Planta (1984)161:246-248
P l a n t a 9 Springer-Verlag 1984
Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.) Dietrich Ernst and Dieter Oesterhelt Max-Planck-Institut fiir Biochemie, D-8033 Martinsried, Federal Republic of Germany
Abstract. A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4.10-8 to 4.10-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5.10 -8 to 5" 10- 7 M), higher concentrations (5" 10- 6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation. Key words: Cell culture - Cell growth - Cytokinin and embryogenesis - Embryogenesis in cell culture - Pimpinella.
These results indicate a possible relationship between embryo development and cytokinin level. However, i) endogenous cytokinins in the carrot culture, which might show an influence on cell development together with the externally applied cytokinins, are not known, and ii) a direct comparison of embryonic and proembryonic cultures has not been reported up to now. This prompted us to investigate the influence of an exogenous application of cytokinins on a proembryonic cell suspension culture, as well as during embryogenesis in the same culture. Two cytokinins, isopentenyladenine (i6Ade) and isopentenyladenosine (i6Ado), were tested because they were found to be present in high concentrations in the cell culture of Pimpinella anisum (Ernst et al. 1984). The general question to be answered here is whether cytokinins merely promote growth or whether they stimulate embryogenesis as well. Material and methods
Introduction
Chemicals. 2,4-Diehlorophenoxyacetic acid (2,4-D), isopentenyladenosine (i6Ado) and isopentenyladenine (i6Ade) were from Sigma (Munich, FRG), all other chemicals were obtained from Merck (Darmstadt, FRG).
Cytokinins are known to occur in plant tissue cultures, and to exhibit a change in concentration during cell growth (Mackenzie and Street 1972; Short and Torrey 1972; Nishinari and Syono 1980; Ernst et al. 1984). Recently we have demonstrated a change in cytokinin content during somatic embryogenesis in an anise cell culture (Ernst et al. 1984). Bennici and Cionini (1979) demonstrated an increase of in-vitro growth in Phaseolus coccineus embryos by the addition of zeatin or zeatin riboside. Fujiumura and Komamine (1975) reported a promotive effect on embryo development in a carrot cell culture by the application of zeatin.
Plant material and culture methods. The isolation and tiae general growth conditions of the anise culture were as described (Ernst et al. 1983, 1984). The culture was grown at 27 ~ C under continuous illumination (fluorescent lamps, 1000 lx). The proembryonic culture ( + 2,4-D) was incubated in 250-ml Delong flasks with 50 ml B5-medium (Gamborg 1975) on a gyratory shaker (140 rpm). The embryonic culture was incubated in Petri dishes (60 mm diameter, 15 rnm deep) with 10 ml of the same medium ommitting 2,4-D, and maintained at 60 rpm under the same conditions. The cytokinins, i6Ado or i6Ade, were added by sterile injection (0.2 gm Acrodisc-filter, Gelman, Ann Arbor, USA) to the culture. The final concentrations of the cytokinins ranged from 4.10 -6 to 5"10 -8 M.
Abbreviations: 2,4-D = 2,4-dichlorophenoxyacetic acid; i6Ade = isopentenyladenine; i6Ado - isopentenyladenosine
Cell and embryo counting, i) Proembryos: to 5-ml samples of cell suspension 1 ml of 90% trichloroacetic acid (w/v) was add-
D. Ernst and D. Oesterhelt: Cytokinins, growth and somatic embryogenesis in Pimpinella cells
247
ed and the cells were counted as described by Huber et al. (1978). ii) Embryos: the number of embryos formed was counted under a light microscope (2540 x ), according to the morphological embryo stages: globular, heart-shaped and torpedo-shaped.
