Skip to main content
SpringerLink
Log in

High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation

  • Protocol
  • Published:
Plant Molecular Biology Reporter Aims and scope Submit manuscript

Abstract

An efficient method for the transformation and regeneration of fertile transgenic rice (Oryza sativa L.) plants is presented. In this protocol seed calli from the varietiesNipponbare andTaipei 309 were used to produce rice suspension cultures in General Medium. Protoplasts were isolated from suspension cells (8 × 106 protoplasts perg fresh weight), then were incubated with sterile DNA in the presence of MaMg solution, followed by addition of PEG to a final concentration of 25%. A hygromycin phosphotransferase (hph) gene under the plant transcriptional regulatory signals was used as a selectable marker gene. Hygromycin-resistant colonies were selected in the presence of 95 μM hygromycin B with apparent frequencies of 2×10−4 and 5×10−4 forNipponbare andTaipei 309, respectively. Plantlets were regenerated from resistant colonies in Murashige and Skoog plant regeneration medium. Among 628 transgenic plants grown to maturity in the greenhouse, two-thirds bore viable seeds.

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

Similar content being viewed by others

Abbreviations

2,4-D:

2,4-dichlorophenoxyacetic acid

BAP:

6-benzylaminopurine

CaMV 35S :

caulifower mosaic virus35S promoter

IAA:

indole-3-acetic acid

MES:

2-[N-morpholinolethanesulfonic acid

PEG:

poly-ethylene glycol 8000

hph :

gene coding for hygromycin B phosphotransferase

nos :

gene coding for nopaline synthetase

tml :

gene fortumor morphology large

References

  • Cao, J., M. C. Rush, M. Nabors, Q. J. Xie, T. P. Croughan, and E. M. Nowick. 1990. Development and inheritance of soma clonal variation in rice. In:Biological Nitrogen Fixation Associated with Rice Production. New Delhi: Oxford and IBH Publishing (in press).

    Google Scholar 

  • Chen, Y. 1986. Anther, and pollen culture of rice. In:Haploids of Higher Plants in vitro. (eds. H. Hu, and H. Y. Yang). pp. 1–25. Beijing: China Academic Publishers and Springer-Verlag.

    Google Scholar 

  • Hansen, J. B. and R. H. Olsen. 1978. Isolation of large bacterial plasmids and characterization of the P2 incompatibility goup plasmids pMG1 and pMG5. J. Bacteriol. 135:227–238.

    PubMed  CAS  Google Scholar 

  • Harms, Ch. T. and I. Potrykus. 1978. Fractionation of plant protoplast types by isoosmotic density gradient centrifugation. Theor. Appl. Genet. 53:57–63.

    Google Scholar 

  • Hauptmann, R. M., V. Vasil, P. Ozias-Akins, Z. Tabaeizadeh, S. G. Rogers, R. T. Fraley, R. B. Horsch and I. K. Vasil. 1988. Evaluation of selectable markers for obtaining stable transformants in theGramineae. Plant Physiol. 86:602–606.

    PubMed  CAS  Google Scholar 

  • Hayashimoto, A., Z. Li and N. Murai. 1990. A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants. Plant Physiol. 93: 857–863.

    Article  PubMed  CAS  Google Scholar 

  • Krens, F. A., L. Molendijk, G. J. Willems and R. A. Schilperoort. 1982.In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296:72–74.

    Article  CAS  Google Scholar 

  • Kyozuka, J., Y. Hayashi and K. Shimamoto. 1977. High frequency plant regeneration from rice protoplasts by novel nurse culture methods. Mol. Gen. Genet. 206:408–413.

    Article  Google Scholar 

  • Li, Z. and N. Murai. 1990. General medium for efficient plant regeneration from rice (Oryza sativa L.) protoplasts. Plant Cell Reports 9:216–220.

    Google Scholar 

  • Luo, Z. X. and R. Wu. 1989. A simple method for the transformation of rice via the pollentube pathway. Plant Molec. Biol. Report. 7:69–77.

    Article  Google Scholar 

  • MacDonald, R. I.. 1985. Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose. Biochemistry 24:4058–4066.

    Article  PubMed  CAS  Google Scholar 

  • Negrutiu, I., R. Shillito, I. Potrykus, G. Biasini and F. Sala. 1987. Hybrid genes in the analysis of transformation conditions. I. Setting up a simple, method for direct gene transfer in plant protoplasts. Plant Mol. Biol. 8:363–373.

    Article  CAS  Google Scholar 

  • Paszkowski, J., R. D. Shillito, M. Saul, V. Mandak, T. Hohn, B. Hohn and I. Potrykus. 1984. Direct gene transfer to plants. EMBO J. 3:2717–2722.

    PubMed  CAS  Google Scholar 

  • Raineri, D. M., P. Bottino, M. P. Gordon and E. W. Nester. 1990.Agrobacterium-mediated transformation of rice (Oryza sativa L.). Bio/Technol. 8:33–38.

    Article  CAS  Google Scholar 

  • Shimamoto, K., R. Terada, T. Izawa and H. Fujimoto. 1989. Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338:274–276.

    Article  CAS  Google Scholar 

  • Toriyama, K., Y. Arimoto, H. Uchiyama and K. Hinata. 1988. Transgenic rice plants after direct gene transfer into protoplasts. Bio/Technol. 6:1072–1074.

    Article  CAS  Google Scholar 

  • Yamada, Y., Y. Zhi-Qi and T. Ding-Tai. 1986. Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.). Plant Cell Reports 5:85–88.

    Article  Google Scholar 

  • Yang, kX. R., J. R. Wang, H. L. Li and Y. F. Li. 1980. Studies on the general medium for anther culture of cereals and increasing of the frequency of green pollen plantlets—induction ofOryza sativa subsp. hsien. Acta Phytophysiol. Sinica 6:67–74 (in Chinese with English abstract).

    Google Scholar 

  • Zhang, W. and R. Wu. 1988. Efficient regeneration of transgenic plants from rice protoplasts and correctly-regulated expression of the foreign gene in plants. Theor. Appl. Genet. 76:835–840.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Plant Pathology and Crop Physiology College of Agriculture, Louisiana State University, 70803-1702, Baton Rouge, LA, USA

    Zhijian Li, Mark D. Burow & Norimoto Murai

Authors
  1. Zhijian Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark D. Burow
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Norimoto Murai
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Z., Burow, M.D. & Murai, N. High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation. Plant Mol Biol Rep 8, 276–291 (1990). https://doi.org/10.1007/BF02668764

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02668764

Keywords

Search

Navigation