Annals of Botany 90: 31-36, 2002
© 2002 Annals of Botany Company
Fluorescence in situ Hybridization Analysis of Alien Genes in Agrobacterium-mediated Cry1A(b)-transformed Rice
1 Key Lab of MOE for Plant Developmental Biology, Wuhan University, Wuhan 430072, P. R. China, 2 Institute of Biotechnology, Hainan University, Haikou 570228, P. R. China and 3 Department of Chemistry, Faculty of Science, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5
* For correspondence. E-mail ycsong{at}whu.edu.cn
Received: 8 January 2002; Returned for revision: 5 March 2002; Accepted: 10 April 2002
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ABSTRACT |
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The transgene in Agrobacterium-mediated Cry1A(b)-transgenic rice plants has been detected and its chromosomal location determined by fluorescence in situ hybridization (FISH). Eight of the nine transgenic lines tested showed hybridization signals. Signals were located on regions of the chromosome in which fraction length (FL) values varied from 26·2 (near the centromere) to 95·2 (distal regions). No signal was found on regions where the fraction length was less than 26·2, while six of the nine signals detected were located on regions with FL values of 75·3 or over. This demonstrates that Agrobacterium-mediated genes can integrate into multiple sites distributed in different parts of the chromosome, but that distal regions are the preferred sites and regions near the centromeres are colder for T-DNA integration. The donor DNA of the transformation was divided into two parts, labelled separately as probes for two-colour FISH. Results show that the transformed DNA sequences remained linked in the recipient genome. The relationship between integration position and transgene silencing, known as the ‘position effect’, is discussed.
Key words: Agrobacterium-mediated transformation, transgenic rice, fluorescence in situ hybridization, FISH, position effect, transgene silencing.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In rice, lepidopteran and stem borers cause high annual yield losses (Herdt, 1991; Teng and Revilla, 1996). One means of transferring resistance to these pests is transformation of Bacillus thuringiensis (Bt) crystal insecticidal protein genes encoding a toxin to the pests. Using a modified Agrobacterium-mediated transformation method for the Cry1A(b) gene, we produced a number of transgenic rice plants (Cheng et al., 1998).
Although gene transfer technology is developing rapidly in plants (Christou et al., 1989; Cheng et al., 1998), the processes involved in integration, stability and expression of transgenes within the recipients are not well known. Whether or not transgenes integrate into specific chromosomes or chromosome regions of the genome has not yet been determined. One of the obstacles in the application of transformation is that expression of the alien genes that are transferred into plants varies from generation to generation or among different transgenic plants, or may even be silenced. Silencing of the Cry1A(b) gene and variability in its expression have been observed in different transformed lines.
One important factor that might affect transgene expression is the influence of chromosome position of the integrated alien genes, known as the ‘position effect’ (Frello et al., 1995; Pedersen et al., 1997). The position effect has been studied extensively in plants, including rice, using molecular and genetics methods (Itoch et al., 1997; Kumpatla et al., 1997, 1998; Kohli et al., 1998). Fluorescence in situ hybridization (FISH), which can identify the physical position of transgenes within the genome, has become one of the most powerful tools used to study the position effect and determine whether there are any ‘hot spots’ for the integration of transgenes (Jiang et al., 1994; Leggett et al., 2000). However, to the best of our knowledge, FISH analysis has not yet been reported for Agrobacterium-mediated transgenic rice.
In this study, the Cry1A(b) gene has been detected and localized onto particular chromosomes by FISH in nine transgenic rice lines. In addition, Cry1A(b) insecticidal protein expression analysis, mRNA in situ hybridization and the relationship between transgene locations and expression level have been analysed in these lines.
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MATERIALS AND METHODS |
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Plant materials
The tested materials included nine transgenic rice (Oryza sativa indica ‘RM’) lines, which were produced by Agrobacterium-mediated transformation with the chimeric gene pKUB. pKUB contained the ubiquitin and CaMV35S promoters, Cry1A(b), Gus (ß-glucuronidase) and HPH (hygromycin phosphotransferase) genes and the nos-km terminator. All of the lines tested were the third generation (T3) of the transformed plants, which had already been tested with Southern and Northern blots (Cheng et al., 1998). Non-transformed plants were used as controls.
mRNA in situ hybridization
Cry1A(b) fragments from pKUB were sub-cloned into pGEM-4Z in both orientations. Digoxigenin-labelled single-stranded antisense probes and control sense probes were synthesized by in vitro transcription using standard procedures (Bouget et al., 1995; Chen et al., 1999). Leaf tissue was collected from transgenic rice plants and prepared for in situ hybridization. After pre-hybridization, the sections were hybridized with antisense RNA or sense RNA. Immunological reactions were tested with anti-Dig-AP (Roche Molecular Biochemicals, Mannheim, Germany) after several rinses. Colour reactions were tested with nitro blue tetratolium salt and bromo-4-choro-3-indoly-l-phosphate.
