Efficient generation of marker-free transgenic rice plants using an improved transposon-mediated transgene reintegration strategy.

Plant Physiology Preview. Published on November 5, 2014, as DOI:10.1104/pp.114.246173

Running head: Transposon-mediated transgene reintegration system

Corresponding Author: Shaohong Qu Institute of Virology and Biotechnology Zhejiang Academy of Agricultural Sciences Hangzhou, Zhejiang 310021, China Tel: +86 571 86419018 Fax: +86 571 86404256 Email: [email protected]

Journal research area: Breakthrough Technologies

1 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

Copyright 2014 by the American Society of Plant Biologists

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy1

Xiaoqing Gao2, Jie Zhou2, Jun Li2, Xiaowei Zou, Jianhua Zhao, Qingliang Li, Ran Xia, Ruifang Yang, Dekai Wang, Zhaoxue Zuo, Jumin Tu, Yuezhi Tao, Xiaoyun Chen, Qi Xie, Zengrong Zhu, and Shaohong Qu*

Institute of Virology and Biotechnology (X.G., J.Z., J.L., J.Z., X.Z., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China; Institute

of Genetics and

Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); and Institute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Z., Z.Z.), Zhejiang University, Hangzhou, Zhejiang 310058, China

One Sentence Summary: An improved transposon-mediated transgene reintegration system was developed to efficiently generate marker-free transgenic rice plants.


1

This work was financially supported by the Ministry of Agriculture Genetically

Modified

Organisms

Breeding

Major

Projects

of

China

(Grants

Nos.

2012ZX08009001 and 2011ZX08010-002-007) and by the Aid Program for Key Science and Technology Innovative Research Team in Zhejiang Province (Grant No. 2011R50023). 2

These authors contributed equally to the article.

* Address correspondence to [email protected] The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shaohong Qu ([email protected]).


Abstract:

Marker-free

transgenic

plants

can

be

developed

through

transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize Ac/Ds transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest (GOI) was linked to green fluorescent protein (GFP) in T-DNA, and a GFP-aided counter-selection against T-DNA was used together with PCR-based positive selection for the GOI to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) ð-endotoxin gene into the Ds elements and transformed transposon vectors into rice cultivars via Agrobacterium. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to TAIL-PCR to determine Ds(Bt) reintegration positions, RT-PCR and ELISA to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis (Walker). Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

DNA vectors used in plant transformation usually contain selectable marker genes, such as those that confer antibiotic or herbicide resistance, to facilitate the selection of transformed cells (Sundar and Sakthivel, 2008). However, the retention of such marker genes in transgenic plants is undesirable because of their possible toxicity or allergenicity to humans and their unpredictable hazards to the environment (Miki and McHugh, 2004; Ramessar et al., 2007; Yau and Stewart, 2013). Therefore, the production of marker-free transgenic crops is crucial to avoid these risks and promote their commercial deployment. Several strategies have been developed to remove selectable markers from 4 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

transgenic plants while retaining the gene of interest (GOI) (Ebinuma et al., 2001; Cotsaftis et al., 2002; Wang et al., 2011; Kapusi et al., 2013; Yau and Stewart, 2013; Oliva et al., 2014). Transposition is an advantageous strategy for marker gene removal, because it allows intact transgene insertion with defined boundaries and requires only a few primary transformants (Cotsaftis et al., 2002). Transposon-mediated transgene reintegration was initially used by Goldsbrough et al. (1993) to reposition a Ds transposon-based β-glucuronidase (GUS) reporter gene in transgenic tomato. In this study, the Ds(GUS) element in T-DNA was tandem-linked to both Ac transposase (AcTPase) and NptII selectable marker to allow the transposition and segregation of Ds from T-DNA-based AcTPase and NptII. Cotsaftis et al. (2002) used the same strategy to reposition a Ds-based Bt endotoxin gene in transgenic rice and recovered marker-free plants resistant to insects. However, these studies employed Southern blot analysis to identify stable insertions of Ds elements in T1 and T2 transgenic plants. Southern blot analysis is labor intensive and systematically inefficient for screening a large number of transgenic populations. Ac/Ds elements tend to reintegrate at genome sites closely linked to the donor site (Dooner et al., 1991; Smith et al., 1996; Machida et al., 1997; Walbot, 2000; Lazarow et al., 2013), and only a fraction of somatic transpositions can be transmitted to the next generation (Kolesnik et al., 2004). Therefore, transposon vectors available for marker-free transformation need to be improved to realize the efficient selection of unlinked germinal transpositions. For recovering stable Ds insertions, a Ds element must be transposed to a new site that is sufficiently distant from the T-DNA site harboring AcTPase and selectable marker to break the linkage between the two sites. In transposon tagging studies, counter selection against Ds donor sites and AcTPase using an IAAH marker or green fluorescent protein (GFP) was established to enrich unlinked Ds transpositions from segregating transgenic populations (Sundaresan et al., 1995; Kolesnik et al., 2004; Qu et al., 2008). Meanwhile, plants that carry reintegrated Ds elements are identified by another selectable marker within the Ds element. This dual selection scheme can exclude Ds elements that remain tightly linked to T-DNA donor sites because of proximal transposition and select only plants that carry transposition to distal sites in 5 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

the genome. Therefore, marker-free transformation can be realized by employing a counter-selection scheme in transposon tagging to eliminate T-DNA-bearing transgenic progeny and by screening reintegrated Ds elements that carry GOI through the PCR assay of T-DNA-free progeny. Michelmore et al. (1991) established bulked segregant analysis (BSA) to efficiently identify individual plants with polymorphic RFLP or RAPD markers from segregating populations. BSA is realized by mixing DNA samples of multiple plants having the same phenotype, detecting polymorphism between the bulks, and analyzing individual plants among the bulks differing in the markers. The principle behind BSA is to group informative individuals so that a particular locus can be studied against a randomized genetic background of unlinked loci. The DNA pool approach in BSA may be extended to screening segregating transgenic populations for the efficient selection of marker-free GOI-bearing plants. In the present study, we improved the transposon-mediated transgene reintegration strategy by redesigning the in cis Ac/Ds vector (Qu et al., 2008) that was originally used for transposon tagging. A Ds-based GOI was linked to GFP in T-DNA, and a GFP-aided counter-selection against T-DNA was used together with PCR-based positive selection for the Ds(GOI) to screen marker-free progeny. For Ds(GOI) detection, the pooled DNA of multiple GFP-negative plants was assayed by the PCR amplification of GOI sequence, and individual plants in GOI-positive pools were further analyzed to identify GOI-positive plants. This study efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

RESULTS Construction of an in cis Ac/Ds Transposon Vector for Marker-Free Transformation 6 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

We constructed an in cis Ac/Ds transposon vector (pLJ26, Fig. 1A), in which the Ds element is linked to immobilized AcTPase, hygromycin phosphotransferase (HPT) and GFP. The vector carries both Ds and AcTPase to mobilize the Ds element after transforming T-DNA into plant cells. A GOI is cloned into a unique AscI site in the Ds element, which functions as a carrier to reposition the GOI to unlinked sites. The intermediate cloning vector pLJ16 (Fig. 1A) was constructed to assemble the DNA fragments of the GOI. The multiple cloning site in pLJ16 was designed in such a way that the multiple cloning site sequence was flanked by two sites of AscI, a rare-cutting restriction enzyme with an 8 bp recognition sequence. The GOI in pLJ16 can be released by AscI and be inserted into the Ds element in pLJ26. The HPT gene in pLJ26 functioned as a transformation selectable marker. The GFP gene carried by the vector was used for two purposes. First, the GFP in plant transformation functioned as a fluorescent reporter that allows the direct visualization of transformed cells. Second, GFP in T1 or other segregating generations of transgenic plants served as a counter-selectable marker that eliminates the progeny carrying T-DNA-based AcTPase, HPT, and GFP itself. Ds elements that remain closely linked to T-DNA sites because of proximal transposition were excluded by counter-selection against GFP-positive (GFP+) plants, and progeny carrying transposition to more distal sites in the genome can be retained. In the final step of selection, marker-free transgenic plants were recovered from GFP-negative (GFP−) progeny through the PCR assay of the GOI (Fig. 1B). To test the efficacy of the vector system described above, we cloned two versions of the Bacillus thuringiensis (Bt) ð-endotoxin gene, Bt-1 and Bt-2, into the Ds elements in pLJ26 to construct pZJ53 and pZJ54 (Fig. 1C). The Bt-1 and Bt-2 genes both have the coding sequence of the Bt ð-endotoxin, but Bt-2 contains an additional 5′ coding sequence of a chloroplast transit peptide.

