing, we analyzed promoter methylation levels in the transgenic cassette by bisulfite sequencing. seedlings from line ice showed a transitional loss of hygromycin resistance and we separated hygromycin sensitive and hygromycin resistant seedlings to compare nos promoter methylation levels within a 294 bp fragment. among these isogenic seedlings, the resistant phenotypes were consistent with the methylation levels and sensitive seedlings had increased methylation levels, particular in the chg and chh sites. interestingly, the ctg at the 84th position was entirely methylation free in resistant seedlings, but to 100% methylated in sensitive seedlings. since this site is located directly downstream of a ccaat box it appears to be particularly important for the transcription process. hypermethylation of the 35s promoter for the methylation analysis of the 35s promoter, individual reverse primers were designed for the two different expression cassettes which allowed amplification of nearly the entire 35s promoter sequence. within the 346 bp fragment a total of cg, chg and chh sites were found as potential targets for methylation. to allow the direct comparison of promoter methylation differences in t and t seedlings, all seeds were germinated on hygromycin-free media. the analysis of line pna indicated in the t stage seedlings methylation levels of % cg and % chg methylation, respectively. in comparison, the methylation rates of t stage seedlings were more than fold increased with % cg and % chg methylation. at the asymmetric sites, the chh methylations levels were fold increased from % in t to % in t seedlings. the clearly increased levels of 35s promoter methylation were consistent with the observed loss of gene expression in this generation. de novo cytosine methylation is only acquired during vegetative growth to trace methylation changes of the 35s promoter at different times during plant growth, we sequentially sampled leaf material and 60 days post germination. the three silencing affected lines showed in both generations much higher 35s promoter methylation rates compared to the control lines. the line pna. indicated the lowest methylation levels and showed throughout the sampling period over both generations mean rates of only % in cg, % in chg and % in chh methylation. these extremely low methylation levels indicate a complete dna conversion during the bisulfite treatment and therefore a negligible false positive signal due to incomplete conversion. the second control was investigated until the t generation and showed in all three generations consistent low rates of 35s promoter methylation. these two stable expressing lines indicated no tendency for an increase in 35s promoter methylation after a generational change or during vegetative growth. in contrast, the unstable lines ice, pna and pna all showed increasing levels of 35s promoter methylation during growth. as a consequence the methylation levels deviated strongly between seedlings and flowering plants within the same generation. for instance, the chg methylation levels of line pna indicated only % in seedlings but % in flowering plants. this resembles an absolute methylation increase during plant development of more than 77% within only 45 days. the most rapid cytosine methylation increase was observed between seedlings and rosette-stage plants, where the cg and chg levels changed within 15 days with a velocity of more than 3% per day. although the ice line started initially with higher methylation levels in seedling stage, it followed a similar trend and all three independent lines showed a similarly dramatic increase in methylation over time. during the growth of t plants, the promoter methylation levels increased only slightly and reached a plateau-like level at around 90% for cg and chg sites and ca. 30% for chh sites. surprisingly, at the generational transition low differences could be observed between the t and t plants. the mean methylation levels of the t seedlings were highly similar to the levels found in the flowering t plants. even the comparison of the individual frequencies at the individual positions indicated no methylation resetting or enhancement across the generational change. it should be explicitly mentioned that the t generation seeds were not collected from exactly the same plants used here as t generation. both seed generations had been collected beforehand and both generations were grown simultaneously adjacent to each other in the glasshouse. regardless, the intensity of the methylation increase was highly reproducible and the patterns from both generations matched perfectly. among all analyzed clones certain asymmetric positions were only methylated at very low frequencies. in particular, the cytosines at the 14th and 160th position showed, for instance, 0% methylation in both generations. we grouped the asymmetric chh sites into low, medium and high methylated positions and found that the overall methylation preference was nucleotide-specific with higher probability at certain positions compared to others. these findings were similar to the site-specific preferences of asymmetric positions found in a genome-wide analysis of the epigenome in arabidopsis. the epigenetic status of the transgene was equally inherited by parental lines since we commonly combine phenotypes of transgenic plants by crossing, we wanted to determine whether the heredity of a silenced transgene might be parent-of-origin-specific. we performed reciprocal crosses between wild-type and transgenic lines and tested the hemizygous offspring for hygromycin resistance. the crosses with the silencing affected lines all showed high levels of hygromycin sensitivity, independent of the direction of the cross revealing equal inheritance of the silenced allele through both female and male gametes. the crossings with the stable expressing control lines always retained their hygromycin resistance. although crossings had, in certain cases, the potential to reduce silencing, we did not observe a reduction compared to plants produced from self-pollinations. equivalent transgene inactivation in ir-lines unwanted transgene inactivation is not restricted to sense expression lines and has been reported frequently for inverted repeat constructs, which can also lose their in trans silencing ability. in the process of producing several hundred ir-lines for the targeted silencing of endogenous n. attenuata genes involved in plant defense against herbivores, we have observed several incidents of resistance marker loss in several ir-lines over the