Assay for Cry1A(b) proteins and histochemical assay of Gus
Leaf and root samples, each weighing about 15 mg, were ground and extracted with the protein extraction buffer contained in the Eniverologix Cry1Ab/Cry1Ac plate kit (Eniverologix, Sunnyvale, CA, USA). Untransformed plants were used as a negative control. Extract (100 µl) was added to test wells coated with anti-Dig-AP (Roche Molecular Biochemicals) raised against the Cry1A(b) toxin. Any residues present in the sample extract bind to the antibodies and are detected after addition of horseradish peroxidase-labelled Cry1A(b) antibody. After a simple wash step, the results of the assay were visualized in a colour development step; colour development was proportional to the concentration of Cry1A(b) in the sample extract. For spectrophotometric measurement, three samples of known concentration contained in the kit were used. Using the samples provided with the kit as controls, the Cry1A(b) concentration in the test samples was calculated. Results are given in ppm. Instructions given in the Eniverologix Cry1A(b)/Cry1Ac plate kit were followed. Data are means of three replicates.
Gus activity was measured as described by Jefferson (1987).
DNA probes and chromosome preparation for FISH
pKSB was about 5·5 kb in size and carried the 35S promoter, the Cry1A(b) gene (1·87 kb) and the nos-km terminator. pHctinG was about 8 kb and carried the Gus and HPH genes. The rice centromere probe, pRCS2, a tandem repeat sequence was kindly provided by Professor Jiming Jiang, University of Wisconsin, Madison, USA. The probes were labelled with digoxigenin and/or biotin, respectively, following the nick translation protocol detailed in the kit (Sino-American Biotechnology Company, Luoyang City, China).
Root tips were excised from actively growing transgenic rice plants and immediately fixed in ethanol : acetic acid (3 : 1) at 4 °C overnight. After a wash in distilled water, root tips were treated with a mixture of 2 % pectinase (Yakult Honsha Co., Ltd, Tokyo, Japan) and 2 % cellulase (Yakult Honsha Co., Ltd) at 28 °C for approx. 3 h. Finally, the treated root tips were squashed on slides and dried over a flame (Song et al., 1995).
In situ hybridization and fluorescence detection
Hybridization was performed as outlined by Song et al. (1995), with an additional 5 µg ml–1 pepsin treatment for 10 min. Biotinylated probes were detected with goat anti-biotin FITC conjugate (Sigma, St Louis, MO, USA), followed by rabbit anti-goat biotin conjugate (Sigma) and finally with goat anti-biotin FITC conjugate (Sigma). Digoxigenin-labelled probes were detected by mouse anti-digoxigenin (Roche Molecular Biochemicals), followed by digoxigenin-conjugated sheep-antimouse (Roche Molecular Biochemicals) and finally with rhodamine anti-digoxigenin (Roche Molecular Biochemicals). Each detection step took place at 37 °C for 30 min and slides were washed with phosphate buffered saline between each step. After immunological reactions, the slides were counterstained with 1 µg ml–1 DAPI or 8 µg ml–1 PI. Chromosomes were examined with an Olympus BX60 fluorescence microscope equipped with Sensys 1401E cooled CCD camera. Red, green and blue images were captured in black and white with G, B and UV excitation filters, respectively. The images were combined and pseudo-coloured using software V++ (Digital Optics, Auckland, New Zealand). For each line tested, at least 30 cells were analysed.
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RESULTS |
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Assay for Cry1A(b) proteins and Gus activity
Of the nine transgenic lines tested, seven tested positive for Cry1A(b) protein; lines 11–3 and 19–3 did not (Fig. 1; Table 1). Plants showed much variation in Cry1A(b) protein expression levels. The Cry1A(b) protein content was 2·4 ppm in leaves of line 27–2, while in leaves of lines 11–3 and 19–3 it was too low to be detected. The content varied from 0·01 to 0·63 ppm in other plants tested. As shown in Fig. 1, expression levels of Cry1A(b) in the roots were similar to those in the leaves. This was expected because both the transformed promoters ubiquitin and CaMV35S were constitutive. The control plant, which was not transformed, reacted negatively for Cry1A(b) protein. With the exception of lines 11–3, 19–3 and the control, the other lines tested displayed Gus activity (Table 1).