Rice Transformation and Molecular Analysis of Transgenic Plants The calli of rice (Oryza sativa L. ssp. japonica) cultivars Zhejing-22 and 7 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

Kongyu-131 were co-cultivated with Agrobacterium strains that harbor the Ac/Ds vectors pZJ53 and pZJ54, respectively, and then subjected to transformation selection. After 27 d of selection culture, hygromycin-resistant (Hyg+) secondary calli that grew from the co-cultivated calli showed fluorescence when assayed under a GFP detector (Fig. 2A). Transformation efficiency, defined as the percentage of co-cultivated calli that produce Hyg+GFP+ secondary calli, was calculated. The transformation efficiencies of the pZJ53/Zhejing-22, pZJ53/Kongyu-131, and pZJ54/Zhejing-22 transformants were 37.0% (163/440), 46.4% (128/276), and 19.1% (70/367), respectively. The Hyg+GFP+ microcalli were subcultured (Fig. 2B) until grown to a size large enough for rice DNA extraction. DNA samples of nine pZJ54-transformed Hyg+GFP+ calli were assayed by PCR using specific primers (P1 and P2, Fig. 1B) to detect transposon empty donor sites (EDS). A 1.8 kb EDS-PCR band was amplified for all the nine calli (Fig. 2C), suggesting that the Ds(Bt) element had excised after its transformation into rice cells. Transformed plants (T0) were regenerated from independent Hyg+GFP+ calli. Up to 43.8% (14/32), 40.8% (31/76), and 75.8% (50/66) of the pZJ53/Zhejing-22, pZJ53/Kongyu-131, and pZJ54/Zhejing-22 calli were regenerated into plants, respectively. Leaf tissue samples were collected from each plant for DNA extraction and EDS-PCR. Ds excision was detected in 100% (14/14) of the young pZJ53/Zhejing-22 plants (Fig. 3A). Meanwhile, 45 pZJ54/Zhejing-22 plants at the tillering stage were analyzed by PCR (Fig. 3B; Supplemental Fig. S1). Two plants (#2 and #17, Fig. 3B) were not amplified, and among the pZJ54/Zhejing-22 plants that were amplified, 60.5% (26/43) were positive in EDS-PCR. We cloned the 1.8 kb EDS-PCR products of seven pZJ54/Zhejing-22 transformants (two calli in Fig. 2C and five plants in Fig. 3B) into a TA-cloning vector and sequenced two to six clones per transformant to characterize the Ds excision events. In total, 22 EDS clones were analyzed through multiple sequence alignment (Supplemental Fig. S2). For each of the #2 and #3 calli and each of the #19, #24, and #29 T0 plants, more than one EDS clone showed polymorphic Ds excision sites. This result suggested that independent excisions occurred in multiple T-DNAs 8 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

or in different cell lineages. Thirty-one pZJ53/pZJ54 T0 plants were analyzed using PCR with primers specific to the T-DNA ends, and multiple T-DNA copies linked in direct tandem repeats were detected in 32.3% (10/31) of the plants (Supplemental Figs. S3 and S4). The EDS bands of the #4 and #41 pZJ54/Zhejing-22 T0 plants were smaller than expected (Fig. 3B; Supplemental Fig. S1). Sequence analysis of the two small EDS bands of the #4 pZJ54/Zhejing-22 plant revealed the insertion of a vector backbone sequence and the deletion of Ds adjacent sequences (Supplemental Figs. S5 and S6), which represent DNA rearrangements associated with T-DNA transformation and Ds excision, respectively. The transposon full donor sites (FDS), the Ds-based Bt gene, and the T-DNA-based GFP gene were amplified by PCR from 14 pZJ53/Zhejing-22 transformants (Fig. 3A) and 28 pZJ54/Zhejing-22 transformants (Fig. 3B). FDS, Bt, and GFP were amplified in most of the plants. Ds excision in the plants positive for both EDS and FDS (EDS+FDS+) can be associated with a fraction of somatic cells or a fraction of T-DNA loci or T-DNA copies. The Ds elements in the plants positive for EDS but negative for FDS (EDS+FDS−) excised completely from transposon donor sites. The #13 pZJ53/Zhejing-22 and #29 pZJ54/Zhejing-22 plants displayed the EDS+FDS− pattern but not Bt amplification. This result suggested that the excised Ds did not reintegrate into the rice genome. The #6, #20, and #22 pZJ54/Zhejing-22 transformants that possessed the EDS−FDS− pattern and exhibited Bt or GFP amplification might contain sequence deletion or rearrangement in T-DNA. The #6 and #33 pZJ54/Zhejing T0 plants (Fig. 3B; Supplemental Fig. S1) with GFP bands larger than expected might contain rearranged GFP sequences. These results can be attributed to the sequence alterations caused by T-DNA transformation and Ds transposition (see Discussion). Taken together, the Ds(Bt) elements in most of the transformed rice calli and T0 plants showed transposition activity.


Recovery of Marker-Free Transgenic Plants by Genetic Screening The pZJ53 and pZJ54 vectors were initially tested by transformation using the Zhejing-22 cultivar. Bioassays against the striped stem borer Chilo suppressalis (Walker) showed that Bt-1 in the pZJ53 transformants conferred a higher level of insect resistance than Bt-2 in the pZJ54 transformants (see below for the results of insect bioassay with pZJ53 plants). Therefore, we focused on the pZJ53 transformants and screened their progeny to generate marker-free plants with insect resistance (Table I). After detecting high-frequency somatic transpositions in the Zhejing-22 transformants, we did not assay the pZJ53/Kongyu-131 transformants by EDS-PCR and FDS-PCR but directly screened the Kongyu-131 T1 progeny to recover marker-free Ds(Bt) plants. High-frequency somatic transposition is important to generate Ds elements reintegrated at different genome sites. However, a transposed Ds must be transmitted to the next generation via germinal cells, and the genetic distance between the reintegrated Ds and its donor T-DNA must be large enough to recombine and segregate marker-free progeny. We established a procedure for the genetic screening of marker-free transgenic plants, in which the T-DNA-based HPT, GFP, AcTPase, and the unexcised Ds were counter-selected by eliminating the GFP+ progeny, and the reintegrated Ds(GOI) was recovered through the PCR assay of the GOI in the GFP− plants. The rice seeds of each T1 line were germinated, and the seedlings were assayed under a GFP fluorescence detector to distinguish fluorescent from non-fluorescent plants. Non-fluorescent plants were selected, and their leaf samples bulked (five plants per bulk) for DNA purification. The bulked rice DNA was then amplified using Bt-specific primers. A total of 693 non-fluorescent T1 plants (15.1 plants per line) were analyzed to detect Ds(Bt) elements. For the bulks that showed Bt amplification, individual plants were further analyzed through the PCR assay of the Bt, HPT, and GFP sequences (Fig. 4). Marker-free T1 plants (Bt+HPT−GFP−) were obtained from 26.1% (12/46) of the pZJ53/pZJ54 transgenic lines (Table I).


Rice lines were randomly selected from each of the vector/cultivar combinations in Table I to screen marker-free Ds(Bt) plants. Among the 24 Zhejing-22 lines whose parents (T0) were assayed by EDS-PCR, the #13 and #43 pZJ54/Zhejing-22 lines displayed the EDS− pattern (Fig. 3B; Supplemental Fig. S1) and did not segregate marker-free T1 progeny. The parents of other Zhejing-22 lines, including seven Zhejing-22 lines that segregated marker-free plants, showed the EDS+ pattern (Fig. 3; Supplemental Fig. S1). The parents of four Zhejing-22 lines with marker-free segregants were analyzed using both EDS-PCR and FDS-PCR. The parents of the #7 and #30 pZJ54/Zhejing-22 lines were EDS+FDS+, and those of the #4 pZJ53/Zhejing-22 and #16 pZJ54/Zhejing-22 lines were EDS+FDS− (Fig. 3). The segregation ratio of fluorescent plants to non-fluorescent plants in each T1 line was investigated through the chi-square test (Table I). Twenty-three (50%) lines segregated with a 3:1 Mendelian pattern of inheritance and the other 23 (50%) lines with non-Mendelian ratios of distortion toward non-fluorescent plants. We isolated Ds flanking sequences from three marker-free lines and T-DNA flanking sequences from the T0 parental plants by TAIL-PCR and determined the genome positions of transposed Ds elements and their donor T-DNAs. These analyses were performed to ascertain whether or not the marker-free plants were derived from unlinked transposition. As shown in Table II, the transposed Ds elements and their donor T-DNA sites were located on different rice chromosomes. Since the marker-free lines for TAIL-PCR were selected on a random basis, the reintegrated Ds elements obtained through our selection scheme tend to be distributed within unlinked genomic regions. Of the 12 T1 lines that segregated marker-free plants (Table I), 11 segregated multiple marker-free plants. For the three lines whose Ds flanking sequences were analyzed by TAIL-PCR, multiple marker-free siblings carried the same reintegrated Ds elements (Table II), suggesting that the marker-free plants were principally derived from transpositions at the early stages of plant development. Non-fluorescent segregants from a total of 28 lines were characterized by PCR amplification of the HPT and GFP genes (Table I). Non-fluorescent plants showing 11 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

HPT+GFP−, HPT−GFP+, or HPT+GFP+ pattern were identified in 28.6% (8/28) of the lines, which can be attributed to the HPT and GFP sequence rearrangement or to the GFP silencing in the T1 generation (see Discussion). For the three rice lines whose Ds(Bt) reintegration sites had been determined (Table II), their T3 progeny were analyzed by Southern hybridization using HPT, GFP, and Ds probes. As revealed by the absence of hybridization with HPT and GFP (Supplemental Fig. S7, A and B), the rice lines did not contain T-DNA vector sequence. The three lines showed single Ds hybridizing bands that were derived from the 5′Ds sequence and the rice genome sequence flanking each Ds insertion (Supplemental Fig. S7, C). Therefore, the marker-free rice lines that were analyzed contained single-copy reintegrated Ds(Bt) elements.

Analysis of Transposition Activity in T1 Plants Eight to forty GFP+ plants from each of the 18 pZJ53-transformed T1 lines were assayed by EDS-PCR or by EDS-PCR and FDS-PCR to explore whether or not the Ds(Bt) elements in the T1 plants were still capable of transposition (Table III and Fig. 5). Ds excision sites were amplified for all the 11 GFP+ plants of the #4 pZJ53/Zhejing-22 line (Table III and Fig. 5A). Marker-free Ds plants were previously recovered from the same T1 population of the #4 pZJ53/Zhejing-22 line (Table I). We also analyzed the GFP+ plants from 17 pZJ53/Kongyu-131 T1 lines that had not segregated marker-free Ds plants (Table III; Figs. 5B and 5C). Approximately 47.1% (8/17) of the pZJ53/Kongyu-131 lines showed new excisions, and 30% to 100% of the plants in each line were EDS+. Therefore, the Ds(Bt) elements in the T1 rice lines can transpose, and the T-DNAs in the T1 plants with high-frequency transposition can be utilized as Ds-launching pads to generate new Ds insertions.

