Theoretical and Applied Genetics

, Volume 106, Issue 8, pp 1396–1408 | Cite as

Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics

  • C. Sallaud
  • D. Meynard
  • J. van Boxtel
  • C. Gay
  • M. Bès
  • J. P. Brizard
  • P. Larmande
  • D. Ortega
  • M. Raynal
  • M. Portefaix
  • P. B. F. Ouwerkerk
  • S. Rueb
  • M. Delseny
  • E. Guiderdoni
Article

Abstract

We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice (Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance (hpt), green fluorescent protein (gfp) and β-glucuronidase (gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75–95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30–40% of the T0 plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T1 progenies showed that 30–50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.

Keywords

Functional genomics Gene transfer Insertional mutagenesis Oryza sativa L. T-DNA 

Notes

Acknowledgements

This paper is dedicated to the late memory of Prof. Harry C. Hoge from Leiden University. The authors wish to thank Dr. Eric Huttner and Dr. Pascual Perez for valuable discussion in the course of this study. The technical help of Martine Bangratz as well as of Pierre Larmande for the bioinformatics is also acknowledged. We thank Dr. Alexander Johnson for reviewing the language. The French National Plant Genomics initiative Génoplante and the EU-funded BIOTECH CT 97-2132 "Rice transposon mutagenesis" programmes have supported this study.

