Summary
Protoplasts isolated enzymatically from leaves of tobacco (Nicotiana tabacum), stem callus of petunia (Petunia hybrida) and roots of corn (Zea mays) seedings were grown aseptically in microchambers, liquid droplets, or in stationary suspension cultures. Cell wall regenration occurred within 2 days in petunia, 6 to 7 days in tobacco, and 10 days in corn protoplasts. In petunia protoplasts, cell divisions ensued immediately after cell wall formation, and a colony of 30 to 40 cells was produced in 10 to 15 days after culture. Tobacco protoplasts took a longer time for the initiation of cell division activity and the formation of a cell colony. Plating of cell colonies derived from petunia and tobacco protoplasts, separately, resulted in larger sized cell masses, each of which formed a callus tissue when rransferred to fresh nutrient media. Auxins and cytokinins were essential for the differentiation, of shoots from protoplast-derived callus tissues of petunia and tobacco. Transfer of individual shoots to simpler nutrient media resulted in the formation of adventitious roots. Such plantlets of petunia and tobacco have flowered and set seeds in the greenhouse. Complete abortion of pollen was seen in plants derived from protoplasts of cytoplasmic male sterile plants of petunia.
Corn root protoplasts elongated following cell wall regeneration, accumulated a few large lipid globules, and remained viable for over 10 weeks in culture. Cell wall regeneration and a low percentage of cell division were seen in protoplasts isolated from corn leaves. Adjacent protoplasts occasionally fused to form two-nucleate protoplasts.
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References
Cocking, E. C.. 1960. A method for the isolation of plant protoplasts and vacuoles. Nature 187: 927–929.
Gregory, D. W., and E. C. Cocking. 1965. The large-scale isolation of protoplasts from immature tomato fruits. J. Cell Biol. 24: 143–146.
Ruesink, A., and K. V. Thimann. 1966. Protoplasts: preparation from higher plants. Science 154: 280–281.
Takebe, I., Y. Otsuki, and S. Aoki. 1968. Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol. 9: 115–124.
Schenk, R. U., and A. C. Hildebrandt. 1969. Production of protoplasts from plant cells in liquid culture using purified commercial cellulases. Crop Sci. 9: 629–631.
Power, J. B., and E. C. Cocking. 1970. Isolation of leaf protoplasts: macromolecule uptake and growth substance response. J. Exp. Bot. 21: 64–70.
Kao, K. N., W. A. Keller, and R. A. Miller. 1970. Cell division in newly formed cells from protoplasts of soybean. Exp. Cell Res. 62: 338–340.
Mishra, A. K., and J. R. Colvin. 1969. The formation of wall-like envelopes by isolated tomato fruit protoplasts. Protoplasma 67: 295–305.
Cocking, E. C., and E. Pojnar. 1969. An electron microscopic study of the infection of isolated tomato fruit protoplasts by tobacco mosaic virus. J. Gen. Virol. 4: 305–312.
Burgess, J., J. W., Watts, E. N. Fleming, and J. M. King, 1973. Plasmalemma fine structure in isolated tobacco mesophyll protoplasts. Planta (Berl.) 110: 291–301.
Ruesink, A. 1973. Surface membrane properties of isolated protoplasts. In “Protoplastes et fusion de cellules somatiques végétales.” Colloq. Int. Centre Natl. Rech. Sci. 212: 41–49.
Willison, J. H. M. 1973. Fine structural changes occurring during the culture of isolated tomato fruit protoplasts. In “Protoplastes et fusion de cellules somatiques végétales,” Colloq. Int. Centre Natl. Rech. Sci. 212: 215–241.
Cocking, E. C. 1970. Virus uptake, cell wall regeneration and virus multiplication in isolated plant protoplasts. Int. Rev. Cytol. 28: 89–124.
Coutts, R. H. A. 1973. Viruses in isolated protoplasts, a potential model system for studying nucleo-protein replication. In “Protoplastes et fusion de cellules somatiques végétales.” Colloq. Int. Centre Natl. Rech. Sci. 212: 353–366.
Aoki, S., and I. Takebe. 1969. Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucleic acid. Virology 39: 439–448.
Hess, D., I. Potrykus, G., Donn, J. Durand, and F. Hoffmann. 1973. Transformation experiments in higher plants: prerequisites for use of isolated protoplasts (isolation from mesophyll and callus cultures, uptake of proteins and DNA, and regeneration of whole plants). In “Protoplastes et fusion de cellules somatiques végétales.” Colloq Int. Centre Natl. Rech. Sci. 212: 343–351.
