Plant Cell Rep DOI 10.

1007/s00299-009-0704-4

GENETIC TRANSFORMATION AND HYBRIDIZATION

Unintended consequence of plant transformation: biolistic transformation caused transpositional activation of an endogenous retrotransposon Tos17 in rice ssp. japonica cv. Matsumae
R. Wu W. L. Guo X. R. Wang X. L. Wang T. T. Zhuang Jihong Liu Clarke B. Liu

Received: 9 February 2009 / Revised: 3 April 2009 / Accepted: 15 April 2009 Springer-Verlag 2009

Abstract Genetic instability could be provoked as an unintended consequence of genetic engineering in plants. Here, we report that the rice endogenous long terminal repeat (LTR) retrotransposon Tos17 was transpositionally activated only in transgenic calli and their regenerated plants produced by biolistic transformation in rice (Oryza sativa L.) ssp. japonica cv. Matsumae. Moreover, the transpositional activity of Tos17 was sustained after plant regeneration in the T0 generation, and produced new germinal insertions. In contrast, the element remained totally quiescent in calli and regenerated plants from tissue culture of this genotype. Nonetheless, transcriptional induction and cytosine demethylation of Tos17 were found to have occurred with no signicant difference in both kinds of calli, tissue culture alone and transgenic. This suggests that callus culture is likely to have played an important role in destabilizing Tos17 in the direction towards transpositional activation, but that biolistic transformation is the direct causal factor.

Keywords Genetic transformation Particle bombardment Tos17 Transpositional activity DNA methylation Rice

Introduction Plant transformation technology holds great promise in molecular plant breeding for the improvement of agronomically important traits, such as increased yield, better quality and enhanced resistance to pathogens and insects (Bukovinszki et al. 2007; Eapen 2008; Larkin and Harrigan 2007; Sinclair et al. 2004). Despite the fact that production and global cultivation of genetically modied (GM) crops are continuously increasing, with an enhancement of 12% in acreage on a global basis (James 2007), the question still remains as to whether genetic engineering approaches can be targeted precisely to the modication of the targeted trait(s) only, or whether unintended genetic and epigenetic effects can be associated with the process (Lu and Snow 2005). Although this issue is important not only with regard to the genetic stability of transgenic plants but also to their safety assessments, it has not often been studied (Filipecki and Malepszy 2006). Genetic changes at the DNA level used to be considered as the primary cause of observed phenotypic variations in transgenic plants (Frugis et al. 2001). However, epigenetic variations, which do not entail a change in the primary nucleotide sequence, but are due to modications of DNA and/or histones, may also bear de novo heritable phenotypic effects. Furthermore, some epigenetic variations like alterations in cytosine methylation may produce immediate genetic effects through, for example, silencing of the transgene and/or host gene and activation of transposable elements (TEs), enhanced mutation rate and genomic

R. Wu and W. L. Guo contributed equally to this work. Communicated by A. Atanassov. R. Wu W. L. Guo X. R. Wang X. L. Wang T. T. Zhuang B. Liu (&) Key Laboratory of Molecular Epigenetics of MOE, Institute of Genetics and Cytology, Northeast Normal University, 130024 Changchun, China e-mail: baoliu6677@yahoo.com.cn J. L. Clarke (&) Plant Health and Protection Division, Norwegian Institute for Agricultural and Environmental Research, Hoegskoleveien 7, 1432 Aas, Norway e-mail: jihong.liu-clarke@bioforsk.no