adenosine (i6Ado) on cell growth in a proembryonic anise culture (50 ml). The initial cell density was 8.104 cells ml-L The results are the average of two replicate experiments, n.d. =not determined
Results and discussion
Cytokinin (x 10-s M)
lsopentenyladenosine and i6Ade (4.10-s to 4.10 -6 M) were added to the anise cell culture containing 2,4-D. We used this range of hormone concentration, as the endogenous pool of i6Ado was about 1.5' 10 -7 mol kg-1 fresh weight during logarithmic growth (Ernst et al. 1984). Concentrations of 4.10-6 M showed no remarkable enhancement of the cell density after an incubation time of 48h or 90h, whereas concentrations of 4.10-7 M and 4.10-s M both increased the cell density by 46% (for i6Ade) and by 24% (for i6Ado) after 48 h of growth (Table 1). An enhancement of about 10% was still evident after 90 h of incubation. A similar effect was reported by Mackenzie et al. (1972) for the growth of a sycamore cell culture with cytokinin concentrations ranging from 10- 8 to 10- 6 M. Addition of i6Ado or i6Ade in concentrations from 5.10 -8 to 5.10 -7 M to the embryonic anise cell culture without 2,4-D caused an increased number of embryos after 6 d, whereas a concentration of 5.10- 6 M had no effect (Table 2). Similar results were obtained by Fujiumura and Komamine (1975, 1980) when embryo formation was measured in a carrot cell culture. However, these authors investigated only embryo formation and not the influence of cytokinins on the proembryonic stage. We further investigated the possibility of cytokinin sensitivity during the various stages of cell growth. Isopentenyladenosine or i6Ade (5"10 -s M) was added to the cultures at different times after the induction of embryogenesis. The maximum enhancement of embryo formation was obtained by adding i6Ado or i6Ade at day two of the culture medium (Table 3). At this time no embryos were visible in the culture; the cells are still in the logarithmic phase of cell growth (Ernst et al. 1984). From Table 3, it is obvious that embryo formation is increased only by a cytokinin application during the first 3 d (up to 46%). Addition of i6Ado or i6Ade after day three of culture produced no enhancement in the number of embryos formed. Furthermore, we found no difference in the percentage of individual embryo stages. Irrespective of the time of application, about 78-82% globular and 18-22% heart- and torpedoshaped embryos were present after a culture period of 6d.
Table 1. Effect of isopentenyladenine (i6Ade) and isopentenyl-
Cell density (x 105 cells m1-1) 48 h
90 h
400 (i6Ade) 40 (i6Ade) 4 (i6Ade) 400 (i6Ado) 40 (i6Ado) 4 (i6Ado)
1.20 1.71 1.62 1.26 1.46 1.49
n.d. 2.09 2.09 n.d. 2.05 2.04
Control
1.17
1.86
Table 2. Effect of isopentenyladenine (i6Ade) and isopentenyl-
adenosine (i6Ado) on somatic embryogenesis in an anise cell culture. The cytokinins were added at the beginning of the incubation time and embryos were counted after 6 d. The initial cell density was 1.2.105 cells m1-1 in a 10-ml culture volume. The results are the average of three independent experiments. Standard deviation: between 10% and 20% Cytokinin ( x 10-a M)
Embryos per Petri-dish
500 (i6Ade) 50 (i6Ade) 5 (i6Ade)
3675 4388
500 (i6Ado) 50 (i6Ado) 5 (i6Ade)
3 964 4572 4635
Control
3838
4631
Table 3. Effect of isopentenyladenine (i6Ade) and isopentenyl-
adenosine (i6Ado) on anise embryo formation. The initial cell density was 1.2" 105 cells m1-1 in a 10-ml culture volume. The cytokinins (5.10 -8 M) were added at different times to the culture and the embryos counted 6 d after the induction of embryogenesis. The results represent the average of three independent experiments. Standard deviation: between 10% and 20%. (Control: 3838 embryos per Petri-dish) Time (d) of cytokinin addition