past decade. most recently, this was observed in the t generation of the ir-acx line, which normally shows a reduced ability to accumulate jasmonic acid after wounding due to the in trans silencing of the endogenous acx gene. consistently with our previous observations, the t seedlings of ir-acx also lost the ability to grow on hygromycin containing media. to test the general applicability of a cell-culture induced transgene reactivation we included this ir-line as a candidate for the secondary regeneration process. restored gene expression after secondary regeneration most methods used to arrest the progress of transgene silencing include the use of cytidine analogs or viral methylation inhibitors, but these substances can cause severe growth and developmental abnormalities. the cell culture step of the plant transformation procedure itself has been shown to be a significant source of methylation changes and we evaluated whether the addition of a secondary cell culture step could interfere with the somatic acquirement of de novo methylation for the recovery of phenotypes in transformed but epigenetically silenced n. attenuata lines. explant cultures were created from hypocotyls of transgenic homozygous t seedlings of lines pna, ice and ir-acx and were called “secondary regenerants”. the offspring of the secondary regenerants showed a large variability in hygromycin resistance. most strikingly 41% of the regenerated plants produced offspring with full resistance to hygromycin and only 24% showed a resistance loss as seen after conventional propagation of these lines. to test if these phenotypically “recovered” plants also were restored in the expression of the transgene, we isolated rna from rosette-stage plants. the gene expression analysis indicated much higher gene expression levels after the secondary regeneration, compared to conventionally propagated plants of the same line. most of the regenerated lines now showed gene expression levels very similar to those of the stable expressing lines. the transgene activity of the ir-acx line was not tested by gene expression analysis, but instead we determined the ability to suppress ja accumulation after simulated herbivory, as it would be performed during an experiment. all offspring from the tested ir-acx regenerants showed suppressed ja accumulation, compared to wild-type plants. this indicated an actively expressed ir-construct and a recovered in trans silencing ability of the endogenous acx gene after secondary regeneration. to explore the durability of this recovery, we germinated the subsequent generation on hygromycin containing media. here the progression of marker gene silencing returned with the characteristic highly variable plant-to-plant pattern of hygromycin sensitivity. lines with low or variable gene expression levels had the highest probability of losing the resistance in the subsequent generation indicating a negative correlation between strength of transgene expression and the subsequent loss of the resistance marker. finally, at least one line from each of the pna and ice regenerants, but seven of the ir-acx regenerants showed enduring resistance up to the t generation. discussion erratic occurrence of unwanted transgene silencing this study summarizes our experience in the overall occurrence of transgene silencing during the screening of n. attenuata plants and provides guidance in identifying and avoiding unstable plant lines. erratic occurrence and variegated phenotypes are commonly reported phenomena of transgene silencing and have been shown in many different plant species. this was recently illustrated for n. benthamiana plants, transformed with a 35s:gfp construct. these plants showed erratic and non-uniform gfp expression phenotypes, which differed strongly among isogenic sibling plants, but also among tissues from the same plant. if no visual marker is used, as in our case, the accurate selection based on the resistance marker turns out to be extremely important. here, the miscellaneous inactivation pattern could be found in the intermediate resistance stages of seedlings or so called “gradual silencing”. we frequently found intermediate resistant seedlings together with a non-mendelian distribution. we hypothesize that the gene silencing starts in the 35s promoter and then gradually spreads into the nos promoter of the resistance marker, as discussed in mishiba et al.. here the advantage of a head-to-head orientation of both promoters becomes clear, as it places them in close vicinity and a loss of the resistance marker would provide an accurate harbinger of the forthcoming silencing within the expression cassette. the following three indicators were our major criteria for the early detection of plant lines affected by unwanted gene silencing: unusual segregation rates with >50% of sensitive seedlings, intermediate phenotypes of seedlings with unclear levels of resistance and large differences in gene expression among isogenic plants. we suggest from our experience that testing the subsequent generations for resistance would be the easiest way to ensure stable transgene expression in n. attenuata. it is generally advisable to keep the number of generations as small as possible in transgenic plants, since with each new generation the probability of silencing increases. these selection criteria are independent of the mechanism responsible for the transgene silencing process, whether it be by tgs or ptgs. as long as the selected plant lines show uniform levels of gene expression and mendelian pattern of inheritance for the resistance marker, they could be considered as “stable” and used for further experiments. sense transgene silencing in nicotiana attenuata the intensity of transgene silencing can vary greatly among different plant species. in transgenic gentian plants the 35s enhancer sequence showed progressive methylation, independently of copy number and position of insertion. all tested gentian lines showed strong de novo methylation, whereas the same construct was methylated with much lower rates in n. tabacum. even among closely related nicotiana species the spontaneous silencing of a transgene was associated with higher methylation levels in n. benthamiana than in n. tabacum. similar observations were made with unstable transgene expression in n. plumbaginifolia. these reports are consistent with the hypothesis of a more rigorous gene silencing machinery in wild diploid plant species, than in the cultivated tetraploid crop. gene silencing by dna methylation is a natural defense mechanism against