Ohyama, K., O. L. Gamborg, J. P. Shyluk, and R. A. Miller. 1973.; Studies on transfromation: uptake of exogenous DNA by plant protoplasts. In “Protoplastes et fusion de cellules somatiques végétales.” Colloq. Int. Centre Natl. Rech. Sci. 212: 423-428.
Mayo, M. A., and E. C. Cocking. 1969. Detection of pinocytotic activity using selective staining with phosphotungstic acid. Protoplasma 68:231–236.
Potrykus, I. 1973. Transplantation of chloroplasts into protoplasts ofPetunia. Z. Pflanzenphysiol. 70: 364–366.
Potrykus, I., and F. Hoffmann. 1973. Transplantation of nuclei into protoplasts of higher plants Z. Pflanzenphysiol. 69: 287–289.
Davey, M. R., and E. C. Cocking. 1972. The uptake of bacteria by isolated plant protoplasts. Nature 239: 455–456.
Power, J. B., S. E. Cummins, and E. C. Cocking. 1970. Fusion of isolated plant protoplasts. Nature 255:1016–1018.
Cocking, E. C. 1971. Fusion of isolated protoplasts: a first step towards the somatic hybridization of plants. In “Les cultures de tisus de plantes.” Colloq. Int. Centre Natl. Rech. Sci. 193: 303–317.
Eriksson, T. 1971. Studies on fusion and growth of protoplasts from higher plants grownin vitro. In “Les cultures de tissue de plantes.” Colloq. Int. Centre Natl. Rech. Sci. 193: 297–302.
Schenk, R. U., and A. C. Hildebrandt. 1971. Production, manipulation and fusion of plant cell protoplasts as steps toward somatic hybridization. In “Les cultures de tissue de plantes”. Colloq. Int. Centre Natl. Rech. Sci. 193: 319–331.
Carlson, P. S., H. H. Smith, and R. D. Dearing. 1972. Parasexual interspecific plant hybridization. Proc. Natl. Acad. Sci. U.S.A. 69:2292–2294.
Carlson, P. S. 1973. The use of protoplasts for genetic research, Proc. Natl. Acad. Sci. U.S.A. 70:598–602.
Davey, M. R., and K. C. Short. 1973. The isolation, culture and fusion of legume and cornflower leaf protoplasts. In “Protoplastes et fusion de cellules somatiques végétales.” Colloq. Int. Centre Natl. Rech. Sci. 212: 437–453.
Evans, P. K., J. Woodcock, and A. G. Keates. 1973. Steps towards cell fusion: studies on cereal protoplasts and protoplasts of haploid tobacco. In “Protoplastes et fusion de cellules somatiques végétales.” Colloq. Int. Centre Natl. Rech. Sci. 212: 469–477.
Power, J. B., and E. M. Frearson. 1973. The inter- and intranspecific fusion of plant protoplasts: subsquent developments in culture, with reference to crown gall callus and to-bacco and petunia leaf systems. In “Protoplastes et fusion de cellules somatiques végétales”. Colloq. Int. Centre Natl. Rech. Sci. 212: 409–421.
Vasil, V., and A. C. Hildebrandt. 1965. Differentiation of tobacco plants from single, isolated cells in microcultures. Science 150: 889–892.
Vasil, I. K., and V. Vasil. 1972. Totipotency and embryogenesis in plant cell and tissue cultures. In Vitro 8: 117–127.
Takebe, I., G. Labib, and G. Melchers. 1971. Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwisenschaften 58: 318–320.
Ohyama, K., and J. P. Nitsch. 1972. Flowering haploid plants obtained from protoplasts of tobacco leaves. Plant Cell Physiol. 13: 229–236.
Grambow, H. J., K. N. Kao, R. A. Miller, and O. L. Gamborg. 1972. Cell division and plant development from protoplasts of carrot cell suspension cultures. Planta (Berl.) 103: 348–355.
Kameya, T., and H. Uchimiya. 1972. Embryoids derived from isolated protoplasts of carrot. Plant (Berl.) 103: 356–360.
Durand, J., I. Potrykus, and G. Donn. 1973. Plantes issues de protoplasten de Petunia. Z. Pflanzenphysiol. 69: 26–34.