Functional Characterization of the Ds-Based Bt Gene in Marker-Free Transgenic Rice Plants We investigated the transcription of the Ds-based Bt gene in transgenic rice plants 12 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

via semi-quantitative RT-PCR analysis. Bt transcripts were detected in the pZJ54/Zhejing-22 T0 plants (Supplemental Fig. S8). Comparable levels of Bt transcripts were detected in the marker-free T1 plants segregated from three pZJ53-transformed rice lines (Fig. 6), in which the Ds(Bt) elements were integrated at independent genome sites. ELISA using an antibody directed against the Bt protein was performed on the leaf protein extracts of marker-free T2 plants from three transgenic lines (Table IV). All the marker-free Ds(Bt) plants expressed the Bt protein at levels comparable with the positive control T51-1, a previously characterized Bt-transgenic rice line (Tu et al., 2000). The insecticidal effect of the Bt protein in transgenic rice was tested in bioassays against rice striped stem borers. At 5 d after the treatment, the larval mortality rates of two pZJ53/Zhejing-22 transformants (T0) containing the Bt-1 gene reached up to 100% and were significantly higher than those of the pZJ54/Zhejing-22 transformants containing the Bt-2 gene as well as the untransformed control (Supplemental Table S1 for the results of pZJ53 T0 plants). The corrected larval mortality rates of the marker-free T2 lines 11-2 (pZJ53/Kongyu-131) and 4-2 (pZJ53/Zhejing-22) reached 64.1% and 67.9%, respectively, at 4 d after feeding (Table V). These data indicated that the marker-free plants containing the Bt-1 gene were highly toxic to the rice pest striped stem borer. Ideally, the Ds-based GOI in the marker-free transgenic plants reintegrates into the plant genome without insertional mutation of endogenous genes. As shown in Figure 7, the Ds(Bt) elements in the marker-free plants from the 11-2 and 14-2 pZJ53/Kongyu-131 lines were reintegrated into the intergenic regions of the rice genome, and the Ds(Bt) in the 4-2 pZJ53/Zhejing-22 line were reinserted into the coding region of the rice gene Os04g57320. Thus, we selected one homozygous T2 plant without obvious morphological alterations from each of the 11-2 and 14-2 lines, and evaluated their T3 progeny by field plot experiment (Supplemental Table S2). The 11-2 and 14-2 T3 lines flowered at the same time as the untransformed Kongyu-131 cultivar. The tiller numbers, plant heights, panicle numbers, and flag leaf lengths of 13 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

the 11-2 plants were comparable with those of Kongyu-131. The 14-2 line showed lower plant heights, shorter flag leaves, and more panicles compared with Kongyu-131 and the 11-2 line. Therefore, the 11-2 marker-free line exhibiting normal agronomic traits and containing the Ds-based Bt gene with optimized transgene insertion and expression can be used to develop commercialized Bt rice cultivars.


DISCUSSION Transposon-mediated transgene reintegration can be identified through Southern blot analysis of transgenic plants using probes specific to the sequences of transposable elements or their donor sites in T-DNA (Goldsbrough et al., 1993; Cotsaftis et al., 2002). However, the Southern hybridization approach is labor intensive for screening a large number of transgenic progeny. In the present study, we improved the transposon-mediated transgene reintegration strategy by redesigning an in cis Ac/Ds transposon tagging vector (Qu et al., 2008) and using a two-step scheme for the recovery of marker-free Ds(Bt) plants. GFP fluorescent plants were initially eliminated from T1 populations by GFP-aided counter-selection. Theoretically, 75% of the T1 plants from a single-locus transgenic line carry T-DNA and can be sorted by removing fluorescent plants. Germinating T1 seeds were assayed using a portable GFP fluorescence detector that was equipped with a blue light source, which is safer for users compared with UV light source in a regular GFP detector (Chen et al., 2005). This approach allows the high-throughput genetic analysis of transgenic plants and thus reduces the workload of screening transgenic progeny. In the second selection step, a DNA pool approach similar to the bulked segregant analysis (Michelmore et al., 1991) was used for the PCR assay of Ds insertions in the non-fluorescent T1 plants. The DNA mixture of multiple plants was initially amplified, and individual plants of the Ds(Bt) positive bulks were assayed to identify Ds(Bt) plants. Marker-free (T-DNA-free) transgenic rice plants were obtained from 26.1% of the primary transformants. The somatic activity of Ds elements in transgenic rice can be monitored by constructing P-Ubi:Ds:GUS cassette and conducting the histochemical GUS assay of rice tissues (Qu et al., 2009). In the present study, we examined Ds excisions by the PCR amplification of EDS sequences. Ds excision was detected in 100% of the transformed calli or plantlets and in 60.5% of the adult T0 plants. Similar frequencies were reported in the Ac/Ds tagging systems of rice (Chin et al., 1999; Nakagawa et al., 2000; Greco et al., 2001; Kolesnik et al., 2004). The high frequencies of Ds excision


in transformed calli and young plants might be due to their highly active cell division and DNA replication, which enhance transposition (Ros, 2001). Among the transformants assayed in EDS-PCR, the #4 pZJ54/Zhejing-22 transformant contained Ds-induced deletion of flanking sequences. Similar results were observed in maize, wherein the Ds elements often leave behind a deletion of adjacent sequences when they excise (Scott et al., 1996). In addition, the EDS-PCR products of the #4 pZJ54/Zhejing-22 transformant contained a vector backbone sequence, which might have been co-transferred with T-DNA during rice transformation. Previous studies reported that 45% of Agrobacterium-transformed rice lines contain a vector backbone sequence (Kim et al., 2003). The T-DNAs carrying unexcised Ds elements were detected by amplifying the FDS sequences. For transformants with multiple T-DNA loci, FDS was possibly amplified from one locus while EDS was amplified from another locus. Similarly, for transformants containing multiple T-DNA copies, FDS was possibly amplified from one T-DNA copy while EDS was amplified from another copy. The average number of T-DNA copies per rice transformant was estimated to be 1.8 to 2 (Sallaud et al., 2003). We designed outward-directed primers specific to the T-DNA ends and amplified the multiple T-DNA copies co-integrated in direct repeats at a single locus in 32.3% of the pZJ53/pZJ54 transformants. Moreover, as revealed by the variegated GUS pattern induced by Ds excision (Qu et al., 2009), it was possible that Ds excision occurred in a fraction of somatic cells while the Ds in other cells remained unexcised or that independent excisions occurred in different cells. Therefore, the EDS+FDS+ pattern might be associated with multiple T-DNAs or with different cell types in the tissue assayed by PCR. The amplification results of the Ds-based Bt gene in the T0 plants showed that a small number of excised Ds elements did not reintegrate in the rice genome. Previous studies reported that 37% of Ac/Ds transformed rice plants contain Ds excision and that only 25% of the transformants carry Ds reinsertion (Cotsaftis et al., 2002). Belzile et al. (1989) investigated the genetic transmission of the autonomous Ac element in tomato and estimated that Ac is lost in 10% of primary transformants. 16 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

To characterize somatic transpositions and investigate the sequence alterations induced by transposition and T-DNA transformation, we analyzed the Ac/Ds transformants through the PCR amplification of EDS, FDS, and T-DNA components. Of the 28 pZJ54/Zhejing-22 T0 plants analyzed by the amplification of the EDS, FDS, Bt, and GFP sequences, three showed the EDS−FDS− pattern with Bt or GFP amplification. Approximately 28.6% of the rice lines were non-fluorescent T1 plants that showed the PCR pattern of HPT−GFP+, HPT+GFP−, or HPT+GFP+. Meanwhile, the GFP transgene in 50% of the pZJ53/pZJ54 lines showed non-Mendelian segregation patterns. These variations can be attributed to T-DNA transformation and Ds transposition. First, the frequent T-DNA truncations and transgene rearrangements in transgenic plants (Cotsaftis et al., 2002; Kim et al., 2003; Rai et al., 2007) may affect the PCR amplification of T-DNA sequences. Second, Ds-induced deletion, a loss of Ds elements, or Ds reinsertion within the T-DNA may change the PCR target sequence and prevent the amplification of EDS, FDS, Ds(Bt), GFP, or HPT. Large-scale analysis of Ds insertion flanking sequences in rice showed that 12% of the Ds insertions were found within the T-DNA construct; 4% of these insertions were found in GFP (Kolesnik et al., 2004). In addition, Ds-disrupted GFP sequences can still be amplified if Ds reinsertion is outside the PCR target region. Another possibility for the non-fluorescent progeny showing the HPT−GFP+ or HPT+GFP+ pattern is the silencing of the GFP transgene. Transgene expression is influenced by the transgene copy number and the T-DNA organization within the transgenic locus, and epigenetic silencing of transgenes can occur at the transcriptional or posttranscriptional level (Depicker et al., 2005). Ds transposition can be investigated by PCR and Southern blot analysis of the genomic DNA from Ac/Ds transformed T0 plants (Cotsaftis et al., 2002). The rice DNA for Southern hybridization or PCR analysis was purified from only a small amount of tissue, and the excision events detected by either method merely reflected the somatic activity of the Ds elements in a small portion of the plant. Kolesnik et al. (2004) reported that only a relatively small number of somatic excisions can be transmitted via germinal cells to the next generation. Moreover, unlinked Ds 17 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