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mRNA in situ hybridization
With the exception of line 11–3 and the control (non-transformed plants), the other lines showed hybridization signals for an antisense RNA transcribed from the Cry1A(b) gene (Table 1; Fig. 2). No signal was detected for any plants hybridized with sense RNA. The signal intensity varied noticeably among the lines tested. Strong signals were observed in lines 27–2 and 25–1, and very weak ones in line 19–3; the other lines showed intermediate results. The Cry1A(b) mRNA signal was mainly localized to the mesophyll cells of the seedlings. Few signals were observed in epidermal cells or in vascular bundle sheaths.
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FISH of tested transgenes
All of the transformed lines except line 11–3 hybridized with the Cry1A(b) probe and showed signals on one or two chromosomes (Fig. 3). In this study, all the signal positions were determined by the double signals located on the sister chromatids so that the results could be confirmed easily. Because karyotype analysis was difficult, we could not determine exactly which chromosome pair(s) showed signals in the eight lines. In some of the plants tested, the rice centromere probe, pRCS2, was used, making recognition of the centromeres and long or short arms easier and more precise (Fig. 3H). Data for the detected chromosomes, including arm ratio and fraction length (FL; percentage of the distance from the detection site to the centromere divided by the length of the detected arm), are summarized in Table 1. The hybridization signals were observed on long arms in lines 12–1, 19–1, 20–2, 25–1, 19–3 and 27–2, on short arms in lines 20–1, and on both long and short arms in line 25–2. The FL values of the detection sites were 26·2, 46·4, 68·0, 75·3, 82·5, 89·5, 90·6, 92·0 and 95·2, respectively (Table 1; Fig. 3), indicating that alien genes could be introgressed into both long and short arms of rice chromosomes. No signal was detected on regions where FL was less than 26·2, while six of the nine signals detected were located on regions where FL was 75·3 or over, demonstrating that terminal or sub-terminal regions of chromosomes are probably the preferred sites for integration of T-DNA in the rice genome.
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As mentioned above, the clones used to transform the tested plants included genes Cry1A(b), Gus and HPH. To check the way in which these genes were integrated, a biotin-labelled pHctinG probe (for Gus and HPH) and a digoxigenin-labelled pKSB probe [for Cry1A(b)] were used for two-colour FISH. Results showed that in all four lines tested (12–1, 25–1, 25–2 and 19–1), the insert including the DNA sequences of probes pHctinG and pKSB was integrated into the hybridization sites with no disjunction. Overlapping signals of pHctinG (green) and pKSB (red) were observed (Fig. 3E–G). In addition, the co-hybridization results also provided another important basis for confirming the location of our transgenes.
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DISCUSSION |
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Our study has identified many T-DNA integration sites in rice. These are interspersed in different regions of chromosomes or on different chromosomes, but they are not found in regions close to centromeres. Our results are consistent with those of other researchers, whose genetic mapping has indicated that T-DNA insertion sites are widely dispersed in plant chromosomes (Deroles et al., 1988; Heberle-Baors et al., 1988), and basically support the illegitimate recombination mode of Agrobacterium-mediated gene transfer (Mayerhofer et al., 1991). However, the distribution of T-DNA insertion sites is not arbitrary. Although the position of the centromere could not be determined exactly on some of our colour images, we could identify its position on the black and white images because these were much clearer. We found that no alien genes were integrated in regions where FL was less than 26·2, and six of the nine signals detected were located on regions where FL was 75·3 or over, demonstrating that Agrobacterium-mediated genes were integrated preferentially in the distal regions instead of in regions close to centromeres. This has also been shown in barley, tomato, tobacco, onion, oat and Petunia (Ambros et al., 1986; Wang et al., 1995; ten Hoopen et al., 1996; Pedersen et al., 1997; Leggett et al., 2000; Khrustaleva et al., 2001). Some studies have shown cold and hot spots for recombination in chromosomes (Leggett et al., 2000; Cheng et al., 2001). Based on our results, regions where FL is less than 26·2 are colder, while those where FL is 75·3 or over are more active than other regions for recombination. As reported previously, the frequency of homologous recombination also showed similar variation to that reported for integration of alien genes from centromeres to chromosome ends. This indicates that the process of alien gene integration resembles that of homologous recombination, even though T-DNA is generally non-homologous to rice genomic DNA. Therefore, it could be deduced that T-DNA recognition sites occur along chromosomes; these are seldom in regions close to centromeres, but occur more often in distal regions than in other regions. The other possibility is that distal regions are the most decondensed parts of cereal chromosomes and contain a large number of actively transcribed genes that are active during the cell cycle of cells that divide rapidly. Therefore, such regions would be more susceptible to alien gene integration. Heterochromatic regions, however, are presumably less amenable for access of foreign genes, since they are condensed during the cell cycle (Pedersen et al., 1997). Besides the two possibilities mentioned above, it should be pointed out that even if alien genes were integrated into regions near the centromeres, they may have been discarded during selection, since their expression would probably be affected by heterochromatin, causing it to decrease to some extent. A decrease in gene activity induced by heterochromatin has been detected in many cases (Meyer and Sadeler, 1996; Ansari et al., 1999; Jakowitsch et al., 1999).