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rearrangements (Matzke and Matzke 1998; Matzke et al. 2000; Meyer and Heidmann 1994). Most of the genetic transformation protocols currently in use require an in vitro tissue culture process including callus formation and plant regeneration. The tissue culture process itself is known to be mutagenic, and may induce an array of mutations collectively known as somaclonal variation (Evans 1989). Although mechanisms underlying the somaclonal variation phenomenon remain to be fully elucidated, several studies have indicated that the disruption of epigenetic cellular controls at the callus stage is likely to be a major cause (Grandbastien 1998; Kubis et al. 2003; Tanurdzic et al. 2008). Thus, a primary type of somaclonal variation appeared to have an epigenetic basis, particularly the alteration of intrinsic cytosine DNA methylation patterns (Kaeppler et al. 2000; Tanurdzic et al. 2008; Zhang et al. 2009). Because transcriptional and transpositional activity of TEs is repressed by DNA hypermethylation (Kaeppler and Phillips 1993; Kubis et al. 2003; Tanurdzic et al. 2008), it is conceivable that their activity might be derepressed under tissue culture conditions. Indeed, several studies have shown that transpositional activation of otherwise quiescent mobile elements occurred as a result of tissue culture (Hirochika 1993; Hirochika et al. 1996; Kikuchi et al. 2003). Apart from the mutagenic effects of tissue culture, the transformation process may impose an additional stress, as both the transformation process (including transgene integration and also mechanical wounding in cases where bombardment was used) and possible regulatory effects on adjacent genes exerted by the transgene might cause epigenetic variations, such that the methylation states and expression patterns of the transgene, as well as its immediate or broader anking regions, may be affected. In fact, several animal studies have documented that random foreign gene integration into the nuclear genome may cause genome-wide epigenetic perturbations including alteration in cytosine DNA methylation and transcriptional activation of endogenous TEs (Heller et al. 1995; Muller et al. 2001). Furthermore, transgene insertion mediated by the TE Sleeping Beauty has led to both activation of the transposon and alteration in DNA methylation at the integration sites (Park et al. 2005; Ivics et al. 2007). Similar observations were reported in plants. For example, introgression of alien chromatin via sexual hybridization in rice has elicited simultaneous mobilization of several TEs, which was accompanied by alterations in their methylation states (Liu and Wendel 2000; Liu et al. 2004a; Shan et al. 2005). Moreover, the transpositional frequencies of some TEs were enhanced in transgenic plant lines relative to the tissue culture regenerants alone (Bhatt et al. 1998; Hirochika et al. 1996). However, to our knowledge, it is unknown whether the transformation process may impose

an effect that is qualitatively distinct from that of tissue culture in eliciting activities of TEs and their associated epigenetic alterations. Hirochika et al. (1996) discovered that a rice endogenous TE (a retrotransposon), Tos17, can be transpositionally activated by tissue culture in certain genotypes of rice, and this element has since become a powerful mutagen for tagging and functional analysis in rice genes (Hirochika et al. 2004; Miyao et al. 2003). We reported previously that Tos17 was transpositionally activated in a rice recombinant inbred line, RIL (RZ35) under tissue culture, derived from the japonica rice cultivar Matsumae; the element nevertheless remained immobile in tissue cultures of Matsumae itself (Liu et al. 2004b). This observation veried that transpositional activation of Tos17 by tissue culture is genotype-dependent, and raised an interesting question as to the genetic basis of its control, given the close relationship between the RIL (RZ35) and its parental cultivar Matsumae. The present study was aimed at testing the possibility of whether transpositional activity of Tos17 in rice (cv. Matsumae) can be induced by other means, and if so, what are the associated epigenetic effects? Here, we report that Tos17 can indeed be induced to transpose in Matsumae by biolistic transformation using particle bombardment. Moreover, the transpositional activity of Tos17 was maintained after plant regeneration, which differed from results of previous studies. Methylation analysis of Tos17 indicated that despite demethylation in calli being likely associated with the elements transcriptional induction, alteration in cytosine methylation was unlikely to have played a decisive role in its transpositional activation.

Materials and methods Plant material and tissue culture Seeds of rice (O. sativa L.) ssp. japonica, cv. Matsumae, were used for callus induction, transformation and plant regeneration. Calli were induced from germinating seeds of Matsumae using the medium described previously by Liu et al. (2004b). Plants were regenerated from embryogenic calli on the same medium without 2,4-D, and were grown to maturity and selfed to produce progenies. Seed plants (germinated from seeds) were used as control. Genetic transformation using particle bombardment The biolistic transformation of rice ssp. japonica, cv. Matsumae was carried out on the NB medium as described by Chen et al. (1998) with minor revisions: callus induction and subculture were performed on NB medium supplemented with 2 mg/L 2,4-D, 3% sucrose, and 0.75%

