Skip to main content
SpringerLink
Log in

Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes

  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The isolation of male and female gametes is a precondition for the micromanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature and vigorous. Sperm cells are isolated from pollen tubes grown on cut styles using the “in vivo/in vitro” technique. Embryo sacs are isolated 2 days after anthesis using brief treatments of minimal concentrations of cell-wall-digesting enzymes on ovules of emasculated flowers. Egg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the minimum required for the pollen tube to penetrate the ovule and effect fertilization in vivo.

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

References

  • Chaubal R, Reger BJ (1992) The dynamics of calcium distribution in the synergid cells of wheat after pollination. Sex Plant Reprod 5:206–213

    Google Scholar 

  • Dumas C, Knox RB, McConchie CA, Russell SD (1984) Emerging physiological concepts in fertilization. What's New Plant Physiol 15:17–20

    Google Scholar 

  • Fame JE, Digonnet C, Dumas C (1994) An in vitro system for adhesion and fusion of maize gametes. Science 263:1598–1600

    Google Scholar 

  • Holm PB, Knudsen S, Mouritzen P, Negri D, Olsen FL, Roue C (1994) Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell. Plant Cell 6:531–543

    Google Scholar 

  • Hu SY, Li LG, Zhu C (1985) Isolation of viable embryo sacs and their protoplasts ofNicotiana tabacum. Acta Bot Sin 27:337–344

    Google Scholar 

  • Huang BQ, Pierson ES, Russell SD, Tiezzi A, Cresti, M (1992) Video microscopic observations of living, isolated embryo sacs ofNicotiana and their component cells. Sex Plant Reprod 5:156–162

    Google Scholar 

  • Kovács M, Barnabas B, Kranz E (1995) Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures. Plant Cell Rep 15:178–180

    Google Scholar 

  • Kranz E, Lörz H (1994) In vitro fertilization of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH. Zygote 2:125–128

    Google Scholar 

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

    Google Scholar 

  • Kranz E, Bautor J, Lörz H (1991b) Electrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process, fusion of sperm cells with synergids and central cells, and cell reconstitution in maize. Sex Plant Reprod 4:17–21

    Google Scholar 

  • Kranz E, Wiegen P, Lörz H (1995) Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes. Plant J 8:9–23

    Google Scholar 

  • Leduc N, Matthys-Rochon E, Dumas C (1995) Deleterious effect of minimal enzymatic treatments on the development of isolated maize embryo sacs in culture. Sex Plant Reprod 8:313–317

    Google Scholar 

  • Mo YS, Yang HY (1992) Isolation and fusion of sperm cells in several bicellular pollen species. Acta Bot Sin 34:688–697

    Google Scholar 

  • Picton JM, Steer MW (1983) Evidence for the role of Ca2+ ions in tip extension in pollen tubes. Protoplasma 115:11–17

    Google Scholar 

  • Russell SD (1993) The egg cell: development and role in fertilization and early embryogenesis. Plant Cell 5:1349–1359

    Google Scholar 

  • Shivanna KR, Xu H, Taylor P, Knox RB (1988) Isolation of sperms from the pollen tubes of flowering plants during fertilization. Plant Physiol 87:647–650

    Google Scholar 

  • Sun MX, Yang HY, Zhou C (1993) A new method for embryo sac isolation and in site fusion of egg and synergid protoplasts inNicotiana tabacum. Acta Bot Sin 35:893–900

    Google Scholar 

  • Theunis CH, Pierson ES, Cresti M (1991) Isolation of male and female gametes in higher plants. Sex Plant Reprod 4:145–154

    Google Scholar 

  • Tian HQ, Russell SD (1997) Micromanipulation of male and female gametes ofNicotiana tabacum. II. Preliminary attempts for in vitro fertilization and egg cell culture. Plant Cell Rep (in press)

  • Wu Y, Zhou C, Koop HU (1993) Enzymatic isolation of viable nucelli at the megaspore mother cell stage and in developing embryo sacs inNicotiana tabacum. Sex Plant Reprod 6:171–175

    Google Scholar 

  • Zhou C (1985) Isolation and identification of viable embryo sacs in several angiosperm species. Acta Bot Sin 27:258–262

    Google Scholar 

Download references

Author information

Author notes

  1. H. Q. Tian

    Present address: Department of Botany and Microbiology, University of Oklahoma, 73019, Norman, OK, USA

Authors and Affiliations

  1. Department of Biology, Wuhan University, Wuhan, China

    H. Q. Tian & S. D. Russell

Authors
  1. H. Q. Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. D. Russell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. D. Russell.

Additional information

Communicated by G. Philipps

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tian, H.Q., Russell, S.D. Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes. Plant Cell Reports 16, 555–560 (1997). https://doi.org/10.1007/BF01142323

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01142323

Key words

Search

Navigation