Skip to main content
SpringerLink
Log in

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

  • Published:
Theoretical and Applied Genetics Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2A–I.
Fig. 3.
Fig. 4.

Similar content being viewed by others

References

  • 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 138:177–190

    Article  CAS  Google Scholar 

  • Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266

    CAS  PubMed  Google Scholar 

  • Chen L, Zhang S, Beachy RN, Fauquet CM (1998) A protocol for consistent, large scale production of fertile transgenic rice plants. Plant Cell Rep 18:25–31

    Google Scholar 

  • 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 14:537–545

    Google Scholar 

  • 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 71:3672–3676

    CAS  PubMed  Google Scholar 

  • 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 5:659–668

    Google Scholar 

  • 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 100:832–839

    CAS  Google Scholar 

  • Davenport RJ (2001) Rice genome. Syngenta finishes, consortium goes on. Science 291:807

    CAS  PubMed  Google Scholar 

  • 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 39:323–334

    Article  Google Scholar 

  • Devic M, Albert S, Delseny M, Roscoe TJ (1997) Efficient PCR walking on plant genomic DNA. Plant Physiol Biochem 35:331–339

    CAS  Google Scholar 

  • Dong JJ, Teng WM, Buchholz WG, Hall TC (1996) Agrobacterium-mediated transformation of Javanica rice. Mol Breed 2:267–276

    CAS  Google Scholar 

  • Feldmann KA (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J 1:71–82

    CAS  Google Scholar 

  • 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 391:806–811

    CAS  PubMed  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Plant cell cultures. 1. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158

    CAS  PubMed  Google Scholar 

  • Glaszmann JC (1987) Isozymes and classification of Asian rice varieties. Theor Appl Genet 74:21–30

    CAS  Google Scholar 

  • 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 296:92–100

    CAS  PubMed  Google Scholar 

  • 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 6:271–282

    CAS  Google Scholar 

  • Hirochika H (2001) Contribution of the Tos17 retrotransposon to rice functional genomics. Curr Opin Plant Biol 4:118–122

    Article  CAS  PubMed  Google Scholar 

  • 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 303:179–180

    CAS  Google Scholar 

  • Hood EE, Gelvin SB, Melchers L.S, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    CAS  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    CAS  Google Scholar 

  • 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 22:561–570

    CAS  PubMed  Google Scholar 

  • Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhnacer for plant genes. Nature 236:1299–1302

    CAS  Google Scholar 

  • Kempin SA, Lijegren SJ, Block SJ, Rounsley SD, Yanofsky MF, Lam E (1997) Targeted disruption in Arabidopsis. Nature 389:802–803

    Article  CAS  PubMed  Google Scholar 

  • Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283–2290

    CAS  PubMed  Google Scholar 

  • Li L, Qu R, de Kochko A, Fauquet C, Beachy RN (1993) An improved rice transformation system using the biolistic method. Plant Cell Rep 12:250–255

    Google Scholar 

  • 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 159:1751–1763

    CAS  PubMed  Google Scholar 

  • Mengiste T, Paszkowski J (1999) Prospects for the precise engineering of plant genomes by homologous recombination. Biol Chem 380:749–758

    CAS  PubMed  Google Scholar 

  • Metzlaff M, O'Dell M, Cluster PD, Flavell RB (1997) RNA-mediated RNA degradation and chalcone synthase A silencing in Petunia. Cell 88:845–854

    CAS  PubMed  Google Scholar 

  • Murashige F, Skoog T (1962) A reversed medium for rapid growth and bioassays with tobacco tissue culture. Physiol plant 15:473–497

    CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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 14:1113–1121

    CAS  Google Scholar 

  • 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 112:893–900

    CAS  PubMed  Google Scholar 

  • 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 11:2263–2270

    CAS  PubMed  Google Scholar 

  • 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 28:141–145

    Article  CAS  PubMed  Google Scholar 

  • Rashid H, Yokoi S, Toriyama K, Hinata K (1996) Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep 15:727–730

    Google Scholar 

  • 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 44:32–37

    Article  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbour

    Google Scholar 

  • 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 30:94–97

    Article  CAS  PubMed  Google Scholar 

  • Sasaki T, Burr B (2000) International Rice Genome Sequencing Project: the effort to completely sequence the rice genome. Curr Opin Plant Biol 3:138–141

    CAS  PubMed  Google Scholar 

  • 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 18:992–1002

    Article  CAS  PubMed  Google Scholar 

  • Siebert PD, Chenchick A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23:1087–1088

    CAS  PubMed  Google Scholar 

  • 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 11:1853–1866

    CAS  PubMed  Google Scholar 

  • Springer PS (2000) Gene traps: tools for plant development and genomics. Plant Cell 12:1007–1020

    CAS  PubMed  Google Scholar 

  • Takano M, Kanegae H, Shinomura T, Miyao A, Hirochika H, Furuya M (2001) Isolation and characterization of rice phytochrome A mutants. Plant Cell 13:521–534

    Google Scholar 

  • Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20:1030–1034

    Article  CAS  PubMed  Google Scholar 

  • 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 11:1841–1852

    CAS  PubMed  Google Scholar 

  • 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 43:67–82

    Google Scholar 

  • 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 14:525–535

    CAS  PubMed  Google Scholar 

  • Yin Z, Wang GL (2000) Evidence of multiple complex patterns of T-DNA integration into the rice genome. Theor Appl Genet 100:461–470

    Article  CAS  Google Scholar 

  • 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 296:79–92

    CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Biotrop Programme, Cirad-Amis, Avenue Agropolis, 34398 Montpellier Cedex 5, France

    C. Sallaud, D. Meynard, J. van Boxtel, M. Bès, P. Larmande & E. Guiderdoni

  2. INRA-ENSAM, Biotrop Programme, Cirad-Amis, Avenue Agropolis, 34398 Montpellier Cedex 5, France

    C. Gay & M. Portefaix

  3. Genetrop, Ird, BP5045, 34032 Montpellier Cedex 01, France

    J. P. Brizard

  4. Laboratoire Génome et Développement des Plantes, UMR5096, CNRS/UP, 52, Avenue de Villeneuve, 66860 Perpignan Cedex, France

    D. Ortega, M. Raynal & M. Delseny

  5. Rice Research Group, Institute of Molecular Plant Science, Leiden University, P.O. Box 9505, 2300 RA Leiden, the Netherlands

    P. B. F. Ouwerkerk & S. Rueb

Authors
  1. C. Sallaud
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Meynard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. van Boxtel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Gay
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Bès
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. P. Brizard
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. P. Larmande
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. D. Ortega
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M. Raynal
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M. Portefaix
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. P. B. F. Ouwerkerk
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. S. Rueb
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. M. Delseny
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. E. Guiderdoni
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to E. Guiderdoni.

Additional information

Communicated by C. Möllers

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sallaud, C., Meynard, D., van Boxtel, J. et al. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106, 1396–1408 (2003). https://doi.org/10.1007/s00122-002-1184-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00122-002-1184-x

Keywords

Search

Navigation