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Isolation of male and female gametes in higher plants

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Summary

The availability of generative cells, sperm cells, embryo sacs and egg cells from angiosperm plants in isolated conditions has opened up many prospects: study of the mechanism of recognition and fusion between gametes of opposite sex and detailed observation of the process of fertilization, biochemical and genetic analysis of gamete-specific components and genetic engineering in combination with in vitro fertilization. This review provides a list of about ninety publications, in which the isolation of male or female angiosperm gametes and the closely related generative cells and embryo sacs is reported. The species used are summarized in two tables. A description is given of the diverse isolation techniques, which consist of enzymatic digestion, bursting of pollen by osmotic shock, squashing, grinding and micro-dissection. Viability of isolated cells and yield, two important aspects of biotechnological manipulation, are emphasized. A critical evaluation of the most significant results obtained so far with isolated material is presented together with notes on prospects for the future.

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References

  1. Allington PM (1985) Micromanipulation of the unfixed cereal embryo sac. In: Chapman GP, Mantell SH, Daniels RM (eds) The experimental manipulation of ovule tissues. Longman, New York London, pp 39–51

    Google Scholar 

  2. Bednara J, Willemse MTM, Van Lammeren AAM (1990) Organization of the actin cytoskeleton during megasporogenesis in Gasteria verrucosa visualized with fluorescent labelled phalloidin. Acta Bot Neerl 39:43–48

    Google Scholar 

  3. Bradley MV (1948) An aceto-carmine squash technique for mature embryo sacs. Stain Technol 23:29–40

    Google Scholar 

  4. Cass DD (1973) An ultrastructural and Nomarski-interference study of the sperms of barley. Can J Bot 51:601–605

    Google Scholar 

  5. Cass DD (1990) Studies of isolated embryo sacs and sperm cells of maize and winter wheat. Am J Bot 77 suppl:5–6

    Google Scholar 

  6. Cass DD, Fabi GC (1988) Structure and properties of sperm cells isolated from the pollen of Zea mays. Can J Bot 66:819–825

    Google Scholar 

  7. Derksen J, Pierson ES, Traas JA (1985) Microtubules in vegetative and generative cells of pollen tubes. Eur J Cell Biol 38:142–148

    Google Scholar 

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

    Google Scholar 

  9. 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 85:876–878

    Google Scholar 

  10. Enaleeva NKh, Dushaeva NA (1975) Cyto-embryological studies in Nicotiana tabacum L. by means of enzymatic maceration. In: Apomixis and cyto-embryology of plants. Saratov University Press, Saratov, 3:171–175

    Google Scholar 

  11. Erdelská O (1974) Contribution to the study of fertilization in the living embryo sac. In: Linskens HF (ed) Fertilization in higher plants. North-Holland Publ, Amsterdam, pp 191–195

    Google Scholar 

  12. Erdelská O (1983) Microcinematographical investigation of the female gametophyte, fertilization and early embryo and endo-sperm development. In: Erdelská O, Čiamporová M, Lux A, Pretová A, Tupý J (eds) Fertilization and embryogenesis in ovulated plants. VEDA, Bratislava, pp 49–54

    Google Scholar 

  13. Forbes I (1960) A rapid enzyme-smear technique for the detection and study of plural embryo sacs in mature ovaries of several Paspalum species. Agron J 52:300–301

    Google Scholar 

  14. Gaff DF, Okang'o-Ogola O (1971) The use of non-permeating pigments for testing the survival of cells. J Expt Bot 22:756–758

    Google Scholar 

  15. Geltz NR, Russell SD (1988) Two-dimensional electrophoretic studies of the proteins and polypeptides in mature pollen grains and the male germ unit of Plumbago zeylanica. Plant Physiol 88:764–769

    Google Scholar 

  16. Herr JM (1971) A new clearing-squash technique for the study of ovule development in angiosperms. Am J Bot 58:785–790

    Google Scholar 

  17. Heslop-Harrison J, Heslop-Harrison Y (1989) Myosin associated with the surface of organelles, vegetative nuclei and generative cells in angiosperms pollen grains and tubes. J Cell Sci 94:319–325

    Google Scholar 

  18. Heslop-Harrison J, Heslop-Harrison Y, Shivanna KR (1984) The evaluation of pollen quality and a further appraisal of the fluorochromatic (FCR) test procedure. Theor Appl Genet 67:367–375