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agarose. After a number of subcultures with an interval of 30 days, calli (between 1 and 3 mm in diameter) were plated in the center of Petri dishes containing osmotic medium (NB supplemented with 0.256 M each of mannitol and sorbitol) for 4 h prior to bombardment. Particle bombardment was performed using a Bio-Rad PDS-1000/ He Biolistic Particle Delivery System (Bio-RAD, USA) to introduce the plasmid vector pCAMBIA1301 (Cambia, Australia) into the rice calli. Sixteen to twenty hours after the bombardment, calli were transferred onto pre-selection medium (NB supplemented with 30 mg/L hygromycin B), and incubated in the dark for 1417 days. Subsequently, the differentiated calli were placed on the selection medium (NB with 50 mg/L hygromycin B) for a further 1417 days. Resistant calli were then transferred to preregeneration medium (NB supplemented with 2 mg/L 6-BA, 1 mg/L NAA, 5 mg/L ABA and 50 mg/L hygromycin B) and cultured in the dark for 7 days, followed by plant regeneration on medium NB supplemented with 3 mg/L 6-BA, 0.5 mg/L NAA and 50 mg/L hygromycin B, for 1421 days under a 16 hour photoperiod until shoots appeared. Regenerated plants were subsequently transferred into a greenhouse and self-pollinated to produce progenies. Southern blot analysis Genomic DNA was extracted from leaves of individual plants (seed plants, callus-regenerated plants and transgenic plants) and calli (derived from tissue-cultured or transgenic plants) using the CTAB method as described by Rogers and Bendich (1988). To detect if the plasmid vector was integrated into the genome of Matsumae and the activation of Tos17, puried genomic DNA was digested with HindIII, and hybridized with two separate probes. Probe 1 was a 700 bp fragment of hyg B gene generated by PCR amplication with primers hyP1: 50 -GAGGCTATT CGGCTATGACTG-30 and hyP2: 50 -ATCGGGAGCGGC GATACCGTA-30 , whereas Probe 2 was a 666 bp internal fragment amplied using RTP1: 50 -GCTACCCGTTCTTG GACTAT-30 and RTP2: 50 -CTGAAATCGGAGCACTG ACA-30 from the reverse transcript (RT)/RnaseH region of Tos17. The DIG DNA Labeling Kit (Roche Diagnostics GmbH, Germany) was used for labeling the probes. To assay cytosine-methylation states at the CCGG site within Tos17, genomic DNA was digested with BstXI in combination with either HpaII or MspI, followed by hybridization utilizing the same probe (Probe 2) as described above. Digested DNA product was separated on 1% agarose gel and transferred onto Hybond-N? nylon membranes (Amersham Biosciences) by alkaline transfer. The signal was detected with DIG Luminescent Detection Kit (Roche Diagnostics GmbH, Germany).

Reverse transcriptase (RT)-PCR analysis Total RNA was isolated from calli (tissue-cultured or transgenic) and young leaves of individual plants derived from seed plants, tissue culture regenerated plants, and transgenic plants, by the Trizol Reagent (Invitrogen, California, USA) according to the manufacturers instructions. For RT-PCR, the RNA was further treated with DNase I (Invitrogen, USA) reverse transcribed by the SuperScript Rnase H- Reverse Transcriptase (Invitrogen, USA), and subjected to RT-PCR analysis using the reversetranscriptase-encoding region-specic primers that amplify a 666 bp fragment, as described above. A pair of primers (actinP1:50 -CGTCTGCGATAATGGAACTG-30 and actinP2: 50 -TCTGGGTCATCTTCTCACGA) designed to specically amplify a 341 bp fragment from cDNA or a 424 bp fragment from genomic DNA of the rice b-actin gene (Genbank accession X79378) were used as an internal control for RNA input and as a control for tracing DNA contamination. Bisulte sequencing For Tos17 bisulte sequencing, 2 lg of genomic DNA from seed plants, tissue culture-derived calli and their regenerated plants, transgenic calli as well as the regenerated transformants and their Tos17 active and inactive progenies was treated with EZ DNA Methylation-Gold Kit (Zymo Research, USA), according to the manufacturers instructions. Modied DNA was puried by Zymo-Spin IC Column and stored at -20C until used. For each PCR, between 1.0 and 3.0 ll of bisulte-treated DNA was used and the PCR products were cloned into the pMD18-T vector (Takara Biotech Inc., Dalian), and 15 clones for each plant sample were sequenced. The primer sequences (degenerate bases are underlined) used for amplication of Tos17 from untreated and treated DNAs were: 30 -LTR (3BSP1: yaagytaatgtaytgtatagttggyyyatg; 3BSP2: aaacatrcaccrcacctt rarrttatcctc), and 50 -LTR (5BSP1: tggayagatyaagyytaay ttgggaagatatg; 5BSP2: traaaarracartrrarcartrrataaatatraat).