reinsertions are rare events in segregating generations because Ds elements transpose preferentially to linked sites (Kolesnik et al., 2004; Qu et al., 2008). In the present study, we did not observe a direct correlation between the occurrence of somatic transposition and the segregation of marker-free Ds(Bt) plants in the rice lines whose T0 plants were analyzed by EDS-PCR and whose T1 progeny were screened for marker-free segregants. Of the 22 Zhejing-22 T1 lines whose parents were positive in EDS-PCR, only seven segregated marker-free Ds(Bt) plants. These results indicate that EDS-PCR and FDS-PCR are useful for testing the transposability of Ds elements in T-DNA vectors. However, the efficiency of transposon-mediated transgene reintegration should be evaluated using the recovery rate of unlinked germinal transpositions or T-DNA-free Ds(Bt) segregants. Increasing the population size in the T1 generation would enhance the probability of recombination between a locally transposed Ds and its donor T-DNA because of the linked transposition of Ds elements. The current rate of T1 lines segregating marker-free plants can be increased by analyzing more T1 segregants. Early transposition during plant development generates a large sector of cells carrying the transposition, resulting in a high germinal transposition frequency and multiple siblings containing the same transposon insertion (Honma et al., 1993). The marker-free Ds plants in our vector system were principally derived from early transpositions based on multiple marker-free siblings carrying identical Ds reinsertions. This result is consistent with previous reports in Arabidopsis (Honma et al., 1993; Balcells and Coupland, 1994) and in rice (Cotsaftis et al., 2002). In testing the Ac/Ds vectors, we characterized stable Ds insertions from three starter transformants through TAIL-PCR and GOI expression analysis. For generating more marker-free lines with optimized Ds(GOI) insertion and GOI expression, it is necessary to determine the genome positions of Ds insertions from other starter transformants and to analyze their GOI expression. Furthermore, the T-DNA in the T1 plants can be utilized as Ds launching pads to generate new Ds insertions because the Ds elements in the T1 progeny possessed a high transposition frequency. Ds elements remain active in the T4 and T5 generations of transgenic rice plants (Szeverenyi et al., 18 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

2006). Transposon tagging studies using the in cis Ac/Ds vector (Qu et al., 2008) obtained 311 rice lines with independent Ds insertions by screening the T2 progeny of GFP-positive T1 plants derived from 26 primary transformants. To establish T2 populations for the screening of marker-free Ds plants, heterozygous T1 plants are needed to segregate the T-DNA-free progeny. GFP heterozygous seedlings usually show a lower level of green fluorescence than homozygous seedlings and can be selected based on their fluorescence intensity (Qu et al., 2008). We evaluated the agronomic traits of two marker-free lines containing intergenic Ds(Bt) insertions by conducting a field plot experiment. One rice line exhibited a field performance comparable with the untransformed parent cultivar and can be utilized to develop commercialized Bt rice cultivars. The other marker-free line exhibited reduced plant height, shorter flag leaves, and more panicles. For a transgenic line without insertional mutation of endogenous genes, the changed traits may be associated with somaclonal variation. The initial creation of transgenic plants largely relies on in vitro techniques that have long been known to generate somaclonal variation, which can result in undesirable alteration to both qualitative and quantitative traits (Bregitzer et al., 2008). For the transgenic lines that are valuable for plant breeding, their somaclonal variation can be minimized by backcrossing to the untransformed parent cultivar (Bregitzer et al., 2008). For the co-transformation, site-specific recombination, and other T-DNA-based strategies for marker gene removal (Ebinuma et al., 2001; Wang et al., 2011; Kapusi et al., 2013; Yau and Stewart, 2013), the complex transgene integration patterns such as multiple T-DNA loci, tandem-linked T-DNA copies, T-DNA truncations, and co-transferred non-T-DNA vector backbone sequence in transgenic plants (Yin and Wang, 2000; Kim et al., 2003; Rai et al., 2007) may complicate the molecular analysis of transgene integration and affect the stability of transgene expression. In the present study, we validated that the maize Ac/Ds transposable element can be routinely used to generate marker-free transgenic plants in rice. Ac/Ds elements are native genomic sequences from maize in terms of transgenic safety, and the Ds-mediated transgene reintegration strategy requires a limited number of primary transformants and results 19 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

in intact transgene insertion with defined boundaries. Therefore, this vector system can be used for marker-free transformation in rice and possibly in other crops including recalcitrant species in terms of transformation.

MATERIALS AND METHODS Vector Construction The 3′Ds sequence in pWS32 (GenBank Accession AF433043) was amplified using

PCR

LJ13-3′Ds_HindIII-F

primers

(5′-AAGCTTTAGGGATGAAAACGGTCGGT-3′)

and

LJ14-3′Ds_AscI-R

(5′-GGCGCGCCATTCAATTAGCAGGATTCATAAG-3′) and then cloned into pMD19-T (Takara, Japan) to construct pLJ11. An AscI linker (annealed with synthesized

oligos

5′-phosphate-AGCTTGCGGCCGCGGCGCGCCG-3′

and

5′-GATCCGGCGCGCCGCGGCCGCA-3′) was inserted between the BglII and HindIII sites in pCAMBIA-0380 (GenBank Accession AF234290) to construct pWX01. The 5′Ds sequence in pWS32 was released as a 1.8 kb EcoRI fragment and ligated to EcoRI-digested pWX01 to construct pLJ20. The 3′Ds fragment in pLJ11 (released by AscI/SacI digestion), 5′Ds fragment in pLJ20 (released by AscI/HindIII digestion),

and

SacI/HindIII-digested

pCAMBIA-1300

(GenBank

Accession

AF234296) were ligated to each other to construct a 2.1 kb Ds element, which resulted in pLJ21. The Ds in pLJ21 was released as a SacI fragment and ligated to the large fragment (~16 kb in size) of SacI-digested pSQ5 (Qu et al., 2008) to construct the Ac/Ds vector pLJ26. The intermediate cloning vector pLJ16 for assembling GOI components was constructed by inserting an EcoRI/SacI/KpnI/BamHI linker (annealed with 5′-phosphate-AATTCGAGCTCGGTACCG-3′

and

5′-GATCCGGTACCGAGCTCG-3′) between the EcoRI and BamHI sites in pWX01. The pea rbcS-3A poly (A) addition sequence (T3A) in pGLW07 was amplified using PCR primers ZJ_20_T3A_SacI_F (5′-GAGCTCAGCTTTCGTCCGTATC-3′ ) and 20 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

ZJ_21_T3A_EcoRI_R (5′ -GAATTCTCGACACAAAAAGCCTATAC-3′) and then cloned into pMD19-T to construct pZJ09. The T3A sequence was released from EcoRI/SacI-digested pZJ09 and ligated to EcoRI/SacI-digested pLJ16 to construct pZJ11. The rice act-1 promoter driving Cry1Ab/Ac (Bt-1) sequence in pZTBtm was released by KpnI/HindIII digestion and fused to the T3A sequence in pZJ11 to construct pZJ51. Similarly, the Act-1:Bt-2:T3A cassette (pZJ52) was constructed using the Act-1:spBt fragment from pZTBtmsp and the T3A from pZJ11. The Bt-2 coding region contained the sequences for a chloroplast transit peptide and the Bt-1 protein. The Act-1:Bt-1:T3A and Act-1:Bt-2:T3A cassettes were isolated as AscI fragments and inserted into the unique AscI site in the Ds element in pLJ26, which resulted in pZJ53 and pZJ54, respectively. The pZJ53 and pZJ54 vectors were electroporated into the Agrobacterium strain EHA105 for rice transformation.

Rice Transformation Mature rice seeds were cultured on NB medium (Chen et al., 1998) for 3 to 4 weeks to induce embryogenic calli. For rice transformation, the Agrobacterium strain carrying an appropriate binary vector was grown overnight in YEP medium at 28 °C. The Agrobacterium culture was harvested by centrifugation (4000 rpm, 5 min) and resuspended in 30 mL of AAM solution containing 100 μM acetosyringone. Rice calli were soaked in Agrobacterium suspension culture for 10 min, blotted onto four layers of sterile filter paper to remove surface fluids, and dried by hood wind for 15 to 30 min. The calli were transferred onto a filter paper that was placed on NB–As medium (NB medium plus 100 µM acetosyringone), and co-cultivated at 23 °C in the dark for 3 d. Co-cultivated rice calli were washed with sterile water five to six times and with a 400 mg/L carbenicillin solution in the final wash. The calli were dried by blotting on sterile filter paper and blowing with hood wind for 15 to 30 min and were then cultured on selective medium (NB containing 50 mg/L hygromycin, 200 mg/L carbenicillin, and 200 mg/L cefotaxime). After 25 d to 30 d, hygromycin-resistant


(Hyg+) calli were collected and subcultured on fresh selective medium for 15 to 20 d. Hyg+GFP+ secondary calli were transferred to regeneration medium (NB containing 3 mg/L 6-benzyl aminopurine and 0.5 mg/L naphthalene acetic acid). After 20 to 30 d, Hyg+ plantlets were regenerated and transferred to rooting medium (1/2 MS salts, 2% sucrose, and 0.3% phytagel, pH 5.8). After 10 to 15 d, the transformed rice plants (T0) were transplanted into the soil.

GFP Fluorescence Assay The transformed rice calli and plants were examined under a fluorescent protein macro detector set (BLS, Hungary) using an appropriate excitation source (460 to 495 nm) and filter (FHS/EF-2G2, 505 to 515 nm). Fluorescence images of calli in Petri dishes were obtained under the detector using a digital camera (SONY, α550) with a GFP filter.