    Google Scholar 

  19. Hough T, Singh MB, Smart I, Knox RB (1986) Immunofluorescent screening of monoclonal antibodies to surface antigens of animal and plant cells bound to polycarbonate membranes. J Immunol Methods 92:103–107

    Google Scholar 

  20. Hu S-Y, Li LG, Zhu C (1985) Isolation of viable embryo sacs and their protoplasts of Nicotiana tabacum. Acta Bot Sin 27:343–347

    Google Scholar 

  21. Huang BQ, Russell SD (1989) Isolation of fixed and viable eggs, central cells and embryo sacs from ovules of Plumbago zeylanica. Plant Physiol 90:9–12

    Google Scholar 

  22. Huang BQ, Russell SD, Strout GW, Mao LJ (1990) Organization of isolated embryo sacs and eggs of Plumbago zeylanica (Plumbaginaceae) before and after fertilization. Am J Bot 77:1401–1410

    Google Scholar 

  23. Jensen CJ, Buchter-Larsen A, Cass DD, Thorn E, Engell K, Olesen P (1986) Pollen and ovule cultures of barley to isolate, manipulate and transfer sperm cells in in vitro fertilization. In: Horn W, Jensen CJ, Odenbach W, Schieder O (eds) Genetic manipulation in plant breeding, de Gruyter, Berlin New York, pp 473–475

    Google Scholar 

  24. Jongedijk E (1987) A quick enzyme squash technique for detailed studies on female meiosis in Solanum. Stain Technol 62:135–142

    Google Scholar 

  25. Keijzer CJ, Wilms HJ, Van Aelst AC, Leferink ten-Klooster HB (1986) Towards the isolation of sperm cells for androgenic purposes. Horn W, Jensen CJ, Odenbach W, Schieder O (eds) Genetic manipulation in plant breeding, de Gruyter, Berlin New York, pp 311–313

    Google Scholar 

  26. Keijzer CJ, Reinders MC, Leferink-ten Klooster HB (1988) A micromanipulation method for artificial fertilization in Torenia. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants. Springer, Berlin Heidelberg New York Tokyo, pp 119–124

    Google Scholar 

  27. Knox RB, Southworth DA, Singh MB (1988) Sperm cell determinants and control mechanisms of fertilization in plants. In: Chapman GP, Ainsworth CC, Chatham CJ (eds) Eukaryotic cell recognition. Cambridge University Press, Cambridge, pp 175–193

    Google Scholar 

  28. 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 

  29. 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 

  30. Lancelle SA, Cresti M, Hepler PK (1987) Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes of Nicotiana alata. Protoplasma 140:141–150

    Google Scholar 

  31. Li L-G, Hu S-Y (1985) Isolation of embryo sac with the enzyme squash technique. Acta Bot Sin 27:561–567

    Google Scholar 

  32. Matthys-Rochon E, Vergne P, Detchepare S, Dumas C (1987) Male germ unit isolation from three tricellular pollen species. Brassica oleracea, Zea mays and Triticum aestivum. Plant Physiol 83:464–466

    Google Scholar 

  33. Matthys-Rochon E, Detchepare S, Wagner V, Roeckel P, Dumas C (1988a) Isolation and characterization of viable sperm cells from tricellular pollen grains. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants, Springer, Heidelberg New York London, pp 245–250

    Google Scholar 

  34. Matthys-Rochon E, Roeckel P, Dupuis I, Dumas C (1988b) Les spermatozoïdes polliniques: mise au point d'une méthode d'isolement et de purification de spermatozoïdes à partir de pollens tricellulés de Maïs et de Colza. Ann Sci 23:104–107

    Google Scholar 

  35. McConchie CA, Knox RB (1986) The male germ unit and prospects for biotechnology. In: Mulcahy DL, Bergamini Mulcahy G, Ottaviano E (eds) Biotechnology and ecology of pollen. Springer, New York Berlin Heidelberg, pp 289–296

    Google Scholar 

  36. Miki-Hirosige H, Nakamura S, Takahashi T, Tanaka I (1988) Ultrastructural observations on isolated and cultured protoplasts of lily pollen. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants. Springer, Heidelberg New York Berlin, pp 239–245

    Google Scholar 

  37. Mogensen HL, Wagner VT, Dumas C (1990) Quantitative, three-dimensional ultrastructure of isolated corn (Zea mays) sperm cells. Protoplasma 153:136–140