The insert carried by T-DNA was more than 10 kb in size and it included the ubiquitin and CaMV35S promoters, the genes Cry1A(b), Gus and HPH, and the nos-km terminator. Our results showed that in all four lines (19–1, 20–1, 20–2 and 25–1) tested by two-colour FISH, the signals of pHctinG (green) and pKSB (red) overlapped (Fig. 3E–G), indicating that all these elements could be transformed without separation. Moreover, each plant showing hybridization signals demonstrated simultaneous activity of Cry1A(b), Gus and HPH genes. Thus, not only did all donor elements remain non-disjointed, but they were also structurally stable in Agrobacterium-mediated gene transfer. It is suggested that Agrobacterium-mediated gene transfer may be more advantageous than other direct gene delivery methods, such as micro-projectile bombardment, which always result in partial integration and rearrangements (Jakowitsch et al., 1999).
It has been suggested that alien genes integrated in distal regions of chromosome arms may show high or stable activities, whereas activity of alien genes integrated near the centromeric regions is low or unstable due to the presence of more heterochromatin in the centromeric regions (Iglesias et al. 1997; Pedersen et al., 1997; Ansari and Gartenberg, 1999). Based on protein and FISH analyses in our study, signals of some plants expressing low-level gene activity (lines 12–1, 20–2 and 20–1) were distributed in regions with FL values of 26·2, 75·3 and 95·2, respectively. Thus, transgenes integrated in regions near centromeres, or in the middle or distal parts of chromosome arms, could all be expressed at low levels. Our results therefore show no obvious effect of integration position on alien gene expression. However, no signal was observed in those regions near the centromeres in which FL was less than 26·2. As mentioned above, this was probably the result of ‘position effect’, because expression of the alien gene was probably affected by heterochromatin. In addition, the ‘position effect’ may be only one of the factors contributing to the low expression of transgenes. In this study, for example, the signal was located in a distal region in line 19–3 (Fig. 3B), but the line did not show expression of the transgene (Fig. 1), demonstrating that the reasons behind gene silencing are complex. The position effect of transgenes must be investigated further using pachytene FISH or fibre FISH combined with molecular tecniques.
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ACKNOWLEDGEMENTS |
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We thank Dr Bruce (Eniverologix Inc., USA) for kindly providing the Cry1A(b)/Cry1Ac plate kit (Cat. No. AP 003). We also thank Professor Jiming Jiang (University of Wisconsin, Madison) for his kind advice. This work was supported by the National Natural Science Foundation of China [No. 30(70376)] and the Research Fund for the Doctoral Program of Higher Education [No. 20(980112)].
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LITERATURE CITED |
|---|
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-
Ambros PF, Matazke MA, Matzke AJM. 1986. Detection of a 17-kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization. Chromosoma 94: 11–18.[CrossRef]
Ansari A, Gartenberg MR. 1999. Persistence of an alternate chromatin structure at silenced loci in vitro. Proceedings of the National Academy of Sciences of the United States of America 96: 343–348.
Bouget FY, Gerttila S, Quatrano RS. 1995. A new nonradioactive in situ hybridization protocol for Fucus (Phaeophytal) embryos. Journal of Phycology 31: 1027–1030.[CrossRef][Web of Science]
Chen SR, Lu YT, Yang HY. 1999. In situ localization of calmodulin mRNA in the developing tobacco anthers. Chinese Science Bulletin 44: 142–145.
Cheng XY, Sardana R, Kaplan H, Altossar I. 1998. Agrobacterium-transformed rice plants expressing synthetic CryIA(b) and CryIA(c) gene are highly toxic to striped stem borer and yellow stem borer. Proceedings of the National Academy of Sciences of the United States of America 95: 2767–2772.
Cheng ZK, Presting GG, Buell CR, Wing RA, Jiang J. 2001. High-resolution pachytene chromosome mapping of Bacterial Artificial Chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosome 10 of rice. Genetics 157: 1749–1757.
Christou P, Swain WF, Yang NS, McCabe DE. 1989. Inheritance and expression of foreign genes in transgenic soybean plants. Proceed ings of the National Academy of Sciences of the United States of America 86: 7500–7504.