viruses, transposons and other form of “invasive elements”. plants have a more complex and sophisticated gene silencing apparatus than animals do and make use of cytosine methylation at multiple sites in combination with histone modifications and harbor a vast variety of small rnas. plants even have a signal transmission pathway for small rnas, which can act as mobile signals to direct rddm systemically. the very active systemic spreading of the silencing signal through the phloem was first observed in the solanaceous plants, tobacco and tomato and later demonstrated also for arabidopsis. from our initial independent sense expression n. attenuata lines we omitted 43% after three generations due to indications of gene silencing. n. attenuata has a highly sophisticated suite of defenses against herbivores and it might be, that this plant also has an active methylation apparatus to protect its genome against genetic manipulations, which michael wassenegger once aptly called a “gene silencing-based resistance against transgene overexpression”. factors influencing transgene silencing – the gene dosage effect factors which have often been shown to increase the probability of transgene silencing are the transgene copy number and the strength of expression. in addition, t-dna rearrangements, read-through transcripts or improperly terminated or non-polyadenylated mrna are also associated with transgene silencing. certainly position effects and integration into heterochromatin have been frequently reported in association with local gene silencing, but integrations into euchromatin can be similarly silenced and more recent studies suggest that overall, the insertion position plays only a minor role. strong viral promoters, such as the 35s cauliflower mosaic virus promoter, were thought to produce “aberrant rna” after exceeding a certain threshold of expression. the progressive methylation of the 35s promoter and the observed downregulation of the transgene in the lines ice, pna and pna might be mainly induced by the presence of two t-dna copies in close proximity to each other. these complex insertions at a single locus can trigger transgene silencing as shown in earlier studies. any form of repeated t-dna arrangement appears to increase the overall silencing probability. but despite the intensity, the methylation increase occurred relatively late in these lines and the loss of the resistance marker was not revealed until the t generation. we hypothesize that the expression of the two t-dna copies remains below a threshold level when plants are hemizygous. once homozygous in the t these thresholds are exceeded and the sum of the four t-dna copies likely initiate the silencing process. this scenario would explain why also all hemizygous t sibling plants of these lines were inconspicuous and showed no abnormal segregation. the attempt to reactivate a silenced transgene by crossing with wild-type plants, to create hemizygous offspring with reduced t-dna copy number, was partly successful in arabidopsis and petunia. in our case the sensitivity in crossings did not differ from self-pollination, probably because the methylation levels had already accumulated past the silencing threshold in flowering t plants. similar as reported for n. tabacum hybrids, we found no evidence of a specifically maternal or paternal contribution to the inactivation process. further monitoring of the crosses could be still interesting if after ongoing propagation, demethylation might occur, as has been seen in other backcrosses with wild-type plants. successive increase of de novo methylation during development usually, epigenetic modifications were considered to be stable in somatic cells and during normal plant development. most substantial epigenetic changes have been reported during gamete formation and embryogenesis in plants. progressive demethylation events that could be observed in endosperm tissue were interpreted as a way to reinforce transposon methylation in the embryo. since transgene silencing has been often described as a sudden switch of the phenotype between plant generations, a similar mechanism might have been responsible for enhancing transgene methylation during the reproductive phase. our observation of a high variability in rosette-stage plants lead to the hypothesis that epigenetic changes might start already early during vegetative growth and increase with different velocities amongst individual plants. other studies suggested a somatic inactivation as well, pointing to evidence of diminishing expression of a reporter gene during development. however, in these studies, methylation levels were not analyzed in different stages of plant development. our methylation kinetic showed a strong somatic increase during growth, but nearly no changes between the generations, resembling a continuous inheritance of the methylation status to the offspring. the recent model of a methylation reinforcement during the reproductive stage, as seen for transposons, seems to be not applicable to the de novo methylation of transgenes. successive analysis of methylation changes have largely been restricted to tissue cultures or micropropagated plants. in a long-term callus cultures of pearl millet, a gradual decrease in gus activity could be associated with increased methylation levels, 18 month after transformation. in potato, a successive increase of gene silencing could be shown during a 5 year period of vegetative propagation. in contrast, we found within only 15 days of normal plant development an absolute increase of 50% in total cg methylation. developmental methylation increases reported in flax and arabidopsis were only observed after treatment with dna demethylating agents and therefore more a remethylation to the former status. likewise, the demethylated genome of arabidopsis ddm mutants showed remethylation after complementation with the wild-type allele. however, it required multiple generations to reach approximately wild-type levels and methylation changes in different plant stages were not compared. a plant stage dependent transgene expression is particular problematic if certain phenotypes can be only observed in late developmental stages. for ecological field experiments in which plant fitness measurements play a central role, it is important to ensure transgene functionality over the entire plant life during a field season. indeed, the strong transgene silencing effects we saw in our lines can be the result of an orchestrated combination of different transcriptional and posttranscriptional effects, which