    Google Scholar 

  38. Mól R (1986) Isolation of protoplasts from female gametophytes of Torenia fournieri. Plant Cell Rep 3:202–206

    Google Scholar 

  39. Murthy UR, Schertz KF, Bashaw EC (1979) Apomictic and sexual reproduction in Sorghum. Indian J Genet Plant Breed 39:271–278

    Google Scholar 

  40. Nielsen JE, Olesen P (1988) Isolation of sperm cells from the trinucleate pollen of sugar beet (Beta vulgaris). In: Wilms HJ, Keijzer CJ (eds) Plant sperm cells as tools for biotechnology. Pudoc, Wageningen, pp 111–122

    Google Scholar 

  41. Paliwal RL (1953) A new technique for the microdissection of embryo-sacs of the Santalaceae. Phytomorphology 3:118–121

    Google Scholar 

  42. Pennell RI, Geltz NR, Koren E, Russell SD (1987) Production and partial characterization of hybridoma antibodies elicited to the sperm of Plumbago zeylanica. Bot Gaz 148:401–406

    Google Scholar 

  43. Roeckel P, Dupuis I, Detchepare S, Matthys-Rochon E, Dumas C (1988) Isolation and viability of sperm cells from corn (Zea mays) and kale (Brassica oleracea) pollen grains. In: Wilms HJ, Keijzer CJ (eds) Plant sperm cells as tools for biotechnology. Pudoc, Wageningen, pp 105–109

    Google Scholar 

  44. Roeckel P, Chaboud A, Matthys-Rochon E, Russell S, Dumas C (1990a) Sperm cell structure, development and organization. In: Blackmore S, Knox RB (eds) Microspores evolution and ontogeny. Academic press, London San Diego New York Boston Sydney Tokyo Toronto, pp 281–307

    Google Scholar 

  45. Roeckel P, Matthys-Rochon E, Dumas C (1990b) Pollen and isolated sperm cell quality in Zea mays. In: Barnabás B, Liszt K (eds) Characterization of male transmission units in higher plant. MTA copy, Budapest, pp 41–48

    Google Scholar 

  46. Russel SD (1984) Ultrastructure of the sperm of Plumbago zeylanica II. Quantitative cytology and three-dimensional organization. Planta 162:385–391

    Google Scholar 

  47. Russell SD (1985) Preferential fertilization in Plumbago: ultrastructural evidence for gamete-level recognition in an angiosperm. Proc Natl Acad Sci USA 82:6129–6132

    Google Scholar 

  48. Russell SD (1986) Isolation of sperm cells from the pollen of Plumbago zeylanica. Plant Physiol 81:317–319

    Google Scholar 

  49. Russell SD (1990) Isolation and characterization of the angiosperm gamete. In: Dale B (ed) Mechanism of fertilization: plants to humans. Springer, Berlin Heidelberg New York, pp 3–15

    Google Scholar 

  50. Russell SD, Huang BQ, Strout GW (1989) Preliminary intermediate voltage electron microscopic observations of the isolated embryo sacs and eggs of Plumbago zeylanica L (Plumbaginaceae). In: Pare J, Bugnicourt M (eds) Some aspects and actual orientations in plant embryology. Université de Picardie, Faculté des Sciences, Picardie, pp 109–119

    Google Scholar 

  51. Russell SC, Cresti M, Dumas C (1990) Recent progress on sperm characterization in flowering plants. Physiol Plant 80:669–676

    Google Scholar 

  52. 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 

  53. Sidorova NV (1985) Characteristics of the ultrastructure of isolated tobacco embryo sacs. Dokl Akad Nauk Ukr SSR (Ser B) Geol Khim Biol Nauki 12:63–66

    Google Scholar 

  54. Solntseva MP, Levkovskii VP (1978) The variant of preparing constant Feulgen's stained slides of total embryo sacs isolated by plant tissue maceration. Both Zh 63:827–831

    Google Scholar 

  55. Solntseva MK, Levkovskii VP (1979) Rapid production of permanent preparations of whole plant embryo sacs and pollen complexes. Cytol Genet 12:489–492

    Google Scholar 

  56. Southworth D (1986) Sperm cell structure in Gerbera jamesonii (Asteracea). In: Williams EG, Knox RB, Irvine D (eds) Pollination '86. University of Melbourne, Melbourne, pp 172–177