Deroles SC, Gardner RC. 1988. Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Molecular Biology 11: 365–377.[CrossRef]
Frello S, Hansen KR, Jensen J, Jorgensen RB. 1995. Inheritance of rapeseed (Brassica napus)-specific RAPD markers and a transgene in the cross B. juncea x (B. juncea x B. napus). Theoretical and Applied Genetics 91: 236–241.
Gheysen G, Villarroel R, Van Montagu M. 1991. Illegitimate recom bination in plants: a model for T-DNA integration. Genes and Development 5: 287–297.
Heberle-Baors E, Charvat B, Thompson D, Schernthaner JP, Barta A, Matzke AJM, Matzke MA. 1988. Genetic analysis of T-DNA insertions into the tobacco genome. Plant Cell Reports 7: 571–574.[CrossRef]
Herdit RW. 1991. Research priorities for rice biotechnology. In: Khush GS, Toenniessen GH, eds. Rice Biotechnology, Wallingford: CABI International, 19–54.
Iglesias V, Moscone E, Papp I, Neuhuber F, Michalowski S, Phelan T. 1997. Molecular and cytogenetic analysis of stably and unstably expressed transgene loci in tobacco. The Plant Cell 9: 1251–1264.[Abstract]
Itoch K, Nakajima M, Shimamoto K. 1997. Silencing of waxy genes in rice containing Wx transgenes. Molecular and General Genetics 255: 351–358.
Jakowitsch J, Papp I, Moscone EA, van der Wunden J, Matzke M, Matzke AJM. 1999. Molecular and cytogenetic characterization of a transgene locus that induces silencing and methylation of homologous promoters. The Plant Journal 17: 131–140.[CrossRef][Web of Science][Medline]
Jefferson RA, Kavanagh TA, Bevan, MW. 1987. GUS fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO Journal 6: 3901–3907.[Web of Science][Medline]
Jiang J, Gill BS. 1994. Nonisotopic in situ hybridization and plant genome mapping: the first 10 years. Genome 37: 717–725.[Medline]
Khrustaleva LI, Kik C. 2001. Location of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tryamide signal amplification The Plant Journal 25: 699–707.
Kohli A, Leech M, Vain P, Laurie D, Christou P. 1998. Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proceedings of the National Academy of Sciences of the United States of America 95: 7203–7208.
Kumpatla S, Hall TC. 1998. Recurrent onset of epigenetic silencing in rice harbouring a multi-copy transgene. The Plant Journal 14: 129–135.
Kumpatla SP, Weimin T, Buchholz WG, Hall TG. 1997. Epigenetic transcriptional silencing. 5-azacytidine mediated reactivation of a complex transgene in rice. Plant Physiology 115: 361–373.[Abstract]
Leggett JM, Perret SJ, Harper J, Morris P. 2000. Chromosome localization of cotransformed transgenes in the hexaploid cultivated oat Avena sativa L. using fluorescence in situ hybridization. Heredity 84: 46–53.[Medline]
Matsumoto S, Ito Y, Takahashi Y, Machida Y. 1990. Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA. Molecular and General Genetics 224: 309–316.
Mayerhofer R, Koncz-Kalman Z, Nawrath C, Koncz C. 1991. T-DNA integration: a mode of illegitimate recombination in plants. EMBO Journal 10: 697–704.[Web of Science][Medline]
Meyer P, Sadeler H. 1996. Homology-dependent gene silencing in plants. Annual Reviews of Plant Physiology. Plant Molecular Biology 47: 23–48.
Pedersen C, Zimny J, Becker D. 1997. Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by florescence in situ hybridization. Theoretical and Applied Genetics 94: 749–757.[CrossRef]
Song YC, Gustafson JP. 1995. The physical location of fourteen RFLP markers in rice (Oryza sativa L.) Theoretical and Applied Genetics 90: 113–119.
ten Hooper R, Robbins TP, Fransz PF, Montijn BM, Oud O, Gerats AGM, Namminga N. 1996. Localization of T-DNA insertion in Petunia by florescence in situ hybridization: physical evidence for suppression of recombination. Plant Cell 8: 823–830.[Abstract]
Teng PS, Revilla IM. 1996. Technical issues in using crop-loss for research prioritization. In: Evenson RE, Herdit RW, Hossain M, eds. Rice Research in Asia: progress and priorities, Wallingford: CABI International, 261–275.
Wang J, Lewis ME, Whallon JH, Sink KC. 1995. Chromosomal mapping of T-DNA inserts in transgenic Petunia by in situ hybridization. Transgenic Research 4: 241–246.
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