Biopolym. Cell. 1991; 7(4):6-20.
Cell Biology
Individual culturing of preselected plant protoplasts and their fusion products in microdroplets
1Kirichenko I. V., 1Gleba Yu. Yu.
  1. Institute of Cell Biology and Genetic Engineering, Academy of Sciences of the Ukrainian SSR
    Kiev, USSR


Successfull development of different plant species single cells, protoplasts and products of preselected single protoplast fusion has been shown using the method of individual culturing in microdroplets developed. The highest plating efficiency among the individual protoplasts reached about 75% (soybean), the lowest one – 2,5% (clover). Electrofusion efficiency was as high as 95%. In this article we describe our individual culturing and electrofusion technology.


[1] Aristotle. Works. M: Mysl, 1981. vol 3. 613 p.
[2] Tempelaar MJ, Duyst A, De Vlas S y., Krol G, Symmonds C, Jones MGK. Modulation and direction of the electrofusion response in plant protoplasts. Plant Sci. 1987;48(2):99–105.
[3] Tempelaar MJ, Jones MG. Directed electrofusion between protoplasts with different responses in a mass fusion system. Plant Cell Rep. 1985;4(2):92-5.
[4] Tempelaar MJ, Jones MG. Analytical and preparative electrofusion of plant protoplast. Oxford surveys of plant mol cell biol. 1985; 2:347-51.
[5] Caplin SM. Mineral Oil Overlay for Conservation of Plant Tissue Cultures. Am J Bot. 1959;46(5):324-9.
[6] Lumiere A, Chevrotier A. Sur la vitalite des cultures dc gonocoques. Compt Rend Acad Sci. 1914;158:1820-1.
[7] Ungermann E. Eine Einfache Methode zur Gewinnung von Dauerkulturen Empfindlicher Bakterienarten und zur Erhaltung der Virulenz Tierpathogener Keime. Arb. Reichsgesundheitsamte. 1918; 51:180-99.
[8] Buell CB, Weston WH. Application of the mineral oil conservation method to maintaining collections of fungous cultures. Am J Bot. 1947;34(10):555-61.
[9] Michael M. Die Konservierung Schwer Haltbarer Bakterienkulturen, Insbesondere des Gonococcus (Modifikation der Ungermannschen Methode). Zbl. Bakteriol., Parasitenk., Infektionskrankh. und Hyd. I. 1921; 86:507-510.
[10] Morton HE, Pulaski EJ. The Preservation of Bacterial Cultures. I. J Bacteriol. 1938;35(2):163-83.
[11] Sherf AF. A method for maintaining Phytomonas sepedonica in culture for long periods without transfer. Phytopathology. 1943; 33:330-2.
[12] Wildy P, Stoker M. Multiplication of solitary HeLa cells. Nature. 1958;181(4620):1407-8.
[13] Vogt M, Dulbecco R. Properties of a HeLa cell culture with increased resistance to poliomyelitis virus. Virology. 1958;5(3):425-34.
[14] Jones LE, Hildebrandt AC, Riker AJ, Wu JH. Growth of Somatic Tobacco Cells in Microculture. Am J Bot. 1960;47(6):468-75.
[15] Rodnight R. Manometric determination of the solubility of oxygen in liquid paraffin, olive oil and silicone fluids. Biochem J. 1954;57(4):661-3.
[16] Gleba YY. Microdroplet culture: tobacco plants from single mesophyll protoplasts. Naturwissenschaften. 1978;65(3):158–9.
[17] Koop H-U, Weber G, Schweiger H-G. Individual culture of selected single cells and protoplasts of higher plants in microdroplets of defined media. Z Pflanzenphysiol. 1983;112(1):21–34.
[18] Koop H-U, Schweiger H-G. Regeneration of Plants from Individually Cultivated Protoplasts using an Improved Microculture System. J Plant Physiol. 1985;121(3):245–57.
[19] Kao KN, Michayluk MR. Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta. 1975;126(2):105-10.