Rice Genomic DNA Extraction Rice leaf tissue was frozen in liquid nitrogen and ground to a fine powder. The frozen tissue was homogenized in DNA extraction buffer (100 mM Tris–HCl, pH 8.0; 10 mM EDTA, pH 8.0; 1 M KCl). The tube was incubated in a water bath at 75 °C for 20 min and then centrifuged at 12,000 rpm for 10 min. The supernatant was transferred into a new tube, and an equal volume of isopropanol was added. The solution was gently mixed and then centrifuged at 12,000 rpm for 5 min. After washing with 70% ethanol and drying in air, the DNA pellet was dissolved in 40 μL of distilled water.

PCR Amplification of Transposon EDS and FDS The transposon EDS and FDS were amplified in PCR reactions using the primers P1

(5'-GCAAAGACCCCAACGAGAAG-3')

(5′-CGGGAGATGCAATAGGTCAG-3′),

and

primers

and P1

P2 and

P3

(5′-TTCCTTGTGGACTTGGATCTG-3′), respectively. The expected sizes of the 22 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

EDS-PCR and FDS-PCR products were 1870 and 711 bp, respectively. The cycling parameters for EDS-PCR were as follows: 95 °C for 5 min; 33 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min; and 72 °C for 7 min. The cycling parameters for FDS-PCR were as follows: 95 °C for 5 min; 33 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; and 72 °C for 7 min. EDS-PCR bands were cloned into pMD18-T, and multiple clones from each band were selected for sequencing. The EDS sequences were analyzed using the online multiple alignment program ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

PCR Amplification of Bt, HPT, and GFP in Transgenic Rice Plants The primers for amplification of the Bt, HPT, and GFP genes were as follows: Bt, 5′-ATCAACAACCAGCAACTTTC-3′

(forward)

and

5′-GTAGATGTGGATGGGAAGTG-3′

(reverse);

HPT,

5′-ATGAAAAAGCCTGAACTCACC-3′ 5′-GGAACCCTAATTCCCTTATCT-3′ 5′-GTGAGCAAGGGCGAGGAG-3′

(forward) (reverse);

and and

(forward)

GFP, and

5′-CTTGTACAGCTCGTCCATGCC-3′ (reverse). The sizes of the amplified Bt, HPT, and GFP sequences were 452, 1116, and 714 bp, respectively. All PCR reactions were performed under the following conditions: 95 °C for 5 min; 33 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; and 72 °C for 7 min.

TAIL-PCR of Ds and T-DNA Flanking Sequences To isolate Ds flanking sequences, three rounds of TAIL-PCR reaction were performed using the conditions described by Liu and Chen (2007) with some modifications. Primary amplification reaction (20 µL) contained 2.0 µL of PCR buffer; 0.5 U Takara Ex Taq; 200 µM each of dATP, dCTP, dGTP, and dTTP; 0.3 µM each of the 5′Ds- or 3′Ds-specific primers (Kolesnik et al., 2004); 1.0 µM each of degenerate primers (LAD-1, LAD2, LAD3, or LAD4, Liu and Chen, 2007); and 20 ng of rice DNA. To isolate T-DNA flanking sequences, TAIL-PCR reactions were performed 23 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

using the primers and thermal cycling conditions described by Liu and Chen (2007). The PCR reactions were conducted in the Veriti® Thermal Cycler (Applied Biosystems, USA). Amplification reaction products were fractionated on 1.0% agarose gels. PCR bands were cut from the gels and purified using a PCR DNA purification kit for sequencing. The Ds or T-DNA flanking sequences were confirmed by PCR using a T-DNA or Ds-specific primer and a flanking sequence-specific primer.

Southern Blot Analysis Southern blot hybridization was conducted using PCR DIG Probe Synthesis Kit and DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) according to the manufacturer’s instructions. Twenty micrograms of rice genomic DNA was digested to completion with 10 U of EcoRI, fractionated on 1% agarose gel and transferred onto Amersham Hybond N++ membrane (GE Healthcare, USA). The HPT, GFP and Ds probes were synthesized using PCR DIG Probe Synthesis Kit. The amplification sizes and primers for the probes were as follows: HPT, 840bp, 5′-GCGTGGATATGTCCTGCGGG-3′ and 5′ -TCTACACAGCCATCGGTCCAG-3′; GFP,

721bp,

5′-CCATGGTGAGCAAGGGCGAG-3′

5′-TACTTGTACAGCTCGTCCATGC-3′;

Ds,

and 822bp,

5′-ACGACTCCATTCCTCAGATGAC-3′

and

5′-TCTAGCTTCTTTAGGCTAACCAC-3′. DNA membranes were hybridized with the probes in DIG Easy Hyb solution at 42°C. After hybridization, the membranes were washed at 65°C, two times with 2× SSC and 0.1% SDS for 10 min and two times with 0.1× SSC and 0.1% SDS for 15 min. Probe hybridization signal was examined by DIG chemiluminescence detection. Membranes were stripped afterwards by washing at 37°C, two times with 0.2N NaOH and 0.1% SDS for 10 min and one time with 2× SSC for 5 min. Further hybridization was performed using DL15000 molecular weight marker (Takara, Japan) that was labeled using DIG High Prime DNA Labeling Kit.


RNA Isolation and RT-PCR Analysis Transcripts of the Ds-based Bt gene in transgenic rice plants were detected in RT-PCR reactions. Rice RNA was isolated using RNeasy Plant Mini Kit (Qiagen, USA) following the manufacturer’s instructions. cDNA was synthesized using the Superscript II® Reverse Transcription System (Invitrogen, USA). For the RT-PCR analysis of T0 plants, a 452 bp sequence was amplified from the Bt cDNA using the primers Bt-forward-T0 (5′-ATCAACAACCAGCAACTTTC-3′) and Bt-reverse-T0 (5′-GTAGATGTGGATGGGAAGTG-3′). For the RT-PCR analysis of the marker-free T1 plants, 500 bp of Bt cDNA sequence was amplified using the primers Bt-forward-T1

(5′-ACAGAAGACCCTTCAATATCGG-3′)

and

Bt-reverse-T1

(5′-ATACCTCACACGAACTCTATATC-3′ ). For the rice actin gene, a 795 bp cDNA sequence

was

amplified

using

the

(5′-GGAACTGGTATGGTCAAGGC-3′)

primers and

Actin-forward Actin-reverse

(5′-AGTCTCATGGATACCCGCAG-3′). The cycling conditions for Bt amplification were as follows: 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s; and 72 °C for 7 min. The cycling conditions for Actin amplification were as follows: 95 °C for 5 min; 28 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; and 72 °C for 7 min.

ELISA of the Bt Protein in Transgenic Rice ELISA was conducted using the DAS ELISA kit to detect the Bt-Cry1Ab/1Ac proteins (Cat No. PSP 06200, Agdia Inc., USA). Rice leaf tissue (5 g) was ground in 5 mL of 1× PBST buffer, and the supernatant was prepared by centrifugation (10000 rpm, 1 min). The supernatant (100 µL) of each sample was added into a well coated with the Bt-Cry1Ab/1Ac antibody in the ELISA plate. The contents were mixed with 100 µL of the enzymatic conjugate and then incubated at 25 °C for 2 h. The ELISA plate was washed seven times in an immunowash microplate washer (Model 1575, Bio-Rad Laboratories, Inc., USA). A 100 µL aliquot of TMB substrate solution was 25 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

added to each well, and the mixed contents were incubated for 20 min. The Bt protein in the samples was detected using the optical density of color development measured at 450 nm in a microplate reader (Model 680, Bio-Rad Laboratories, Inc., USA). All samples were assayed in duplicate, and positive and negative controls (Agdia Inc., USA) were included.

Bioassay of Rice Plants against Striped Stem Borer For each treatment, rice leaves were placed on wet cotton in a finger-shaped test tube to maintain humidity, and then six larvae of striped stem borer at the second instar stage were placed in the tube for feeding. The mouth of each tube was covered with a cotton ball. The tubes were randomly assigned to the treatments. Three independent biological replicates were performed for each treatment. The survival of striped stem borer was monitored on a daily basis, and larvae mortality and leaf damage were continuously recorded for 4 to 5 d. Percent corrected mortality was calculated using Abbott’s formula: % Corrected mortality = [(% Kill in treated – % Kill in control)/(100 – % Kill in control)] × 100.

Field Evaluation of Marker-Free Transgenic Rice Plants The rice seeds of marker-free transgenic lines were imbibed in water at 35 °C for 72 h, wrapped with wet towels, and then incubated at 30 °C for 72 h. The untransformed parent cultivar Kongyu-131 was tested as a control. Germinating seeds were grown in a seedling bed for 25 d, and the young plants were transplanted to a well-isolated paddy field on July 16, 2013. The plot for each rice line consisted of three rows with 12 plants per row, and two replications were arranged for each line. For each plot, ten plants (from the second to the eleventh) in the middle row were selected to evaluate agronomic traits. The average calendar day on which 50% of the plants showed heads was recorded as heading date, and days to heading were recorded as the number of days from seeding to heading. The tiller number, plant height, panicle number, and flag leaf length of each rice plant were evaluated on September 26 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

30, 2013 using the IRRI standard evaluation system for rice (IRRI, 1996).

Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. PCR amplification of the EDS, Ds(Bt), and GFP sequences in the #31 to #45 pZJ54/Zhejing-22 T0 plants. Supplemental Figure S2. Multiple sequence alignment of the Ds excision sites in pZJ54/Zhejing-22 transformants. Supplemental Figure S3. PCR amplification and sequence analysis of direct T-DNA repeats in pZJ53/Zhejing-22 T0 plants. Supplemental Figure S4. PCR amplification and sequence analysis of direct T-DNA repeats in pZJ54/Zhejing-22 T0 plants. Supplemental Figure S5. Sequence of the 1244 bp EDS-PCR product of the #4 pZJ54/Zhejing-22 T0 plant. Supplemental Figure S6. Sequence of the 917 bp EDS-PCR product of the #4 pZJ54/Zhejing-22 T0 plant. Supplemental Figure S7. Southern blot analysis of marker-free transgenic rice plants. Supplemental Figure S8. RT-PCR of the Ds-based Bt gene in pZJ54/Zhejing-22 T0 plants. Supplemental Table S1. Mortality rates of rice striped stem borer fed on young leaves of two pZJ53/Zhejing-22 transformants (T0). Supplemental Table S2. Evaluation of the agronomic traits of two marker-free transgenic rice lines.

ACKNOWLEDGEMENTS The authors would like to thank Dr. Venkatesan Sundaresan for providing plasmid pSQ5 as well as Zhengjie Yuan, Zhonglai Liu, Yali Li, and Haiyan He for their technical assistance. 27 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

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progeny of co-transformed barley plants. Plant Mol Biol 81: 149-60 Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52: 761-773 Kolesnik T, Szeverenyi I, Bachmann D, Kumar CS, Jiang S, Ramamoorthy R, Cai M, Ma ZG, Sundaresan V, Ramachandran S (2004) Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences. Plant J 37: 301-314 Lazarow K, Doll ML, Kunze R (2013) Molecular biology of maize Ac/Ds elements: an overview. Methods Mol Biol 1057: 59-82 Liu YG, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43: 649-650, 652, 654 passim Machida C, Onouchi H, Koizumi J, Hamada S, Semiarti E, Torikai S, Machida Y (1997) Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI. Proc Natl Acad Sci USA 94: 8675-8680 Michelmore RW, Paran I, Kesseli RV (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA 88: 9828–9832 Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Biotechnol 107: 193-232 Nakagawa Y, Machida C, Machida Y, Toriyama K (2000) Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants. Plant Cell Physiol 41: 733-742 Oliva N, Chadha-Mohanty P, Poletti S, Abrigo E, Atienza G, Torrizo L, Garcia R, Dueñas C Jr, Poncio MA, Balindong J et al. (2014) Large-scale production and evaluation of marker-free indica rice IR64 expressing phytoferritin genes. Mol Breed 33: 23-37 Qu S, Desai A, Wing R, Sundaresan V (2008) A versatile transposon-based activation tag vector system for functional genomics in cereals and other monocot plants. Plant Physiol 146: 189-199 Qu S, Jeon JS, Ouwerkerk PB, Bellizzi M, Leach J, Ronald P, Wang GL (2009) Construction and application of efficient Ac-Ds transposon tagging vectors in rice. J Integr Plant Biol 51: 982-992 Rai M, Datta K, Parkhi V, Tan J, Oliva N, Chawla HS, Datta SK (2007) Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice. Plant Cell Rep 26: 1221-1231 Ramessar K, Peremarti A, Gomez-Galera S, Naqvi S, Moralejo M, Munoz P, Capell T, Christou P (2007) Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics. Transgenic Res 16: 261-280 Ros F (2001) Regulation of maize Ac/Ds transposition by replication and DNA 29 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

methylation. PhD thesis. University of Munich, Munich Sallaud C, Meynard D, van Boxtel J, Gay C, Bès M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M et al. (2003) Highly efficient production and characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics. Theor Appl Genet 106: 1396-1408 Scott L, LaFoe D, Weil CF (1996) Adjacent sequences influence DNA repair accompanying transposon excision in maize. Genetics 142: 237-246 Smith D, Yanai Y, Liu YG, Ishiguro S, Okada K, Shibata D, Whittier RF, Fedoroff NV (1996) Characterization and mapping of Ds-GUS-T-DNA lines for targeted insertional mutagenesis. Plant J 10: 721-732 Sundar IK, Sakthivel N (2008) Advances in selectable marker genes for plant transformation. J Plant Physiol 165: 1698-1716 Sundaresan V, Springer P, Volpe T, Haward S, Jones JD, Dean C, Ma H, Martienssen R (1995) Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev 9: 1797-1810 Szeverenyi I, Ramamoorthy R, Teo ZW, Luan HF, Ma ZG, Ramachandran S (2006) Large-scale systematic study on stability of the Ds element and timing of transposition in rice. Plant Cell Physiol 47: 84-95 Tu J, Zhang G, Datta K, Xu C, He Y, Zhang Q, Khush GS, Datta SK (2000) Field performance of transgenic elite commercial hybrid rice expressing bacillus thuringiensis delta-endotoxin. Nat Biotechnol 18: 1101-1104 Walbot V (2000) Saturation mutagenesis using maize transposons. Curr Opin Plant Biol 3: 103-107 Wang Y, Yau Y-Y, Perkins-Balding D, Thomson J (2011) Recombinase technology: applications and possibilities. Plant Cell Rep 30: 267-285 Yau Y-Y, Stewart CN Jr (2013) Less is more: strategies to remove marker genes from transgenic plants. BMC Biotechnol 13: 36 doi:10.1186/1472-6750-13-36


Table I. GFP fluorescence assays and PCR analysis of T1 transgenic rice plants The rice lines of each vector/cv combination were randomly selected but not based on their parental EDS-PCR patterns. Except for the Kongyu-131 transgenic lines, the parents (T0) of the Zhejing-22 transgenic lines were assayed by EDS-PCR. The parents of the #13 and #43 pZJ54/Zhejing-22 lines were EDS-negative (EDS–), and the parents of the other Zhejing-22 lines showed EDS+ patterns (Fig. 3; Supplemental Fig. S1; the parental plants are numbered the same as T1 lines). a, significant level; b, the most significant level; ND, Not determined. GFP fluorescence assays Vector / cv

Line No.

PCR analysis of non-fluorescent plants

Positive

Negative

χ2 3:1

Plants

Bt+

HPT+

GFP+

Bt+HPT-GFP–

9

22

21

11.79 b

8

0

0

0

0

10

46

47

31 b

27

0

0

0

0

11

71

31

1.31

24

15

2

0

13

12

20

37

46.32 b

13

0

0

0

0

13

33

10

0.01

3

0

0

0

0

14

32

39

32.34 b

25

10

0

0

10

15

53

23

0.84

20

0

0

0

0

16

38

5

3.42

1

0

0

0

0

17

39

18

0.99

11

1

1

1

0

18

23

17

5.63 a

8

4

4

0

0

pZJ53 /

20

57

16

0.22

12

0

0

2

0

Kongyu-131

21

56

13

1.09

6

0

0

0

0

22

38

29

11 b

24

3

0

0

3

23

56

19

0.00

11

0

1

0

0

24

10

2

0.11

2

0

0

0

0

25

35

38

27.07 b

23

0

0

0

0

pZJ53 / Zhejing-22

26

49

31

7.35 b

24

0

0

0

0

27

43

55

48.98 b

43

2

2

2

0

28

64

11

3.74

10

0

0

0

0

29

65

26

0.44

22

16

0

0

16

30

25

22

10.79 b

13

1

0

0

1

31

29

34

26.67 b

31

0

0

0

0

1

42

27

6.61 a

27

0

ND

ND

0

2

43

12

0.15

12

0

ND

ND

0

3

29

27

14.88 b

27

0

ND

ND

0

4

21

8

0.01

8

3

0

0

3

5

7

5

0.75

5

0

ND

ND

0

7

30

7

0.44

7

0

ND

ND

0

9

41

28

8.12 b

28

0

ND

ND

0

10

27

39

39.11 b

39

0

ND

ND

0

11

5

7

5.44 a

7

0

ND

ND

0


Table I (cont'd). GFP fluorescence assays and PCR analysis of T1 transgenic rice plants GFP fluorescence assays Vector / cv

pZJ54 / Zhejing-22

Line No.

PCR analysis of non-fluorescent plants

Positive

Negative

χ2 3:1

Plants

7

14

16

11.38 b

11

5

0

0

5

8

23

17

5.63 a

8

0

ND

ND

0

9

27

21

8.03 b

12

0

ND

ND

0

11

44

19

0.64

13

0

ND

ND

0

13

44

20

1.02

16

0

ND

ND

0

16

21

13

2.51

7

5

ND

ND

5

30

41

20

1.58

10

5

0

1

4

31

41

30

10.37 b

18

0

ND

ND

0

36

14

16

11.38 b

11

5

0

0

5

39

44

19

0.64

13

0

ND

ND

0

41

44

20

1.02

16

0

ND

ND

0

42

23

17

5.63 a

8

0

ND

ND

0

43

27

21

8.03 b

12

0

ND

ND

0

44

21

13

2.51

7

5

0

0

5

45

41

20

1.58

10

5

1

1

4

Bt+

HPT+

GFP+

Bt+HPT-GFP–


Table II. Genome positions of the transposed Ds elements and the donor T-DNAs For each rice line, the reintegrated Ds(Bt) elements in the marker-free T1 plants and T-DNA in the T0 parental plant were mapped to the rice genome on the basis of Ds(Bt) and T-DNA flanking sequences. For each of the three lines, the Ds(Bt) in the marker-free T1 plants showed the same integration position. Vector / cv

pZJ53/Kon gyu-131

pZJ53/Zhej ing-22

Line No.

