Skip to main content
SpringerLink
Log in

Optimisation of introducing foreign genes into egg cells and zygotes of wheat (Triticum aestivum L.) via microinjection

  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

An expeditious and highly efficient technique of microinjection has been developed with the aim of introducing exogenous DNA into egg cells and zygotes of wheat. Using a mechanical-dissection method and a novel immobilisation approach enabled us to microinject around 15 egg cells of wheat per hour. Exposing the protoplasts to a high-frequency alternating-current field for immobilisation, a significantly higher transient expression rate of the injected genes (46% and 52% for egg cells and zygotes, respectively) could be achieved than reported thus far for plant protoplasts. Whether this high transformation efficiency is due to the highfrequency electrical field applied for immobilising the protoplasts is not known. The transformation rate appeared to be a factor depending upon the time of egg cell isolation. According to the ultrastructural observations this seems to reflect a variation in competence of the egg cells during in situ development. In order to conduct studies directed towards establishing the optimal timewindow for DNA delivery into the fertilised egg cell, the time course of DNA dynamics during zygotic development has been quantified via quantitative microspectrofluorometry.

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

AC:

alternating current

DAE:

days after emasculation

FDA:

fluorescein diacetate

HAP:

hours after pollination

References

  • Blank M (1973) Biological effects of electromagnetic fields. J Bioenerg 32: 203–210

    Google Scholar 

  • Brinster RL, Chen HY, Trumbauer M, Yagle MK, Palmiter RD (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci USA 82: 4438–4442

    PubMed  Google Scholar 

  • Capecchi MR (1980) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22: 479–488

    PubMed  Google Scholar 

  • Carmichael JS, Friedman WE (1988) Double fertilization inGnetum gnemon: the relationship between the cell cycle and sexual reproduction. Plant Cell 7: 1975–1988

    Google Scholar 

  • Collas P, Balise JJ, Hofman GA, Robl JM (1989) Electrical activation of mouse oocytes. Theriogenology 32: 835–844

    Google Scholar 

  • Crossway A, Oakes JV, Irvine JM, Ward B, Knauf C, Shewmaker CK (1986) Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Mol Gen Genet 202: 179–185

    Google Scholar 

  • Cuthberston KSR, Cobbold PH (1985) Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca++. Nature 316: 541–542

    PubMed  Google Scholar 

  • Dijak M, Smith DI, Wolson TJ, Brown DCW (1986) Simulation of direct embryogenesis from mesophyll protoplasts ofMedicago saliva. Plant Cell Rep 5: 468–470

    Google Scholar 

  • Faure JE, Mogensen HL, Dumas C, Lörz H, Kranz E (1993) Karyogamy after electrofusion of single egg and sperm cell protoplasts from maize: cytological evidence and time course. Plant Cell 5: 747–755

    PubMed  Google Scholar 

  • Griesbach RJ (1983) Protoplast microinjection. Plant Mol Biol Rep 1: 32–37

    Google Scholar 

  • Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL (1985) Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315: 680–683

    PubMed  Google Scholar 

  • Heslop-Harrison J (1972) Sexuality in angiosperms. In: Steward FC (ed) Plant physiology: a treatise, physiology and development from seeds to sexuality. Academic Press, New York, pp 216–239

    Google Scholar 

  • —, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzimatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol 45: 115–120

    PubMed  Google Scholar 

  • Holm PB, Knudsen S, Mouritzen P, Negri D, Olsen FL, Roué C (1994) Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell. Plant Cell 6: 531–534

    PubMed  Google Scholar 

  • Jaenish R, Mintz B (1964) Simian virus 40 sequences in DNA of healthy adult mice derived from preimplantation blastocytes injected with viral DNA. Proc Natl Acad Sci USA 71: 1250–1254

    Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-Glucuronidase as a sensitive and versatile gene fusion marker. EMBO J 6: 3901–3908

    PubMed  Google Scholar 

  • Kao KN, Michayluk MR (1975) Nutritional requirements for growth ofVicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126: 105–110

    Google Scholar 

  • Keller A, Coster GL, Schnabl H, Mahaworasipla TL (1997) Influence of electrical treatment and cell fusion on ceil proliferation capacity of sunflower protoplasts in very low density culture. Plant Sci 126: 79–86

    Google Scholar 

  • Koop HU, Schweiger HG (1985a) Regeneration of plants after electrofusion of pairs of protoplasts. Eur J Cell Biol 39: 46–49

    Google Scholar 

  • — — (1985b) Regeneration of plants from individually cultivated protoplasts using an improved microculture system. J Plant Physiol 121: 245–257

    Google Scholar 

  • Kovács M, Barnabas B, Kranz E (1994) The isolation of viable egg cells from wheat (Triticum aestivum L.). Sex Plant Reprod 7: 31–312

    Google Scholar 

  • Kranz E, Lörz H (1993) In vitro fertilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plants. Plant Cell 5: 739–746

    PubMed  Google Scholar 

  • —, Bauer J, Lörz H (1991) In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex Plant Reprod 4: 12–16

    Google Scholar 

  • Kumlehn J, Schieder O, Lörz H (1997) In vitro development of wheat (Triticum aestivum L.) from zygote to plant via ovule culture. Plant Cell Rep 16: 663–667

    Google Scholar 

  • —, Lörz H, Kranz E (1998) Differentiation of isolated wheat zygotes into embryos and normal plaijts. Planta 205: 327–333