    Google Scholar 

  57. Southworth D, Knox RB (1988) Methods for isolation of sperm cells from pollen. In: Wilms HJ, Keijzer CJ (eds) Plant sperm cells as emerging tools for crop biotechnology. Pudoc, Wageningen, pp 87–95

    Google Scholar 

  58. Southworth D, Knox RB (1989) Flowering plant sperm cells: Isolation from pollen of Gerbera jamesonii (Asteraceae). Plant Sci 60:273–277

    Google Scholar 

  59. Szakács E, Barnabas B (1990) Sperm cell isolation from wheat (Triticum aestivum L.). In: Barnábas B, Liszt K (eds) Characterization of male transmission units in higher plants. MTA copy, Budapest, pp 37–40

    Google Scholar 

  60. Tanaka I (1988) Isolation of generative cells and their protoplasts from pollen of Lilium longiflorum. Protoplasma 142:68–73

    Google Scholar 

  61. Tanaka I, Nakamura S, Miki-Hirosige H (1989) Structural features of isolated generative cells and their protoplasts from pollen of some Liliaceous plants. Gamete Res 24:361–374

    Google Scholar 

  62. Taylor P, Kenrick J, Li Y, Kaul V, Gunning BES, Knox RB (1989) The male germ unit of Rhododendron: quantitative cytology, three-dimensional reconstruction, isolation and detection using fluorescent probes. Sex Plant Reprod 2:254–264

    Google Scholar 

  63. Theunis CH (1990) Ultrastructural analysis of sperm cells of Spinacia oleracea isolated from mature pollen grains. Protoplasma 158:176–181

    Google Scholar 

  64. Theunis CH, Van Went JL (1989) Isolation of sperm cells from Spinacia oleracea from mature pollen. Sex Plant Reprod 2:97–102

    Google Scholar 

  65. Theunis CH, Van Went JL (1990) Isolated sperm cells of Spinacia oleracea. In: Barnabás B, Liszt K (eds) Characterization of male transmission units in higher plants. MTA copy, Budapest, pp 25–29

    Google Scholar 

  66. Theunis CH, Van Went JL, Wilms HJ (1988) A technique to isolate sperm cells of mature spinach pollen. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants. Springer, Berlin Heidelberg New York, pp 233–238

    Google Scholar 

  67. Tyrnov VS, Enaleeva NKh, Knokhlov SS (1975) A study of n vitro isolated embryo sacs in angiosperms. In: These of Reports. XII Int Bot Congress. Nauka, Leningrad, 266

    Google Scholar 

  68. Van Aelst AC, Theunis CH, Van Went JL (1990) Freeze-fracture studies of isolated sperm cells of Spinacia oleracea L. Protoplasma 153:204–207

    Google Scholar 

  69. Van Der Maas HM, Zaal MACM (1990a) Sperm cell isolation from pollen of perennial ryegrass (Lolium perenne L.). In: Barnabás B, Liszt K (eds) Characterization of male transmission untis in higher plants. MTA copy, Budapest, pp 31–35

    Google Scholar 

  70. Van Der Maas HM, Zaal MACM (1990b) Gametoplasts in perennial ryegrass. Acta Bot Neerl 39:399

    Google Scholar 

  71. Van Went JL, Kwee HS (1990) Enzymatic isolation of living embryo sac of Petunia. Sex Plant Reprod 3:257–262

    Google Scholar 

  72. Wagner VT, Song YC, Matthys-Rochon E, Dumas C (1988) The isolated embryo sac of Zea mays: structural and ultrastructural observations. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants. Springer, Heidelberg Berlin New York, pp 125–130

    Google Scholar 

  73. Wagner VT, Dumas C, Mogensen HL (1989a) Morphometric analysis of isolated Zea mays sperm. J Cell Sci 93:179–184

    Google Scholar 

  74. Wagner VT, Kardolus JP, Van Went JL (1989b) Isolation of the lily embryo sac. Sex Plant Reprod 2:219–224

    Google Scholar 

  75. Wagner VT, Song YC, Matthys-Rochon E, Dumas C (1989c) Observations on the isolated embryo sac of Zea mays. Plant Sci 59:127–132

    Google Scholar 

  76. Webb MC, Gunning BES (1990) Embryo sac development in Arabidopsis thaliana. I. Megasporogenesis, including the microtubular cytoskeleton. Sex Plant Reprod 3:244–256