References

  1. Bec S, Chen LL, Ferriere NM, Legavre T, Fauquet C, Guiderdoni E (1998) Comparative histology of microprojectile-mediated gene transfer to embryogenic calli in japonica rice (Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability. Plant Sci 138:177–190CrossRefGoogle Scholar
  2. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266PubMedGoogle Scholar
  3. Chen L, Zhang S, Beachy RN, Fauquet CM (1998) A protocol for consistent, large scale production of fertile transgenic rice plants. Plant Cell Rep 18:25–31Google Scholar
  4. Chen M, Presting G, Barbazuk WB, Goicoechea JL, Blackmon B, Fang G, Kim H, Frisch D, Yu Y, Sun S, Higingbottom S, Phimphilai J, Phimphilai D, Thurmond S, Gaudette B, Li P, Liu J, Hatfield J, Main D, Farrar K, Henderson C, Barnett L, Costa R, Williams B, Walser S, Atkins M, Hall C, Budiman MA, Tomkins JP, Luo M, Bancroft I, Salse J, Regad F, Mohapatra T, Singh NK, Tyagi AK, Soderlund C, Dean RA, Wing RA (2002) An integrated physical and genetic map of the rice genome. Plant Cell 14:537–545Google Scholar
  5. Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71:3672–3676PubMedGoogle Scholar
  6. Chu CC, Wang CC, Sun CS, Hsu C, Kin KC, Yin C, Chy Y, B. FY (1975) Establishment of an efficient medium for anther culture of rice though comparative experiments on the nitrogen source. Sci Sin 5:659–668Google Scholar
  7. Datta K, Koukolikova-Nicola Z, Baisakh N, Oliva N, Datta SK (2000) Agrobacterium-mediated enginering for sheath blight resistance of indica rice cultivars. Theor Appl Genet 100:832–839Google Scholar
  8. Davenport RJ (2001) Rice genome. Syngenta finishes, consortium goes on. Science 291:807PubMedGoogle Scholar
  9. Delseny M, Salses J, Cooke R, Sallaud C, Regad F, Lagoda P, Guiderdoni E, Ventelon M, Brugidou C, Ghesquière A (2001) Rice genomics: present and future. Plant Physiol Biochem 39:323–334CrossRefGoogle Scholar
  10. Devic M, Albert S, Delseny M, Roscoe TJ (1997) Efficient PCR walking on plant genomic DNA. Plant Physiol Biochem 35:331–339Google Scholar
  11. Dong JJ, Teng WM, Buchholz WG, Hall TC (1996) Agrobacterium-mediated transformation of Javanica rice. Mol Breed 2:267–276Google Scholar
  12. Feldmann KA (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J 1:71–82Google Scholar
  13. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811PubMedGoogle Scholar
  14. Gamborg OL, Miller RA, Ojima K (1968) Plant cell cultures. 1. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158PubMedGoogle Scholar
  15. Glaszmann JC (1987) Isozymes and classification of Asian rice varieties. Theor Appl Genet 74:21–30Google Scholar
  16. Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J, Miguel T, Paszkowski U, Zhang S, Colbert M, Sun WL, Chen L, Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92–100PubMedGoogle Scholar
  17. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza-sativa L.) mediated by Agrobacterium and sequence-analysis of the boundaries of the T-DNA. Plant J 6:271–282Google Scholar
  18. Hirochika H (2001) Contribution of the Tos17 retrotransposon to rice functional genomics. Curr Opin Plant Biol 4:118–122CrossRefPubMedGoogle Scholar
  19. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and t-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180Google Scholar
  20. Hood EE, Gelvin SB, Melchers L.S, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218Google Scholar
  21. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405Google Scholar
  22. Jeon JS, Lee S, Jung KH, Jun SH, Jeong DH, Lee J, Kim C, Jang S, Yang K, Nam J, An K, Han MJ, Sung RJ, Choi HS, Yu JH, Choi JH, Cho SY, Cha SS, Kim SI, An G (2000) T-DNA insertional mutagenesis for functional genomics in rice. Plant J 22:561–570PubMedGoogle Scholar
  23. Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhnacer for plant genes. Nature 236:1299–1302Google Scholar
  24. Kempin SA, Lijegren SJ, Block SJ, Rounsley SD, Yanofsky MF, Lam E (1997) Targeted disruption in Arabidopsis. Nature 389:802–803CrossRefPubMedGoogle Scholar
  25. Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283–2290PubMedGoogle Scholar
  26. Li L, Qu R, de Kochko A, Fauquet C, Beachy RN (1993) An improved rice transformation system using the biolistic method. Plant Cell Rep 12:250–255Google Scholar
  27. McElver J, Tzafrir I, Aux G, Rogers R, Ashby C, Smith K, Thomas C, Schetter A, Zhou Q, Cushman MA, Tossberg J, Nickle T, Levin JZ, Law M, Meinke D, Patton D (2001) Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159:1751–1763PubMedGoogle Scholar
  28. Mengiste T, Paszkowski J (1999) Prospects for the precise engineering of plant genomes by homologous recombination. Biol Chem 380:749–758PubMedGoogle Scholar
  29. Metzlaff M, O'Dell M, Cluster PD, Flavell RB (1997) RNA-mediated RNA degradation and chalcone synthase A silencing in Petunia. Cell 88:845–854PubMedGoogle Scholar
  30. Murashige F, Skoog T (1962) A reversed medium for rapid growth and bioassays with tobacco tissue culture. Physiol plant 15:473–497Google Scholar