    Google Scholar 

  • Lawrence WA, Davies DR (1985) A method for the microinjection and culture of protoplasts at very low densities. Plant Cell Rep 3: 33–35

    Google Scholar 

  • Leduc N, Matthys-Rochon E, Rougier M, Mogensen L, Holm PB, Magnard JL, Dumas C (1996) Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro. Dev Biol 177: 190–203

    PubMed  Google Scholar 

  • Mendel RR, Müller B, Schulze J, Kolesnikov V, Zelenin A (1989) Delivery of foreign genes to intact barley cells by high velocity microprojectiles. Theor Appl Genet 78: 31–34

    Google Scholar 

  • Mogensen HL, Holm PB (1995) Dynamics of DNA quantities during zygote development in barley. Plant Cell 7: 487–494

    PubMed  Google Scholar 

  • Morikawa H, Yamada Y (1985) Capillary microinjection into protoplasts and intranuclear localization of injected materials. Plant Cell Physiol 26: 229–236

    Google Scholar 

  • Ochatt SJ, Chand PK, Rech EL, Davey ML, Power JB (1988) Electroporation-mediated improvement of plant regeneration of plantlets from protoplasts derived from cell suspension of barley (Hordeum vulgare). Physiol Plant 85: 289–294

    Google Scholar 

  • Pónya Zs, Tímár I, Szabó L, Kristóf Z, Barnabás B (1999) Morphological characterisation of wheat (T. aestivum L.) egg cell proto-plasts isolated from immature and overaged caryopses. Sex Plant Reprod 11: 357–359

    Google Scholar 

  • Rech EL, Ochatt SJ, Chand PK, Power JB, Davey MR (1987) Electro-enhancement of division of plant protoplast-derived cells. Protoplasma 141: 169–176

    Google Scholar 

  • — — —, Muligan BJ, Davey MR, Power JB (1988) Electroporation increases DNA synthesis in cultured plant protoplasts. Biol Technol 6: 1091–1093

    Google Scholar 

  • Rubin GM, Spradling AC (1982) Genetic transformation ofDrosophila with transposable element vectors. Science 218: 348–353

    PubMed  Google Scholar 

  • Rusconi S, Schaffner W (1981) Transformation of frog embryos with a rabbit b-globin gene. Proc Natl Acad Sci USA 78: 5051–5055

    PubMed  Google Scholar 

  • Sagata N, Watanable N, Vande Woude GF, Ikawa Y (1989) The cmos protooncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature 342: 512–518

    PubMed  Google Scholar 

  • Sambrook J, Fritsch CF, Maniatis T (1989) Purification of DNA recovered from agarose gels. In: Ford N, Nolan C, Ferguson M (eds) Gene cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 6.32–6.33

    Google Scholar 

  • Sauter M, Wiegen P, Lörz H, Kranz E (1998) Cell cycle regulatory genes from maize are differentially controlled during fertilization and first embryonic division. Sex Plant Reprod 11: 41–48

    Google Scholar 

  • Schnorf M, Neuhaus-Url, Galli A, Iida S, Potrykus I, Neuhaus G (1991) An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis. Transgen Res 1: 23–30

    Google Scholar 

  • Steinbiss HH, Stapel P, Topfer R, Hirtz RD, Schell J (1983) Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer Yellow. Protoplasma 116: 223–227

    Google Scholar 

  • Tímár I, Kristóf Z, Barnabas B (1997) Comparative studies on the male and female gametophyte development in three differentTriticum species. Plant Sci 126: 97–104

    Google Scholar 

  • Tischner T, Köszegi B, Veisz O (1997) Climatic programmes used in the Martonvásár Phytotron most frequently in recent years. Acta Agron Hung 45: 85–104

    Google Scholar 

  • Wagner TE, Hoppe PC, Jollick JD, Scholl DR, Hodinka RL, Gault JB (1981) Microinjection of a rabbit b-globin gene into zygotes and its subsequent expression in adult mice and their offspring. Proc Natl Acad Sci USA 78: 6376–6380

    PubMed  Google Scholar 

  • Whittingham DG (1980) Parthenogenesis in mammals. Oxford Rev Reprod Biol 2: 205–231

    Google Scholar 

  • Yamaizumi M, Horwich AL, Ruddle F (1983) Expression and stabilization of microinjected plasmids containing the herpes simplex virus thymidine kinase and polyoma virus DNA in mouse cells. Mol Cell Biol 3: 511–522

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Agricultural Research Institute of the Hungarian Academy of Sciences, P.O. Box 19, H-2462, Martonvásár, Hungary

    Z. Pónya, P. Finy & B. Barnabás

  2. Biological Research Center, Hungarian Academy of Sciences, Szeged

    A. Fehér & D. Dudits

  3. Agricultural Biotechnology Center, Gödöllö

    J. Mitykó

Authors
  1. Z. Pónya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Finy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Fehér
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Mitykó
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. Dudits
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B. Barnabás
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Z. Pónya.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pónya, Z., Finy, P., Fehér, A. et al. Optimisation of introducing foreign genes into egg cells and zygotes of wheat (Triticum aestivum L.) via microinjection. Protoplasma 208, 163–172 (1999). https://doi.org/10.1007/BF01279087

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01279087

Keywords

Search

Navigation