No. of marker-free T1 plants

11

13

14

10

4

3

Ds(Bt)

T-DNA

position

position

Chr10:

Chr01:

4058932

17977177

Chr02:

Chr07:11

28926474

479203

Chr04:

Chr12:

33940231

19381789


Table III. EDS-PCR assays of the GFP+ plants from pZJ53-transformed T1 lines Line 4, #4 pZJ53/Zhejing-22 T1 line. Lines 9 to 31, pZJ53/Kongyu-131 T1 lines. Individual GFP+ plants were selected from each line and subjected to DNA extraction and PCR analyses. Line

GFP+ plants

EDS+

Percentage of

No.

analyzed

plants

EDS+ plants (%)

4

11

11

100

9

22

0

0

10

40

40

100

12

19

17

89.5

13

29

17

58.6

15

10

0

0

16

10

7

70

17

10

0

0

18

10

10

100

20

10

5

50

21

10

3

30

23

10

8

80

24

8

0

0

25

10

0

0

26

10

0

0

27

10

0

0

28

10

0

0

31

10

0

0


Table IV. ELISA assay of the Bt protein in marker-free transgenic rice plants 11-2 and 14-2, marker-free T2 lines from the #11 and #14 pZJ53/Kongyu-131 T0 plants, respectively. 4-2, marker-free T2 line from the #4 pZJ53/Zhejing-22 T0 plant. Kongyu-131 and Zhejing-22, untransformed cultivars. T51-1, a Bt-transgenic rice line (Tu et al., 2000). Mean and standard deviation (SD) were calculated based on three measurements of optical density of the protein sample at 450 nm. Line

11-2

14-2

Kongyu-131

4-2

Zhejing-22

T51-1

Plant No.

1

2

3

1

2

1

1

2

1

1

Mean

0.198

0.196

0.198

0.199

0.188

–0.002

0.188

0.188

–0.001

0.215

SD

0.007

0.003

0.000

0.014

0.017

0.007

0.004

0.008

0.006

0.007


Table V. Mortality rates of rice striped stem borer fed on young leaves of two marker-free T2 lines 11-2, marker-free T2 line from the #11 pZJ53/Kongyu-131 T0 plant; 4-2, marker-free T2 line from the #4 pZJ53/Zhejing-22 T0 plant. Four plants per T2 line were tested, and the Ds-based Bt in each plant was confirmed by PCR. Average mortality (%)

Day after

Kongyu

Corrected average mortality (%) Zhejing

Kongyu-

Zhejing-

treatment

11-2

Day 1

15.6

10.0

33.3

10.0

6.2

0.0

25.9

0.0

Day 2

24.4

10.0

36.7

10.0

16.0

0.0

29.7

0.0

Day 3

33.3

10.0

50.0

10.0

25.9

0.0

44.4

0.0

Day 4

68.9

13.3

73.3

16.7

64.1

0.0

67.9

0.0

-131

4-2

-22

11-2

131

4-2

22


Figure legends Figure 1. Ac/Ds transposon vectors for the generation of marker-free transgenic plants. A, pLJ26, a universal vector for transposon-mediated transgene reintegration; pLJ16, an intermediate cloning vector for assembling the DNA fragments of the gene of interest (GOI). Assembled GOI can be released by AscI and inserted into the unique AscI site within the Ds element in pLJ26. B, Ds-mediated transgene reintegration. In the presence of AcTPase, the Ds-based GOI in transformed cells (T0) excised from T-DNA and reintegrated into an unlinked chromosomal site. Recombination between transposed Ds and its donor T-DNA produced a marker-free T1 plant. The transposon empty donor site (EDS) and full donor site (FDS) can be amplified in PCR using the primer pairs P1/P2 and P1/P3, respectively (Figs. 2C and 3). C, pZJ53 and pZJ54, Ac/Ds vectors for marker-free transformation of the Bacillus thuringiensis (Bt) ð-endotoxin gene. The Bt-1 in pZJ53 and Bt-2 in pZJ54 both have the coding sequence of the Bt ð-endotoxin, but Bt-2 contains an additional 5′ coding sequence of a chloroplast transit peptide (see Materials and Methods). Abbreviations: R and L, T-DNA right and left borders, respectively; AcTPase, Ac transposase; GFP, green fluorescent protein; 3′Ds and 5′Ds, 3′ and 5′ termini of the Ds element; HPT, hygromycin phosphotransferase.

Figure 2. GFP assay and EDS-PCR analysis of transformed rice calli. A, GFP expressed in Hyg+ secondary calli. The calli of rice cv Zhejing-22 were co-cultivated with Agrobacterium harboring the pZJ54 vector, cultured on hygromycin medium for 27 d, and then assayed under a GFP detector. B, Hyg+ secondary calli after 35 d of selection culture. C, Ds excision assay of transformed rice calli. Nine independent Hyg+GFP+ calli of the pZJ54/Zhejing-22 transformation were randomly selected for rice DNA extraction. DNA was amplified using primers P1 and P2 (Fig. 1B) to detect the EDS sequence. For rice calli marked with an asterisk, 1.8 kb EDS-PCR bands were purified and sequenced for further analysis (Supplemental Fig. S2). M, Trans 2K plus II DNA marker (TransGen Biotech, China); 1 to 9, Hyg+GFP+ calli; vector,


pZJ54.

Figure 3. PCR amplification of the Ds empty donor site (EDS), full donor site (FDS), Ds(Bt), and GFP sequences in T0 plants. A, 14 plantlets from the pZJ53/Zhejing-22 transformation. B, 30 adult plants (#1 to #30) from the pZJ54/Zhejing-22 transformation. When the pZJ53-transformed plantlets were transplanted to soil or when the pZJ54-transformed plants grown in soil were at the onset of the tillering stage, 200 to 300 mg of leaf tissue was collected from each plant for rice DNA extraction and PCR amplification. For plants marked with an asterisk, the EDS-PCR bands were sequenced for further analysis (Supplemental Fig. S2). M, GeneRuler 1 Kb DNA ladder (Fermentas, Canada).

Figure 4. PCR amplification of the Bt, HPT, and GFP sequences in marker-free T1 plants. Results of one marker-free plant per T1 line are shown. M, Trans 2K plus DNA marker (TransGen Biotech, China).

Figure 5. EDS-PCR and FDS-PCR analyses of GFP+ plants from three pZJ53-transformed T1 lines. A, All 11 GFP+ plants of the #4 pZJ53/Zhejing-22 line (Table III). B, 24 GFP+ plants from the #10 pZJ53/Kongyu-131 line. C, 24 GFP+ plants from the #13 pZJ53/Kongyu-131 line. M, Trans 2K plus DNA marker.

Figure 6. RT-PCR of the Ds-based Bt gene in marker-free T1 plants. 11-1, 11-2, and 11-3, marker-free T1 plants segregated from the #11 pZJ53/Kongyu-131 line; 14-1 and 14-2, from the #14 pZJ53/Kongyu-131 T1 line; 4-1 and 4-2, from the #4 pZJ53/Zhejing-22 T1 line. The Ds(Bt) elements in the marker-free plants of each line were reintegrated at the same genome site (Table II). Kongyu-131 and Zhejing-22, untransformed rice cultivars.

Figure 7. Rice genes adjacent to Ds(Bt) reintegration sites in three marker-free transgenic lines. A, Ds insertion (Chr10:4058932) in the 11-2 pZJ53/Kongyu-131 line 38 Downloaded from www.plantphysiol.org on November 11, 2014 - Published by www.plant.org Copyright © 2014 American Society of Plant Biologists. All rights reserved.

[2941 bp upstream from the Os10g07552 coding sequence (CDS) and 4264 bp upstream from Os10g07548 CDS]. B, Ds insertion (Chr02:28926474) in the 14-2 pZJ53/Kongyu-131 line (3394 bp upstream from the Os02g47400 CDS and 2309 bp upstream from the Os02g47390 CDS). C, Ds insertion (Chr04:33940231) in the 4-2 pZJ53/Zhejing-22 line (within the Os04g57320 CDS).


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Supplemental Figures

M 2000bp 1500bp 1000bp 750bp 500bp

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45 EDS

Bt

250bp 1000bp 750bp 500bp

GFP

Supplemental Figure S1. PCR amplification of the EDS, Ds(Bt), and GFP sequences in the #31 to #45 pZJ54/Zhejing-22 T0 plants. Rice genomic DNA was isolated from the T0 plants at the onset of the tillering stage and analyzed by PCR using EDS-, Bt-, and GFP-specific primers (see Material and Methods). M, GeneRuler 1Kb DNA ladder (Fermentas, Canada).

Ds(Bt)

Supplemental Figure S2. Multiple sequence alignment of the Ds excision sites in pZJ54/Zhejing-22 transformants. EDS-PCR products of two Hyg+GFP+ calli and five Hyg+GFP+ plants were cloned into the pMD18-T vector and sequenced. The sequences at the Ds empty donor site of six clones per callus and two clones per plant were aligned using ClustalW2 program. C2-1 to C2-6, clones from the #2 callus (Fig. 2C). C3-1 to C3-6, clones from the #3 callus. P16-1, P16-2, P19-1, P19-2, P24-1, P24-2, P29-1, P29-2, P30-1, and P30-2, respective clones from the #16, #19, #24, #29, and #30 plants (Fig. 3B). Nucleotide deletion and substitution are shown in dashes and lowercase letters, respectively. The Ds cloning site in the pZJ54 vector is indicated by an arrow.

