Abstract
Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l-1 NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1–4 cells ml-1, provided the concentration of NAA in the medium was lowered to below 0.3 mg l-1. The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.
A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30–40%, independent of the cell density in the range of 1–4 to 3·104 cells ml-1, has been established. In this medium inositol was the only vitamin stringently required for growth.
Growth of cells at low densities was also possible in a medium initially containing 3 mg l-1 NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [14C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- BA:
-
6-benzyladenine
- EDTA:
-
ethylene diaminetetraacetic acid
- NAA:
-
naphtaleneacetic acid
References
Behrend, J., Mateles, R.I. (1976) Nitrogen metabolism in plant cell suspension cultures. 2. Role of organic acids during growth on ammonia. Plant Physiol. 58, 510–512
Bourgin, J.P. (1978) Valine-resistant plants from in vitro selected tobacco cells. Mol. Gen. Genet. 161, 225–230
Bourgin, J.P., Chupeau, Y., Missonier, C. (1979) Plant regeneration from mesophyll protoplasts of several Nicotiana species. Physiol. Plant. 45, 288–292
Carlson, P.S. (1973) The use of protoplasts for genetic research. Proc. Natl. Acad. Sci. USA. 70, 598–602
Chupeau, Y., Bourgin, J.P., Missonier, C., Dorion, N., Morel, G. (1974) Préparation et culture de protoplastes de divers Nicotiana. C.R. Acad. Sci. 278, 1565–1568
Dravnieks, D.E., Skoog, F., Burris, R.H. (1969) Cytokinin activation of de novo thiamin biosynthesis in tobacco callus cultures. Plant Physiol. 44, 866–870
Eriksson, T. (1965) Studies on the growth requirements and growth measurements of cell cultures of Happlopappus gracilis. Physiol. Plant. 18, 976–993
Fujimoto, W.Y., Subak-Sharpe, J.H., Seegmiller, J.E. (1971) Hypoxanthine-guanine phosphoribosyltransferase deficiency: Chemical agents for mutant cultured fibroblasts in mixed and heterozygote cultures. Proc. Natl. Acad. Sci. USA 68, 1516–1519
Gamborg, O.L., Shyluk, J.P. (1970) The culture of plant cells with ammonium salts as the sole nitrogen source. Plant Physiol. 45, 598–600
Goren, R., Bukovac, M.J. (1973) Mechanism of naphtaleneacetic acid conjugation. No effect of ethylene. Plant Physiol. 51, 907–913
Ham, R.G. (1965) Clonal growth of mammalian cells in chemically defined synthetic medium. Proc. Natl. Acad. Sci. USA 53, 288–293
Heller, R. (1953) Recherches sur la nutrition minérale des tissues végétaux cultivés in vitro. Ann. Sci. Natl. Biol. Vég. 14, 1–223
Kao, K.N., Michayluk, M.R. (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126, 105–110
Leguay, J.J., Guern, J. (1977) Quantitative effects of 2,4-dichlorophenoxyacetic acid on growth of suspension-cultured Acer pseudoplatanus cells. 2. Influence of 2,4-D metabolism and intracellular pH on the control of cell division by intracellular 2,4-D concentration. Plant Physiol. 60, 265–270
Linsmaier, E.M., Skoog, F. (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, 100–127
Logemann, H., Bergmann, L. (1974) Einfluß von Licht und Medium auf die “plating efficiency” isolierter Zellen aus Calluskulturen von Nicotiana tabacum var. “Samsun”. Planta 121, 283–292
Meyer, Y., Abel, W.O. (1975) Budding and cleavage division of tobacco mesophyll protoplasts in relation to pseudo-wall and wall formation. Planta 125, 1–13
Morel, G., Wetmore, R.H. (1951) Fern callus tissue culture. Am. J. Bot. 38, 141–143
Murashige, T., Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497
Nagata, T., Takebe, I. (1971) Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta 99, 12–20
Price, C.A., Clark, H.E., Funkhouser, E.A. (1972) Function of micronutrients in plants In: Micronutrients in agriculture, pp. 231–242, Mortvedt, J.J., ed. Madison Soil science society of America
Raveh, D., Huberman, E., Galun, E. (1973) In vitro culture of tobacco protoplasts: use of feeder techniques to support division of cells plated at low densities. In Vitro 9, 216–222
Rossini, L. (1973) Division of free leaf cells of Calystegia sepium in vitro. Phytomorphology 22, 21–29
Sargent, P.A., King, J. (1974) Investigations of growth-promoting factors in conditioned soybean root cells and in the liquid medium in which they grow: ammonium, glutamine, and aminoacids. Can. J. Bot. 52, 1747–1755
Schilde-Rentschler, L. (1973) Preparation of protoplasts for infection with Agrobacterium tumefaciens In: Protoplastes et fusions de cellules somatiques végétales. Colloq. Int. C.N.R.S. 212, 479–483
Weber, G., Lark, K.G. (1979) An efficient plating system for rapid isolation of mutants from plant cell suspensions. Theor. Appl. Genet. 55, 81–86
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Caboche, M. Nutritional requirements of protoplast-derived, haploid tobacco cells grown at low cell densities in liquid medium. Planta 149, 7–18 (1980). https://doi.org/10.1007/BF00386221
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00386221