together contribute to the downregulation of the transgene. since gene expression levels might not be comparable among different plant stages, the survey of the cytosine methylation levels was the more appropriate method to visualize changes during development. comparable analysis of the timing of gene silencing in chicken cells indicated that histone hypoacetylation and transcriptional shutdown occurs even before the promoter shows hypermethylation. inhibition of transgene silencing cytidine analogs and methytransferase blockers are commonly used treatments to prevent gene silencing in cell cultures. these chemicals can inhibit the transgene methylation process and have been successfully applied in plant, as well as in animal cell cultures. however, a treatment of cell cultures differs substantially from that of an intact organism. the stable co-expression of silencing inhibitors in n. benthamiana and n. tabacum plants resulted in much higher transgene expression levels, but both plant species suffered from abnormal growth and altered leaf morphologies, which would invalidate their use in ecological experiments. although plants are surprisingly able to tolerate even mutations in genes of the dna methylation pathway, the knockdown of the expression of these genes leads to the accumulation of developmental abnormalities. the gene silencing machinery is an important part of the gene regulatory mechanism and their disturbance has global negative effects on development. to date, there is no nuanced method available of selectively recovering only a single silenced transgene without causing substantial collateral damage to genome-wide methylation patterns. reactivation of transgene expression through cell culture to rescue phenotypes for ecological research the cell culture step of the plant transformation process is a common source of unintended side effects. the somaclonal variations that result from the de- and re-differentiation steps of cell culturing can be of genetic or epigenetic origin. since dna methylation patterns were highly variable among regenerated plants, an altered dna methylation machinery during cell culture conditions had been suggested. most studies in different plant species found a genome-wide trend towards hypomethylation after a tissue culture step with even the possibility of restoring the activity of a former deactivated transgene. recently, an epigenome analyses in rice revealed the details of the genome-wide loss of dna methylation after regeneration. we demonstrated for transgenic n. attenuata plants, that a secondary callus regeneration step could be used to recover transgene expression in the offspring of the regenerated plants. in this way, the desired gene expression levels could be achieved, even from plants with progressively silenced constructs. however, the transgene was re-silenced within most of the regenerants after two generations, highlighting the temporary character of the recovery. regardless, the onset of gene silencing was successfully deferred for one generation with plants that produce many seeds, which provides a long-term source of material for further experiments. similar attempts in gentian plants failed and the gene suppression persisted, probably because already silenced leaf tissue was used for the secondary regeneration. therefore we used hypocotyl tissue of t seedlings, which were still resistant and indicated a relative low methylation rate. we hypothesize, that a cell culture induced transgene recovery mainly functions by interfering with the somatic de novo methylation process, rather than actively demethylating a transgene. the offspring of the regenerants were phenotypically normal, making this method suitable for ecological research.  there is considerable interest in the creation of transformed plant lines with stable and heritable phenotypes, but the dynamics of epigenetic mechanisms during plant development can lead to gradual changes within a single generation and “transgene half-life” could compromise long-term experiments. overall, the regulation of cytosine methylation in vegetative tissue seems be more dynamic than previously thought. unlike in animals in which the germline is sequestered, plants develop germ cells directly from somatic cells relative late in their life cycle. any vegetative acquired change of the genome could therefore be potentially submitted to the offspring, giving plants the potential to flexibly adapt to a rapid changing environment. apparently epigenetic processes can play a much greater role in driving plant evolution than previously thought. 
transgenic wild tobacco plants expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. to shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35s promoter methylation during vegetative growth. we focused on three independently transformed lines as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than fold after only one plant generation. a transgene can be epigenetically silenced by a process called rna directed dna methylation, which can be seen as a heritable loss of gene expression. a secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants. a secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits. despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. the levels of de novo methylation were inherited by the offspring without any visible discontinuation. de novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. the unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines.
 genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. a transgene can be epigenetically silenced by a process called rna directed dna methylation, which can be seen as a heritable loss of gene expression. the spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear.  transgenic wild tobacco plants expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. we focused on three independently transformed lines as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than fold after only one plant generation. bisulfite sequencing indicated hypermethylation within the 35s and nos promoters of these lines. to shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35s promoter methylation during vegetative growth. the levels of de novo methylation were inherited by the offspring without any visible discontinuation. a secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants.  the unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. de novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. a secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits. 