Results Transpositional activation of Tos17 occurred in transgenic plants but not in tissue culture regenerants of rice cv. Matsumae Twenty-seven independent putative transformants were obtained from the biolistic transformation experiment. Southern blot analysis using part of the hyg B gene as a probe veried that 15 of the 27 regenerated plants were transformants containing various copies of the transgene

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transposition of Tos17 occurred in at least nine of the 15 transgenic plants (Fig. 2b) regenerated from independently transformed calli (Fig. 1). However, we did not detect any transpositional event of Tos17 in any of the 37 cv. Matsumae plants derived from tissue culture including escapers (6 of the 37 plants are shown in Fig. 2a). Transcriptional activation of Tos17 in calli was accompanied by cytosine demethylation; however, neither was sufcient to induce Tos17 transposition Because transcription is the rst step in retrotransposon transposition (retrotransposition), we investigated the state of Tos17 expression in the various plant materials including calli derived from tissue cultures (including escapers) and transgenics, together with their regenerated plants. The reverse-transcriptase (RT)-PCR analysis showed that the level of Tos17 transcript was up-regulated in both kinds of calli, and even to a greater magnitude in the calli from tissue culture alone. However, the expression of Tos17 became down-regulated and even silenced in most of the regenerated plants (derived from both kinds of calli) (Fig. 3). It was shown previously that transcriptional induction of Tos17 was associated with decreased cytosine methylation within the elements (Liu et al. 2004b). To test

Fig. 1 Validation of genetic transformation of the rice ssp. japonica cv. Matsumae after the biolistic particle bombardment. The HindIIIdigested genomic DNA was probed with a 700 bp fragment of the Hygromycin B gene. The number of hybridization bands represents copy numbers of the transgene. Lane S, seed-plants; lane Reg., regenerated plants from tissue culture-derived calli; lanes T0-1 through T0-15 are 15 separate transgenic plants

(Fig. 1). In further experiments, we included the 15 independent transgenic rice plants and 37 tissue culture regenerants including escapers. We investigated the copy number of Tos17 in both kinds of calli, transgenic and derived from tissue culture alone, together with their regenerants. Our results showed that

Fig. 2 Analysis of transpositional activation of Tos17 in the regenerated plants from tissue culture (a) and transgenic plants (b). Genomic DNA was digested with HindIII and probed with a 666 bp fragment of the reverse transcriptase (RT)/RnaseH region of Tos17 . a lanes Reg. 1-6, six separate regenerants from tissue cultured

(non-transformed) calli; b lanes T0-1 through -15, the 15 separate transgenic plants (the same as in Fig. 1). Transpositional events of Tos17 (arrowed) were detected in nine of the 15 separate transgenic plants but in none of 37 (six are shown) regenerated plants from tissue culture (including escapers)

Fig. 3 Reverse-transcriptase (RT)-PCR analysis of Tos17 steadystate transcript abundance in the two kinds of calli (tissue cultured alone and transgenic), the young leaf tissue of their regenerated plants

and the seed plants. The rice b-actin gene (Genbank accession X79378) was used as an internal control for RNA input

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Fig. 4 Detection of partial CG demethylation at some of the CCGG sites within Tos17 in both kinds of calli, tissue cultured alone and transgenic, and their regenerants, by methylation-sensitive Southern blot analysis. The near full length of Tos17 (4.3 kb) was delineated by BstXI (Liu et al. 2004b); the product was then digested with the methylation-sensitive enzyme HpaII, and probed with the 666 bp

fragment of Tos17 (see Materials and methods). Appearance of the *0.9 kb hybridization bands in both kinds of calli and their regenerated plants denotes CG demethylation at some of the CCGG sites within Tos17. No evidence for CNG methylation was found when the same set of DNAs was digested by MspI (an isoschizomer of HpaII) and hybridized by the same probe

if this was also the case in the transformed calli and transgenic plants, and furthermore, whether there might be some differences in methylation alteration due to the tissue culture alone or the biolistic transformation process, we investigated the methylation state within Tos17 by double enzyme restrictions, i.e. BstXI?HpaII and BstXI?MspI. We found that Tos17 was CG demethylated at some CCGG sites within the element in both transformed and nontransformed calli, and subsequently became partially or completely remethylated in the regenerated plants (Fig. 4). Given that the Southern blotting-based analysis is informative only for the CCGG sites within the element, while in plants (in contrast to mammals) cytosine methylation may occur in any sequence context, including CG, CNG and asymmetric CHH, we performed bisulte sequencing on the terminal portion of both the 50 - and 30 -LTRs together with their immediate contiguous anking regions. Results indicated that: (1) for the 50 -LTR and its anking region, no methylation existed in any of the cytosines (44 CG, 42 CNG and 102 CHH sites assayed) of the cv. Matsumae seed-plants and also no de novo methylation occurred in any of the calli or regenerants (transformed or non-transformed) (data not shown). (2) For the 30 -LTR and its contiguous anking region, a high level of methylation existed at one of the two CG sites, four of the seven CNG sites and two of the 14 CHH sites in the seed plants (Fig. 5a). (3) Within the analyzed 30 -LTR portion, nearly complete loss of methylation at most (but not all) cytosines of CNG and CHH occurred in the non-transformed or transformed calli alike, but no loss of methylation was detected at the CG site (compare Fig. 5b and d with a). (4)