P4

A

T0

3’Ds

GOI

P5

L R AcTPase

HPT

5’Ds T-DNA 1

B

M

1

2

GFP

T-DNA 2

3

5

6

7

9

10

11

14

2000bp 1000bp 500bp

pZJ53 vector

pZJ53 vector

LB RB 27bp+ :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

#3 pZJ53 vector

pZJ53 vector

LB RB 287bp+

#6

▽ 8bp :::::::::::::::::::::::::::::::::::::::::::::::

Supplemental Figure S3. PCR amplification and sequence analysis of direct T-DNA repeats in pZJ53/Zhejing-22 T0 plants. A, Outward-directed primers [P4 (5′-ACTGTCGGGCGTACACAAATCG-3′) and P5 (5′TACGTCAGTGGAGATATCACAT-3′)] specific to T-DNA ends were designed to amplify direct T-DNA repeats. B, Direct repeats were detected in two of the ten pZJ53/Zhejing-22 T0 plants (shown in red). The PCR bands were cloned into pMD18-T and then sequenced. The T-DNA junction in the #3 plant contained deletion of LB, RB, 11 bp upstream of LB, and 27 bp downstream of RB. For the #6 plant, RB, 13 bp in LB, and 287 bp downstream of RB were deleted while an 8 bp filler DNA was inserted (indicated by an arrow). M, Trans 2K plus DNA marker (TransGen Biotech, China).

A

M 4

6 16 20 22 24 29

2000bp 1000bp 500bp

B

M 2

3 9 10 11 12 14 17 21 23 25 26 27 28

2000bp 1000bp 500bp

pZJ54 vector

LB

pZJ54 vector RB 17bp+

#4

▽ 62bp ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

#9

▽ 6bp ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ▽ 1bp :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

#10 #20

::::::::::::::::::::::::::::: :::::::::::::::::::::::::::::::::::::::::::::::::::

#22

:::::::::::::::::::::::::::::::::::::::::::::::

#26 pZJ54 vector

pZJ54 vector +152bp LB

#6 #11

RB 17bp+

▽ 20bp ::::::::::::::::::::::::::::::::: :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

Supplemental Figure S4. PCR amplification and sequence analysis of direct T-DNA repeats in pZJ54/Zhejing-22 T0 plants. A, P4 and P5 (Supplemental Fig. S2A) were used to amplify the junction between tandem-linked T-DNAs in 21 pZJ54/Zhejing-22 plants (Fig. 3B). Direct T-DNA repeats in eight plants (shown in red) were confirmed by sequencing the PCR products. M, Trans 2K plus DNA marker. B, Junctions in the eight plants were analyzed by multiple sequence alignment. For each T-DNA involved, the deletion ranged from 6 to 221 bp, and the filler DNA (indicated by an arrow) ranged from 1 to 62 bp.

GCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACATACCACTT GTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCTACTTACTTTGCCATCTTTCACAAAGATGTT GCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAAT CATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGCCTC CTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTAC AAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGAT GGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGA TTGATGTGAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGA CCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGC TATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAA GGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAG CATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGA CGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCAT TTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCG GGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGT TCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTA GGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTAT CGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCT GACCTATTGCATCTCCCG Supplemental Figure S5. Sequence of the 1244 bp EDS-PCR product of the #4 pZJ54/Zhejing-22 T0 plant. The EDS-PCR bands of the #4 pZJ54/Zhejing-22 T0 plant (Fig. 3B) were cloned into pMD18-T and sequenced. For the 1244 bp EDS-PCR band, Ds excision induced an 828 bp deletion in the flanking Ubi-GFPTnos and CaMV35S-HPT-T35S regions; 418 bp including 43 bp of the 3′ GFP coding sequence and 253 bp of Tnos were deleted from the GFP side, and 410 bp including 359 bp of the 5′ CaMV35S promoter sequence were deleted from the HPT side. Between GFP and HPT is a 202 bp vector backbone sequence that was co-transferred with T-DNA during Agrobacterium-mediated transformation. GFP, in bold letters. CaMV 35S-HPT, in italic letters. Vector backbone sequence, underlined.

GCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACATACCACTT GTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCTACTTACTTTGCCATCTTTCACAAAGATGTT GCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAAT CATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGCCTC CTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTAC AAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGAT GGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGA TTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCT ATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCT CTCGAGCTTTCGCAGATCCGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCG AGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCT CGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTT CTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACAT TGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCG Supplemental Figure S6. Sequence of the 917 bp EDS-PCR product of the #4 pZJ54/Zhejing-22 T0 plant. The 917 bp EDS-PCR band was cloned into pMD18-T and sequenced. On the basis of the sequencing results, Ds excision induced a 1155 bp deletion in the flanking Ubi-GFP-Tnos and CaMV35S-HPT-T35S regions; 418 bp including 43 bp of the 3′ GFP coding sequence and 253 bp of Tnos were deleted from the GFP side, and 737 bp including 686 bp of the 5′ CaMV35S promoter sequence were deleted from the HPT side. Between GFP and HPT is 202 bp of the vector backbone sequence co-transferred with T-DNA. GFP, in bold letters. CaMV 35S-HPT, in italic letters. Vector backbone sequence, underlined.

A

M

1

2

Ds probe

C

HPT probe 3

4

5

6

M

7

1

2

3

4

5

6

7

10K 7.5K

10K 7.5K

5.0K

5.0K

2.5K

2.5K 1.0K

B

D

GFP probe M

1

2

10K 7.5K 5.0K

2.5K

Supplemental Fig. S7

3

4

5

6

T-DNA structure

7 o Ec

RI

o Ec

RI

1.5 kb

R

AcTPase

o Ec

RI

o Ec

RI

o Ec

RI

1.6 kb

GFP GFP probe (721 bp)

3’Ds

Bt-1/2

o Ec

RI

o Ec

RI

1.8 kb

1.8 kb

5’Ds

HPT

Ds probe (822 bp)

HPT probe (840 bp)

L

Supplemental Figure S7. Southern blot analysis of marker-free transgenic rice plants. DNA membranes were hybridized using HPT (A), GFP (B) and Ds (C) probes, respectively, which were labeled with digoxigenin-11-dUTP (see Materials and Methods). The positions of individual probe sequences in T-DNA are shown (D). Lane 1, 0.03ng of EcoRI-digested pZJ53 vector; lanes 2 and 3, untransformed Kongyu-131 and Zhejing-22; lane 4 in A and B, a GFP+ plant from the #31 pZJ53/Kongyu-131 T1 line (Table 1); lane 4 in C, the #31 pZJ54/Zhejing-22 T0 plant (Supplemental Fig. S1); lanes 5 and 6, the #11 and #14 pZJ53/Kongyu-131 marker-free lines (T3); lane 7, the #4 pZJ53/Zhejing-22 marker-free line (T3). For each rice material, 20 µg of EcoRI-digested genomic DNA was loaded. M, DL15000 DNA marker (TaKaRa, Dalian, China). The DNA membranes were hybridized again using DIG-labeled DL15000 after removing the HPT, GFP and Ds probes. As indicated by an arrow, the HPT probe hybridized to the HPT sequence near T-DNA left border (A, lane 4). The GFP probe hybridized to a 1.6 kb T-DNA fragment (B, lanes 1 and 4). The Ds probe hybridized to a 1.5 kb AcTPase/EcoRI fragment and a 1.8 kb 5'Ds/EcoRI fragment in T-DNA (C, lanes 1 and 4), and to reintegrated Ds(Bt) elements in marker-free plants (C, lanes 5, 6 and 7).

Zhejing-22

45

43

34

31

pZJ54/Zhejing-22 T0

452bp

Bt

30 cycles

795bp

Actin

28 cycles

Supplemental Figure S8. RT-PCR of the Ds-based Bt gene in pZJ54/Zhejing-22 T0 plants. The #31, #34, #43, and #45 plants were independent transformants from pZJ54/Zhejing-22 transformation. Zhejing-22, untransformed control.

Supplemental Table S1. Mortality rates of rice striped stem borer fed on young leaves of two pZJ53/Zhejing-22 transformants (T0) #1 and #12, independent pZJ53/Zhejing-22 transformants. For each transformant, three plantlets (i.e. regenerants from the same callus) were tested. Average mortality (%) Day after treatment

pZJ53/Zhejing-22 plants #1

#12

Day 1

5.6

16.7

Day 2

44.4

Day 3

Corrected average mortality (%)

Zhejing-22

pZJ53/Zhejing 22 plants

Zhejing-22

#1

#12

5.6

0.0

11.8

0.0

66.7

11.1

37.5

62.5

0.0

66.7

83.3

22.2

57.2

78.5

0.0

Day 4

88.9

88.9

38.9

81.8

81.8

0.0

Day 5

100.0

100.0

55.6

100.0

100.0

0.0

Supplemental Table S2. Evaluation of the agronomic traits of two marker-free transgenic rice lines 11-2 and 14-2, marker-free T3 lines derived from the #11 and #14 pZJ53/Kongyu-131 T0 plants, respectively. The plants were grown in two replicates (ten plants per replicate). Data in columns 2-5 are the mean ± standard error. Values with different letters are significantly different (P

Transgenic indica rice plants.

Design rules for efficient transgene expression in plants.

An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo.

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing.

Transposition of the maize activator element in transgenic rice plants.

An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.

Virus-induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation.

Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions.

Phytoremediation of Explosives using Transgenic Plants.

Creation of transgenic rice plants producing small interfering RNA of Rice tungro spherical virus.

An improved rice transformation system using the biolistic method.

Analysis of rice Act1 5' region activity in transgenic rice plants.

Efficient construction of unmarked recombinant mycobacteria using an improved system.

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification.

Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition.

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

Generation of Transgenic Rats Using Lentiviral Vectors.

Cas9 system.

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method.

An Efficient Method for Generation of Transgenic Rats Avoiding Embryo Manipulation.

Using inositol as a biocompatible ligand for efficient transgene expression.

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.

Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.