[20] Caboche M. Nutritional requirements of protoplast-derived, haploid tobacco cells grown at low cell densities in liquid medium. Planta. 1980;149(1):7-18.
[21] Endo T, Komiya T, Masumitsu Y, Morikawa H, Yamada Y. An intergeneric hybrid cell line of duboisia hopwoodii and nicotiana tabacum by protoplast fusion. J Plant Physiol. 1987;129(5):453–9.
[22] Gilmour DM, Davey MR, Cocking EC, Pental D. Culture of low numbers of forage legume protoplasts in membrane chambers. J Plant Physiol. 1987;126(4-5):457–65.
[23] Kirichenko IV, Melnikov PV, Gleba YuYu. Regeneration of plants from individually cultivated tobacco protoplasts. Doklady Akad Nauk Ukr SSR. Ser B. 1989; (7):63-5.
[24] Eibs HG, Spielmann H. Preimplantation embryos. I. Laboratory equipment, preparation of media, sampling and handling of the embryos. Meth in prenatal toxicology. Eds D. Neubert et al. Stuttgart: Thieme publ., 1977:210-20.
[25] Cocking EC. Plant Cell Protoplasts-Isolation and Development. Annu Rev Plant Physiol. 1972;23(1):29–50.
[26] Koop HU, Schweiger HG. Regeneration of plants after electrofusion of selected pairs of protoplasts. Eur J Cell Biol. 1985;36(7 Suppl.):46-9.
[27] Schmalhausen II. Organisms growth. Kiev: Medvydav, 1932; 81 p.
[28] Murashige T, Skoog F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol Plant. 1962;15(3):473–97.
[29] Medgyesy P, Menczel L, Maliga P. The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and Isolation of their somatic hybrids. Mol Gen Genet. 1980;179(3):693–8.
[30] Kuchuk NV. Isolation and cultivation of protoplasts from five legume species. Sov Plant Physiol. 1989; 36(4):675-7.
[31] Kirichenko IV, Kuchuk NV., Sakhno LA, Gleba YuYu. Individual culturing of plant protoplasts and cells. Doklady Akad Nauk Ukr SSR. Ser B. 1990; (3):63-5.
[32] Eibs HG, Spielmann H. Preimplantation embryos. II. Culture and transplantation. Meth. in prenatal toxicology. Eds. D. Neubert et al. Stuttgart: Thieme publ., 1977:221-7.
[33] Brinster RL. In vitro cultivation of mammalian ova. Adv Biosci. 1969;N 4:200-33.
[34] Skarzhinska MV. Preparation of hybrid cell clones by culturing single individual merger products isolated protoplasts. Ukr Bot Zh. 1980; 36(6):58-73.
[35] Spangenberg G, Osusky M, Oliveira MM, Freydl E, Nagel J, Pais MS, Potrykus I. Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological, genetic, and molecular characterization. Theor Appl Genet. 1990;80(5):577-87.
[36] Crossway A, Oakes JV, Irvine JM, Ward B, Knauf VC, Shewmaker CK. Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts. Mol Gen Genet. 1986;202(2):179–85.
[37] Kirichenko IV. Fusion of individual pairs preselectioned tobacco protoplasts by electric fields. Doklady Akad Nauk Ukr SSR. Ser B. 1990; (6):74-7.
[38] Morikawa H, Hayashi Y, Hirabayashi Y. et al. Cellular and vacuolar fusion of protoplasts electrofused using platinum microelectrodes. Plant Cell Physiol. 1988; 29(1):189-93.
[39] Sowers AE. The Mechanism of Electroporation and Electrofusion in Erythrocyte Membranes. Electroporation and electrofusion in cell biol. Eds E. Neumann et al. New York; London: Plenum press, 1989:229-56.
[40] Vollet IJ, Roth LE. Cell fusion by nascent-membrane induction and divalent-cation treatment. Cytobiologie. 1974; 9(3):249-62.
[41] Spangenberg G, Koop H-U, Lichter R, Schweiger HG. Microculture of single protoplasts of Brassica napus. Physiol Plant. 1986;66(1):1–8.