    Google Scholar 

  77. Wilms HJ, Keijzer CJ (1985) Cytology of pollen tube and embryo sac development as possible tools for in vitro plant (re)-production. In: Chapman GP, Mantell SH, Daniels RW (eds) The experimental manipulation of ovule tissues. Longman, New York London, pp 24–35

    Google Scholar 

  78. Wilms HJ, Leferink-ten Klooster HB, Van Aelst AC (1986) Isolation of spinach sperm cells: I. Ultrastructure and three-dimensional construction in the mature pollen grain. In: Mulcahy DL, Bergamini Mulcahy G, Ottaviano E (eds) Biotechnology and ecology of pollen. Springer, Heidelberg Berlin New York, pp 307–312

    Google Scholar 

  79. Wu Y, Zhou C (1988) Observation on enzymatically isolated embryo sacs in Adenophora axilliflora: fertilization, zygote and endosperm. Acta Bot Sin 30:210–212

    Google Scholar 

  80. Yang H-Y, Zhou C (1984) Observations on megasporogenesis and megagametophyte development in Paulownia sp. and Sesamum indicum by enzymatic maceration technique. Acta Bot Sin 26:342–346

    Google Scholar 

  81. Yang HY, Zhou C (1989) Isolation of viable sperms from pollen of Brassica napus, Zea mays and Secale cereale. Chin J Bot 1:80–84

    Google Scholar 

  82. Yeung EC, Thorpe TA, Jensen CJ (1981) In vitro fertilization and embryo culture. In: Thorpe TA (ed) Plant Tissue Culture methods and applications in agriculture, Academic Press New York pp 253–271

    Google Scholar 

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

    Google Scholar 

  84. Zhou C (1987a) A study of fertilization events in living embryo sacs isolated from sunflower ovules. Plant Sci 52:147–151

    Google Scholar 

  85. Zhou C (1987b) Cell biological studies on artificially isolated generative cells from angiosperm pollen. Acta Bot Sin 29:117–122

    Google Scholar 

  86. Zhou C (1988) Isolation and purification of generative cells from fresh pollen of Vicia faba L. Plant Cell Rep 7:107–110

    Google Scholar 

  87. Zhou C, Yang H-Y (1982) Enzymatic isolation of embryo sacs in angiosperms: isolation and microscopical observations of fixed materials. Acta Bot Sin 24:403–407

    Google Scholar 

  88. Zhou C, Yang H-Y (1984) The enzymatic isolation of embryo sacs from fixed and fresh ovules of Antirrhinum majus L. Acta Biol Exp Sin 17:141–147

    Google Scholar 

  89. Zhou C, Yang H-Y (1985) Observations on enzymatically isolated, living and fixed embryo sacs in several angiosperm species. Planta 165:225–231

    Google Scholar 

  90. Zhou C, Yang H-Y (1986) Isolation of embryo sacs by enzymatic maceration and its potential in haploid study. In: Hu H, Yang HY (eds) Haploids of higher plants in vitro. Springer, Berlin Heidelberg New York, pp 192–203

    Google Scholar 

  91. Zhou C, Wu X-J (1990) Mass isolation and purification of generative cells from pollen grains. Acta Bot Sin 32:404–406

    Google Scholar 

  92. Zhou C, Orndorff K, Allen RD, DeMaggio AE (1986) Direct observations on generative cells isolated from pollen grains of Haemanthus katherinae Baker. Plant Cell Rep 5:306–309

    Google Scholar 

  93. Zhou C, Orndorff K, Daghlian CP, DeMaggio AE (1988) Isolated generative cells in some angiosperms: a further study. Sex Plant Reprod 1:97–102

    Google Scholar 

  94. Zhou C, Zee SY, Yang HY (1990) Microtubule organization of in situ and isolated generative cells in Zephyranthes grandiflora Lindl. Sex Plant Reprod 3:213–218

    Google Scholar 

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  1. Dipartimento di Biologia Ambientale, Università di Siena, Via P.A. Mattioli 4, I-53100, Siena, Italy

    C. H. Theunis, E. S. Pierson & M. Cresti

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Theunis, C.H., Pierson, E.S. & Cresti, M. Isolation of male and female gametes in higher plants. Sexual Plant Reprod 4, 145–154 (1991). https://doi.org/10.1007/BF00189998

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