  31. 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 41:733–742Google Scholar
  32. Ohira K, Ojima K, Fujiwara A (1973) Studies on the nutrition of rice cell culture. I. A simple defined medium for rapid growth in suspension culture. Plant & Cell Physiol 14:1113–1121Google Scholar
  33. Pang SZ, DeBoer DL, Wan Y, Ye G, Layton JG, Neher M.K., Armstrong CL, Fry JE, Hinchee MA, Fromm ME (1996) An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol 112:893–900PubMedGoogle Scholar
  34. Parinov S, Sevugan M, De Y, Yang WC, Kumaran M, Sundaresan V (1999) Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis. Plant Cell 11:2263–2270PubMedGoogle Scholar
  35. Quackenbush J, Liang F, Holt I, Pertea G, Upton J (2000) The TIGR gene indices: reconstruction and representation of expressed gene sequences. Nucleic Acids Res 28:141–145CrossRefPubMedGoogle Scholar
  36. Rashid H, Yokoi S, Toriyama K, Hinata K (1996) Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep 15:727–730Google Scholar
  37. Saji S, Umehara Y, Antonio BA, Yamane H, Tanoue H, Baba T, Aoki H, Ishige N, Wu J, Koike K, Matsumoto T, Sasaki T (2001) A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes. Genome 44:32–37CrossRefPubMedGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarbourGoogle Scholar
  39. Samson F, Brunaud D, Balzergue S, Dubreucq B, Lepiniec L, Pelletier G, Caboche M, Lecharny A (2002) FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants. Nucleic Acids Res 30:94–97CrossRefPubMedGoogle Scholar
  40. Sasaki T, Burr B (2000) International Rice Genome Sequencing Project: the effort to completely sequence the rice genome. Curr Opin Plant Biol 3:138–141PubMedGoogle Scholar
  41. Sato Y, Sentoku N, Miura Y, Hirochika H, Kitano H, Matsuoka M (1999) Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants. EMBO J 18:992–1002CrossRefPubMedGoogle Scholar
  42. Siebert PD, Chenchick A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088PubMedGoogle Scholar
  43. Speulman E, Metz PL, van Arkel G, te Lintel HB, Stiekema WJ, Pereira A (1999) A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome. Plant Cell 11:1853–1866PubMedGoogle Scholar
  44. Springer PS (2000) Gene traps: tools for plant development and genomics. Plant Cell 12:1007–1020PubMedGoogle Scholar
  45. Takano M, Kanegae H, Shinomura T, Miyao A, Hirochika H, Furuya M (2001) Isolation and characterization of rice phytochrome A mutants. Plant Cell 13:521–534Google Scholar
  46. Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20:1030–1034CrossRefPubMedGoogle Scholar
  47. Tissier AF, Marillonnet S, Klimyuk V, Patel K, Torres MA, Murphy G, Jones JD (1999) Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics. Plant Cell 11:1841–1852PubMedGoogle Scholar
  48. Wang MB, Waterhouse PM (2000) High-efficiency silencing of a beta-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Mol Biol 43:67–82Google Scholar
  49. Wu J, Maehara T, Shimokawa T, Yamamoto S, Harada C, Takazaki Y, Ono N, Mukai Y, Koike K, Yazaki J, Fujii F, Shomura A, Ando T, Kono I, Waki K, Yamamoto K, Yano M, Matsumoto T, Sasaki T (2002) A comprehensive rice transcript map containing 6,591 expressed sequence tag sites. Plant Cell 14:525–535PubMedGoogle Scholar
  50. Yin Z, Wang GL (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100:461–470CrossRefGoogle Scholar
  51. Yu J, Hu S, Wang J, Wong GK, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X, Cao M, Liu J, Sun J, Tang J, Chen Y, Huang X, Lin W, Ye C, Tong W, Cong L, Geng J, Han Y, Li L, Li W, Hu G, Huang X, Li W, Li J, Liu Z, Li L, Liu J, Qi Q, Liu J, Li L, Li T, Wang X, Lu H, Wu T, Zhu M, Ni P, Han H, Dong W, Ren X, Feng X, Cui P, Li X, Wang H, Xu X, Zhai W, Xu Z, Zhang J, He S, Zhang J, Xu J, Zhang K, Zheng X, Dong J, Zeng W, Tao L, Ye J, Tan J, Ren X, Chen X, He J, Liu D, Tian W, Tian C, Xia H, Bao Q, Li G, Gao H, Cao T, Wang J, Zhao W, Li P, Chen W, Wang X, Zhang Y, Hu J, Wang J, Liu S, Yang J, Zhang G, Xiong Y, Li Z, Mao L, Zhou C, Zhu Z, Chen R, Hao B, Zheng W, Chen S, Guo W, Li G, Liu S, Tao M, Wang J, Zhu L, Yuan L, Yang H (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296:79–92PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • C. Sallaud
    • 1
  • D. Meynard
    • 1
  • J. van Boxtel
    • 1
  • C. Gay
    • 2
  • M. Bès
    • 1
  • J. P. Brizard
    • 3
  • P. Larmande
    • 1
  • D. Ortega
    • 4
  • M. Raynal
    • 4
  • M. Portefaix
    • 2
  • P. B. F. Ouwerkerk
    • 5
  • S. Rueb
    • 5
  • M. Delseny
    • 4
  • E. Guiderdoni
    • 1
  1. 1.Biotrop Programme, Cirad-Amis, Avenue Agropolis, 34398 Montpellier Cedex 5, France
  2. 2.INRA-ENSAM, Biotrop Programme, Cirad-Amis, Avenue Agropolis, 34398 Montpellier Cedex 5, France
  3. 3.Genetrop, Ird, BP5045, 34032 Montpellier Cedex 01, France
  4. 4.Laboratoire Génome et Développement des Plantes, UMR5096, CNRS/UP, 52, Avenue de Villeneuve, 66860 Perpignan Cedex, France
  5. 5.Rice Research Group, Institute of Molecular Plant Science, Leiden University, P.O. Box 9505, 2300 RA Leiden, the Netherlands

Personalised recommendations