In Vitro Fertilization with Rice Gametes: Production of Zygotes and Zygote and Embryo Culture

  • Takashi OkamotoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 710)


In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development, and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. A rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes can be performed using electrofusion and the fusion product, a zygote, forms a cell wall and an additional nucleolus. The zygote divides into an asymmetric two-celled embryo and develops into an early globular embryo, as in planta. The embryo further develops into irregularly shaped cell masses and fertile plants can be regenerated from the cell masses. This rice IVF system is a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.

Key words

Egg cell Globular embryo In vitro fertilization Regeneration Sperm cell Two-celled embryo Zygote 


  1. 1.
    Raghavan V (2003) Some reflections on double fertilization, from its discovery to the present. New Phytol 159:565–583CrossRefGoogle Scholar
  2. 2.
    Russell SD (1992) Double fertilization. Int Rev Cytol 40:357–390CrossRefGoogle Scholar
  3. 3.
    Pollock EG, Jensen WA (1964) Cell development during early embryogenesis in Capsella and Gossypium. Am J Bot 51:915–921CrossRefGoogle Scholar
  4. 4.
    Schel JHN, Kieft H, van Lammeren AAM (1984) Interactions between embryo and endosperm during early developmental stage of maize caryopses (Zea mays). Can J Bot 62:2842–2853CrossRefGoogle Scholar
  5. 5.
    Mansfield SG, Briarty LG (1991) Early embryogenesis in Arabidopsis thaliana. II. The developing embryo. Can J Bot 69:461–476CrossRefGoogle Scholar
  6. 6.
    Schulz R, Jensen WA (1968) Capsella embryogenesis: the egg, zygote and young embryo. Am J Bot 55:807–819CrossRefGoogle Scholar
  7. 7.
    Tykarska T (1979) Rape embryogenesis: II. Development of embryo proper. Acta Soc Bot Pol 48:391–421Google Scholar
  8. 8.
    Pritchard NH (1964) A cytochemical study of embryo development in Stellaria media. Am J Bot 51:472–479CrossRefGoogle Scholar
  9. 9.
    Tykarska T (1976) Rape embryogenesis: I. The proembryo development. Acta Soc Bot Pol 45:3–16Google Scholar
  10. 10.
    Lindsey K, Topping JE (1993) Embryogenesis: a question of pattern. J Exp Bot 259:359–374CrossRefGoogle Scholar
  11. 11.
    Haecker A, Gross-Hardt R, Geiges B, Sarkar A, Breuninger H, Herrmann M, Laux T (2004) Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 131:657–668PubMedCrossRefGoogle Scholar
  12. 12.
    Friml J, Vieten A, Sauer M, Weijers D, Schwarz H, Hamann T, Offringa R, Jürgens G (2003) Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis. Nature 426:147–153PubMedCrossRefGoogle Scholar
  13. 13.
    Lukowitz W, Roeder A, Parmenter D, Somerville C (2004) A MAPKK kinase gene regulates extra-embryonic cell fate in Arabidopsis. Cell 116:109–119PubMedCrossRefGoogle Scholar
  14. 14.
    Bowman JL, Eshed Y (2000) Formation and maintenance of the shoot apical meristem. Trends Plant Sci 5:110–115PubMedCrossRefGoogle Scholar
  15. 15.
    Laux T, Wurschum T, Breuninger H (2004) Genetic regulation of embryonic pattern ­formation. Plant Cell 16:S190–S202PubMedCrossRefGoogle Scholar
  16. 16.
    Mayer KF, Schoof H, Haecker A, Lenhard M, Jürgens G, Laux T (1998) Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell 95:805–815PubMedCrossRefGoogle Scholar
  17. 17.
    Sato Y, Hong SK, Tagiri A, Kitano H, Yamamoto N, Nagato Y, Matsuoka M (1996) A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis. Proc Natl Acad Sci USA 93:8117–8122PubMedCrossRefGoogle Scholar
  18. 18.
    Wang YY, Kuang A, Russell SD, Tian HQ (2006) In vitro fertilization as a tool for investigating sexual reproduction of angiosperm. Sex Plant Reprod 19:103–115CrossRefGoogle Scholar
  19. 19.
    Kranz E (1999) In vitro fertilization with isolated single gametes. Methods Mol Biol 111:259–267PubMedGoogle Scholar
  20. 20.
    Dupuis I, Roeckel P, Matthys-Rochon E, Dumas C (1987) Procedure to isolate viable sperm cells from corn (Zea mays L.) pollen grains. Plant Physiol 85:876–878PubMedCrossRefGoogle Scholar
  21. 21.
    Kranz E, Bautor J, Lörz H (1991) In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Sex Plant Reprod 4:12–16Google Scholar
  22. 22.
    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 6:531–543PubMedCrossRefGoogle Scholar
  23. 23.
    Kovács M, Barnabás B, Kranz E (1994) The isolation of viable egg cells of wheat (Triticum aestivum L.). Sex Plant Reprod 7:311–312CrossRefGoogle Scholar
  24. 24.
    Tian HQ, Russell SD (1997) Micromanipulation of male and female gametes of Nicotiana tabacum: I. Isolation of gametes. Plant Cell Rep 16:555–560Google Scholar