Similar to the 30 -LTR itself, no signicant loss of methylation at the three CG sites at the immediate anking region in either transformed or non-transformed calli was detected (compare Fig. 5b, d with a). (5) Regeneration was accompanied by restoration and/or de novo methylation at ecotopic cytosine sites (mainly of the CHH type) of both the 30 -LTR and its anking regions (Fig. 5c, e). Together with the methylation-sensitive Southern blotting results, it is clear that the dynamic changing patterns of cytosine methylation from seed plants to calli and then to regenerants were largely limited to those cytosines with inherent methylation modications, and which were virtually the same between transgenic and non-transgenic conditions. Therefore, it is evident that the loss of cytosine methylation in Tos17 was induced by callus formation, which was likely associated with transcriptional induction of the element (Fig. 3) but not a major causal factor for its transpositional activation. Sustained transpositional activity of Tos17 in the regenerated transgenic plants To test if the biolistic transformation-induced transpositional activity of Tos17 in rice cultivar Matsumae was conned to the callus stage, and hence was immediately repressed upon plant regeneration, or if the element continued to transpose after plant regeneration, we chose two transgenic T0 plants, T0-9 and T0-11, and studied their respective T1 progenies. Southern blot analysis of a limited number of T1 plants derived from each of the two T0 transgenic plants indicated that several T1 progeny-specic

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Plant Cell Rep Fig. 5 Validation of callus culture-induced cytosine demethylation in a portion of the 30 -LTR of Tos17 and its immediate anking region of the element by bisulte sequencing: a the donor seed plants, b tissue culture-derived calli (untransformed), c regenerated plant of b, d transgenic calli, and e transgenic plant regenerated from (d). Notably, plant regeneration appeared to be accompanied by de novo methylation at some ecotopic sites, mainly of the asymmetric (CHH) type within the element. The numbers on the x-axis represent the position of cytosines within the partial 30 -LTR of Tos17 and its immediate anking region

Fig. 6 Sustained Tos17 activity in the regenerated T0 plants from transgenic calli. The genomic DNA of the T1 progenies of two T0 transgenic plants, T0-9 and T0-11, was digested by HindIII, and hybridized with the same Tos17 probe as in Figs. 2 and 4. The solid circles point to the original transpositional events in the T0 plants

(occurred at the callus stage), which showed genetic segregation in the T1 plants. The arrows refer to new transpositional events of Tos17, newly occurred in germinal cells of the T0 plants, which were inherited by the T1 progenies

new transpositions apparently occurred (Fig. 6). Specically, of the ten T1 plants derived from T0-9, eight (80%) showed evidence for new transpositions, and of the six T1 plants derived from T0-11, three (50%) showed new

transpositions. In contrast, and as expected, none of the 12 R1 plants derived from a tissue culture regenerant (Reg-2) (which did not show transpositional activity of Tos17 in the R0 generation) showed transposition in the R1 generation

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Plant Cell Rep Table 1 Sustained transpositional activity of Tos17 after plant regeneration in the R0/T0 generation from tissue culture and biolistically transformed calli, based on analysis of R1 or T1 plants R0 or T0 plant Tissue Transformation culture Reg-2 T0-9 T0-11 12 10 6

No. of R1 or T1 progeny plants tested

No. and frequency (%) of R1 or T1 plants 0 showing new transposition events of Tos17

8 (80) 3 (50)

(Table 1). Because all these progeny plants (T1 and R1) were grown under the same normal conditions, it is inconceivable that the new transposition of Tos17 in the T1 generation plants was caused by some additional unknown factor(s). Instead, it is most likely that the new transpositional events in the transgenic T1 progenies were the result of sustained activity of the element after plant regeneration in the T0 generation. For example, if a retrotransposition event occurred in the male and/or female meiocytes of a given T0 plant, then many of the T1 progenies might inherit the transpositional event (a new band), provided the insertions did not affect the viability of the resulting gametes.

