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
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
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
Bradley MV (1948) An aceto-carmine squash technique for mature embryo sacs. Stain Technol 23:29–40
Cass DD (1973) An ultrastructural and Nomarski-interference study of the sperms of barley. Can J Bot 51:601–605
Cass DD (1990) Studies of isolated embryo sacs and sperm cells of maize and winter wheat. Am J Bot 77 suppl:5–6
Cass DD, Fabi GC (1988) Structure and properties of sperm cells isolated from the pollen of Zea mays. Can J Bot 66:819–825
Derksen J, Pierson ES, Traas JA (1985) Microtubules in vegetative and generative cells of pollen tubes. Eur J Cell Biol 38:142–148
Dumas C, Knox RB, McConchie CA, Russell SD (1984) Emerging physiological concepts in fertilization. What's New Plant Physiol 15:17–20
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 85:876–878
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
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
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
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
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
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
Herr JM (1971) A new clearing-squash technique for the study of ovule development in angiosperms. Am J Bot 58:785–790
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
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
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
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
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
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
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
Jongedijk E (1987) A quick enzyme squash technique for detailed studies on female meiosis in Solanum. Stain Technol 62:135–142
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
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
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
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
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
Lancelle SA, Cresti M, Hepler PK (1987) Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes of Nicotiana alata. Protoplasma 140:141–150
Li L-G, Hu S-Y (1985) Isolation of embryo sac with the enzyme squash technique. Acta Bot Sin 27:561–567
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
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
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
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
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
Mogensen HL, Wagner VT, Dumas C (1990) Quantitative, three-dimensional ultrastructure of isolated corn (Zea mays) sperm cells. Protoplasma 153:136–140
Mól R (1986) Isolation of protoplasts from female gametophytes of Torenia fournieri. Plant Cell Rep 3:202–206
Murthy UR, Schertz KF, Bashaw EC (1979) Apomictic and sexual reproduction in Sorghum. Indian J Genet Plant Breed 39:271–278
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
Paliwal RL (1953) A new technique for the microdissection of embryo-sacs of the Santalaceae. Phytomorphology 3:118–121
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
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
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
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
Russel SD (1984) Ultrastructure of the sperm of Plumbago zeylanica II. Quantitative cytology and three-dimensional organization. Planta 162:385–391
Russell SD (1985) Preferential fertilization in Plumbago: ultrastructural evidence for gamete-level recognition in an angiosperm. Proc Natl Acad Sci USA 82:6129–6132
Russell SD (1986) Isolation of sperm cells from the pollen of Plumbago zeylanica. Plant Physiol 81:317–319
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
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
Russell SC, Cresti M, Dumas C (1990) Recent progress on sperm characterization in flowering plants. Physiol Plant 80:669–676
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
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
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
Solntseva MK, Levkovskii VP (1979) Rapid production of permanent preparations of whole plant embryo sacs and pollen complexes. Cytol Genet 12:489–492
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
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
Southworth D, Knox RB (1989) Flowering plant sperm cells: Isolation from pollen of Gerbera jamesonii (Asteraceae). Plant Sci 60:273–277
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
Tanaka I (1988) Isolation of generative cells and their protoplasts from pollen of Lilium longiflorum. Protoplasma 142:68–73
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
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
Theunis CH (1990) Ultrastructural analysis of sperm cells of Spinacia oleracea isolated from mature pollen grains. Protoplasma 158:176–181
Theunis CH, Van Went JL (1989) Isolation of sperm cells from Spinacia oleracea from mature pollen. Sex Plant Reprod 2:97–102
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
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
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
Van Aelst AC, Theunis CH, Van Went JL (1990) Freeze-fracture studies of isolated sperm cells of Spinacia oleracea L. Protoplasma 153:204–207
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
Van Der Maas HM, Zaal MACM (1990b) Gametoplasts in perennial ryegrass. Acta Bot Neerl 39:399
Van Went JL, Kwee HS (1990) Enzymatic isolation of living embryo sac of Petunia. Sex Plant Reprod 3:257–262
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
Wagner VT, Dumas C, Mogensen HL (1989a) Morphometric analysis of isolated Zea mays sperm. J Cell Sci 93:179–184
Wagner VT, Kardolus JP, Van Went JL (1989b) Isolation of the lily embryo sac. Sex Plant Reprod 2:219–224
Wagner VT, Song YC, Matthys-Rochon E, Dumas C (1989c) Observations on the isolated embryo sac of Zea mays. Plant Sci 59:127–132
Webb MC, Gunning BES (1990) Embryo sac development in Arabidopsis thaliana. I. Megasporogenesis, including the microtubular cytoskeleton. Sex Plant Reprod 3:244–256
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
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
Wu Y, Zhou C (1988) Observation on enzymatically isolated embryo sacs in Adenophora axilliflora: fertilization, zygote and endosperm. Acta Bot Sin 30:210–212
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
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
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
Zhou C (1985) Isolation and identification of viable embryo sacs in several angiosperm species. Acta Bot Sin 27:258–262
Zhou C (1987a) A study of fertilization events in living embryo sacs isolated from sunflower ovules. Plant Sci 52:147–151
Zhou C (1987b) Cell biological studies on artificially isolated generative cells from angiosperm pollen. Acta Bot Sin 29:117–122
Zhou C (1988) Isolation and purification of generative cells from fresh pollen of Vicia faba L. Plant Cell Rep 7:107–110
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
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
Zhou C, Yang H-Y (1985) Observations on enzymatically isolated, living and fixed embryo sacs in several angiosperm species. Planta 165:225–231
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
Zhou C, Wu X-J (1990) Mass isolation and purification of generative cells from pollen grains. Acta Bot Sin 32:404–406
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
Zhou C, Orndorff K, Daghlian CP, DeMaggio AE (1988) Isolated generative cells in some angiosperms: a further study. Sex Plant Reprod 1:97–102
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
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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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DOI: https://doi.org/10.1007/BF00189998