  25. 25.
    Katoh N, Lörz H, Kranz E (1997) Isolation of viable egg cells of rape (Brassica napus L.). Zygote 5:31–33PubMedCrossRefGoogle Scholar
  26. 26.
    Cao Y, Russell SD (1997) Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica. Sex Plant Reprod 10:36–73CrossRefGoogle Scholar
  27. 27.
    Uchiumi T, Komatsu S, Koshiba T, Okamoto T (2006) Isolation of gametes and central cells from Oryza sative L. Sex Plant Reprod 19:37–45CrossRefGoogle Scholar
  28. 28.
    Hoshino Y, Murata N, Shinoda K (2006) Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump. Ann Bot 97:1139–1144PubMedCrossRefGoogle Scholar
  29. 29.
    Kranz E, Lörz H (1993) In vitro fertilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plants. Plant Cell 5:739–746PubMedCrossRefGoogle Scholar
  30. 30.
    Faure J-E, Digonnet C, Dumas C (1994) An in vitro system for adhesion and fusion of maize gametes. Science 263:1598–1600PubMedCrossRefGoogle Scholar
  31. 31.
    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 2:125–128PubMedCrossRefGoogle Scholar
  32. 32.
    Khalequzzaman M, Haq N (2005) Isolation and in vitro fusion of egg and sperm cells in Oryza sativa. Plant Physiol Biochem 43:69–75PubMedCrossRefGoogle Scholar
  33. 33.
    Sun M, Yang H, Zhou C, Koop H-U (1995) Single-pair fusion of various combinations between female gametoplasts and other ­protoplasts in Nicotiana tabacum. Acta Bot Sin 37:1–6Google Scholar
  34. 34.
    Tian HQ, Russell SD (1997) Micromanipula­tion of male and female ­gametes of Nicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture. Plant Cell Rep 16:657–661CrossRefGoogle Scholar
  35. 35.
    Peng XB, Sun MX, Yang HY (2005) A novel in vitro system for gamete fusion in maize. Cell Res 15:734–738PubMedCrossRefGoogle Scholar
  36. 36.
    Antoine AF, Faure J-E, Dumas C, Feijo JA (2001) Differential contribution of cytoplasmic Ca2+ and Ca2+ influx to gamete fusion and egg activation in maize. Nat Cell Biol 3:1120–1123PubMedCrossRefGoogle Scholar
  37. 37.
    Sun M-X, Moscatelli A, Yang H-Y, Cresti M (2002) In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions. Sex Plant Reprod 13:113–117CrossRefGoogle Scholar
  38. 38.
    Kranz E, von 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 8:9–23CrossRefGoogle Scholar
  39. 39.
    Faure J-E, 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 5:747–755PubMedCrossRefGoogle Scholar
  40. 40.
    Scholten S, Lörz H, Kranz E (2002) Paternal mRNA and protein synthesis coincides with male chromatin decondensation in maize zygotes. Plant J 32:221–231PubMedCrossRefGoogle Scholar
  41. 41.
    Hoshino Y, Scholten S, von Wiegen P, Lörz H, Kranz E (2004) Fertilization induced changes in the microtubular architecture of the maize egg cell and zygote – an immunocytochemical approach adapted to single cells. Sex Plant Reprod 17:89–95CrossRefGoogle Scholar
  42. 42.
    Okamoto T, Scholten S, Lörz H, Kranz E (2005) Identification of genes that are up- or down-regulated in the apical or basal cell of maize two-celled embryos and monitoring their expression during zygote development by a cell manipulation- and PCR-based approach. Plant Cell Physiol 46:332–338PubMedCrossRefGoogle Scholar
  43. 43.
    Ito Y, Arikawa K, Antonio BA et al (2005) Rice annotation database (RAD): a contig-oriented database for map-based rice genomics. Nucleic Acids Res 33:D651–D655PubMedCrossRefGoogle Scholar
  44. 44.
    Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y, Onosato K, Hirochika H (2003) Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 15:1771–1780PubMedCrossRefGoogle Scholar
  45. 45.
    Kikuchi S, Satoh K, Nagata T et al (2003) Collection, mapping and annotation of over 28, 000 cDNA clones from japonica rice. Science 301:376–379PubMedCrossRefGoogle Scholar
  46. 46.
    Uchiumi T, Uemura I, Okamoto T (2007) Establishment of an in vitro fertilization ­system in rice (Oryza sativa L.). Planta 226:581–589PubMedCrossRefGoogle Scholar
  47. 47.
    Itoh J, Nonomura K, Ikeda K, Yamaki S, Inukai Y, Yamagishi H, Kitano H, Nagato Y (2005) Rice plant development: from zygote to spiklet. Plant Cell Physiol 46:23–47PubMedCrossRefGoogle Scholar
  48. 48.
    Kumlehn J, Lörz H, Kranz E (1998) Differentiation of isolated wheat zygotes into embryos and normal plants. Planta 205:327–333CrossRefGoogle Scholar
  49. 49.
    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 6:271–282PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.Department of Biological SciencesTokyo Metropolitan UniversityTokyoJapan

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