Discussion We have shown in this study that transpositional activation of a rice endogenous retrotransposon (Tos17) occurred only in transgenic plants regenerated from biolistically bombarded calli of the japonica rice cultivar Matsumae, but not in regenerated plants from tissue culture (including escapers). This result, together with the fact that all these studied plants were grown under the same conditions, suggests that the biolistic transformation process was the causal factor for the transpositional activation of Tos17. Either or both of the two major events involved in the transformation process, i.e. mechanical wounding by gold particles and transgene integration, may be responsible for the element activation. To our knowledge, there has only been one similar previous study of the phenomenon of genetic transformation-induced TE transposition in plants (Bhatt et al. 1998). In that study, it was found that transgene integration caused a signicant increase in the transpositional frequency of an endogenous transposon (Tag1) in Arabidopsis (Bhatt et al. 1998). Although the mechanism for this phenomenon remains to be elucidated, we consider that it may share a similar basis to other stressinduced transposon activation, like tissue culture, pathogen attacks, UV irradiation, interspecic hybridization and polyploidy (reviewed in Madlung and Comai 2004).

Predictability of the targeted trait(s) and genetic stability of GM plants produced via transgenic technology are issues that need to be further investigated (reviewed by Dunwell 2005). The results of this study have apparent implications pertinent to this issue, as unintended genetic instability could be encountered due to transpositional activation of an otherwise quiescent retrotransposon even under the tissue culture conditions in this specic genotype (Matsumae), which might have phenotypic consequences over generations of transgenic plants. The possibility that the transposition of Tos17 in Matsumae was caused by tissue culture alone could be condently ruled out. This is based on the fact that not a single transpositional event of the element was detected in a number of independently regenerated plants of Matsumae, either in the current study (n = 37) or in our previous investigation (Liu et al. 2004b), despite the number of samples analyzed together exceeding at least 10-fold that of the transgenic plants studied. Nevertheless, it is conceivable that tissue culture may still have played some essential role in the element activation, as transcriptional induction, which is an integral rst step in the retrotranspositional process, was apparently accomplished at the stage of callus culture (Fig. 3). Another role of callus culture that has likely facilitated Tos17 transposition is cytosine demethylation (Figs. 4, 5). It has been established that a heavily methylated cytosine state would impede transposition of a TE through at least two mechanisms: by transcriptional repression of genes encoding for enzymes (e.g. transposases) that are required for transposition, and by interfering with binding of the transposase to the excision and/or insertion sites (Rudenko and Walbot 2001). It has been repeatedly shown that formation of calli in nearly all studied plants is accompanied by globally measurable reduction of cytosine methylation levels (Kaeppler et al. 2000; Guo et al. 2007; Li et al. 2007). In the present study, both the methylation-sensitive Southern blotting and bisulte sequencing data showed that the cytosine methylation state of Tos17 was considerably reduced in both types of calli (tissue culture alone and transgenic) (Figs. 4, 5), which might have been responsible for the transcriptional activation of the element (Fig. 3). Taken together, it is reasonable to deduce that the combined effects of callus induction and biolistic transformation have elicited transpositional activation of Tos17 in Matsumae. The sustained transpositional activity of Tos17 induced by transformation is interesting and novel, as this is not the usual characteristic of this element induced by other means (Cheng et al. 2006; Ding et al. 2007; Hirochika et al. 1996). In these previous studies, the elements transpositional activity was found to be rapidly repressed upon plant regeneration (Cheng et al. 2006; Ding et al. 2007; Hirochika et al. 1996). Because different cultivars were

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utilized in our and previous studies, a possible reason for the discrepancy could be the genotype background effect. However, it is notable that in the case of Tag1 in Arabidopsis, the elements transpositional activity was also maintained in the regenerated transgenic plants and even in their progenies (Bhatt et al. 1998). Thus, the possibility arises that the sustained transpositional activity might be a transformation-related phenomenon, which warrants further investigations.
Acknowledgments This study was supported by the Program for Changjiang Scholars and Innovative Research Team (PCSIRT) at the University (#IRT0519) and Bioforsk core funding (#1110222) to Dr. Jihong Liu Clarke. We are grateful to Dr. Rudolf Hegaman, Dr. Sonja S. Klemsdal and two anonymous reviewers for constructive suggestions to improve the manuscript, and to Dr. Nicholas Clarke for linguistic correction.

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