Frearson, E. M., J. B. Power, and E. C. Cocking. 1973. The isolation, culture and regeneration ofPetunia leaf protoplasts. Dev. Biol. 33: 130–137.
Bui-Dang-Ha, D., and I. A. Mackenzie, 1973. The division of protoplasts fromAsparagus officinalis L. and their growth and differentation. Protoplasma 78: 215–221.
Gresshoff, P. M., and C. H. Doy. 1973. Methods for diploid callus culture and the subsequent differentiation of various plant structures. Aust. J. Biol. Sci. 26: 505–508.
Vasil, V., and I. K. Vasil. 1973. Growth and cell division in isolated plant protoplasts in microchambers. In “Protoplastes et fusion de cellules somatiques végétales”. Colloq. Int. Centre Natl. Rech. Sci. 212: 139–149.
Carter, S. B. 1967. Effects of cytochalasins on mammalian cells. Nature 213: 261.
Ladda, R. L., and R. D. Estensen. 1970. Introduction of a heterologous nucleus into enucleated cytoplasms of cultured mouse L-cells. Proc. Natl. Acad. Sci. U.S.A. 67: 1528–1533.
Pollack, R., and R. Goldman. 1973. Synthesis of effective poliovirus in BSC-1 monkey cells enucleated with cytochalasin B. Science 179: 915–916.
Wessells, N. K., B. S. Spooner, J. F. Ash, M. O. Bradley, M. A. Luduena, E. L. Taylor, T. T. Wrenn, and K. M. Yamada. 1971. Microfilaments in cellular and developmental processes. Science 171: 135–143.
Franke, W. W., W. Herth, W. J. VanDer-Woude, and D. J. Morre. 1972. Tubular and filamentous structures in pollen tubes: possible involvement as guide elements in protoplasmic streaming and vectorial migration of secretory vesicles. Planta (Berl.) 105: 317–341.
Mascarenhas, J. P., and J. LaFountain. 1972. Protoplasmic streaming, cytochalasin B, and growth of the pollen tube. Tissue Cell 4: 11–14.
Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.
Vasil, I. K., and A. C. Hildebrandt. 1966. Growth and chlorophyll production in plant callus tissues grownin vitro. Plant (Berl.) 68: 69–82.
Nagata, T., and I. Takebe. 1971. Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta (Berl.) 99: 12–20.
White, P. R. 1963.The Cultivation of Animal and Plant Cells. 2nd ed. Ronald Press, New York.
Potrykus, I., and J. Durand. 1972. Callus formation from single protoplasts ofPetunia. Nature New Biol. 237: 286–287.
Rao, P. S., W. Handro, and H. Harada, 1973. Hormonal control of differentiation of shoots, roots and embryos in leaf and stem cultures ofPetunia inflata andPetunia hybrida. Physiol. Plant. 28: 458–463.
Gamborg, O. L., R. A. Miller, and K. Ojima. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158.
Bawa, S. B., and J. G. Torrey. 1971. “Budding” and nuclear division in cultured protoplasts of corn,Convolvulus and onion Bot. Gaz. 132: 240–245.
Motoyoshi, F. 1971. Protoplasts isolated from callus cells of maize endosperm. Exp. Cell Res. 68: 452–456.
Reinert, J., and S. Hellmann. 1973. Aspects of nuclear division and cell wall formation in protoplasts of different origin. In “Proto-plastes et fusion de cellules somatiques végétales”. Colloq. Int. Centre Natl. Rech. Sci. 212: 273–279.
Maretzki, A., and L. G. Nickell. 1973. Formation of protoplasts from sugarcane cell cultures from protoplasts. In “Protoplastes et fusion de cellules somatiques végétales”. Colloq. Int. Centre Natl. Rech. Sci. 212: 51–63.
Mackenzie, I. A., D. Bui-Dang-Ha, and M. R. Davey. 1973. Some aspects of the isolation, fine structure and growth of protoplasts fromAsparagus officinalis L. In “Protoplastes et fusion de cellules somatiques végétales”. Colloq. Int. Centre Natl. Rech. Sci. 212: 291–299.
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This work was supported by a cooperative agreement with the Agricultural Research Service, United States Department of Agriculture (12-14-100-10, 870) (34).
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Vasil, V., Vasil, I.K. Regeneration of tobacco and petunia plants from protoplasts an culture of corn protoplasts. In Vitro 10, 83–96 (1974). https://doi.org/10.1007/BF02615342
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DOI: https://doi.org/10.1007/BF02615342