0 l 2 3 4
Embryos per Petri-dish i6Ade
i6Ado
4631 4803 5 640 4762 3509
4635 4725 5 499 4617 3936
The results indicate that the addition of cytokinins enhances cell division during this period of growth; more cells are yielded and there is, therefore, the possibility of the formation of more embryos at a later stage. This assumption is supported
248
D. Ernst and D. Oesterhelt: Cytokinins, growth and somatic embryogenesis in Pimp&ella cells
by a number of other facts: i) addition of i6Ado or i6Ade to the embryo culture after day three yielded about the same number of embryos as a control culture (Table 3); ii) addition of i6Ado or i6Ade to a proembryonic culture increased the cell density (Table 1); iii) the endogenous maxima of cytokinins are correlated with logarithmic growth and not with a morphologically visible embryo formation (Ernst et al. 1984); iv) Huber et al. (1978) demonstrated a direct correlation of the number of anise embryos formed in a cell culture with the initial cell density: higher initial cell densities yielded more embryos; v) application of zeatin or zeatin riboside to Phaseolus coccineus embryos increased the embryo length, indicating a cytokinin influence on cell growth (Bennici and Cionini 1979). Our observations and the results from the literature lead to the following conclusion: cytokinins are not involved in embryo development directly, rather they show a stimulating effect on cell division, resulting in an increased number of cells and, therefore, more embryos in the culture.
References
Ernst, D., Sch/ifer, W., Oesterhelt, D. (1983) Isolation and quantitation of isopentenyladenosine in an anise cell culture by single ion monitoring, radioimmunoassay and bioassay. Planta 159, 216-221 Ernst, D., Oesterhelt, D., Schiller, W. (1984) Endogenous cytokinins during embryogenesis in an anise cell culture (Pirnpinella an&urn L.). Planta 161,240-245 Fujiumura, T., Komamine, A. (1975) Effects of various growth regulators on the embryogenesis in a carrot cell suspension culture. Plant Sci. Lett. 5, 359-364 Fujiumura, T., Komamine, A. (1980) Mode of action of 2,4-D and zeatin on somatic embryogenesis in a carrot cell suspension culture. Z. Pflanzenphysiol. 99, 1-8 Gamborg, O.L. (1975) Callus and cell culture. In: Plant tissue culture methods, pp. 1-11, Gamborg, O.L., Wetter, L.R., eds. National Research Council of Canada, Saskatoon, Saskatchewan Huber, J., Constabel, F., Gamborg, O.L. (1978) A cell counting procedure applied to embryogenesis in cell suspension cultures of anise (Pirnpinella an&urn L.). Plant Sci. Lett. 12, 209-215 Mackenzie, I.A., Street, H.E. (1972) The cytokinins of cultured sycamore cells. New Phytol. 71,621-631 Mackenzie, I.A., Konar, A., Street, H.E. (1972) Cytokinins and the growth of cultured sycamore cells. New Phytol. 71, 633-638 Nishinari, N., Syono, K. (1980) Changes in endogenous cytokinin levels in partially synchronized cultured tobacco cells. Plant Physiol. 65, 437-441 Short, C.K., Torrey, J.G. (1972) Cytokinin production in relation to the growth of pea-root callus tissue. J. Exp. Bot. 23, 1099-1105
Bennici, A., Cionini, P.G. (1979) Cytokinins and in vitro development of Phaseolus coccineus embryos. Planta 147, 27-29
Received 12 November 1983 ; accepted 7 February 1984
P l a n t a 9 Springer-Verlag 1984
Effect of exogenous cytokinins on growth and somatic embryogenesis in anise cells (Pimpinella anisum L.) Dietrich Ernst and Dieter Oesterhelt Max-Planck-Institut fiir Biochemie, D-8033 Martinsried, Federal Republic of Germany
Abstract. A cell culture of anise was grown in the presence or absence of 2,4-dichlorophenoxyacetic acid (2,4-D). Application of isopentenyladenine or isopentenyladenosine (4.10-8 to 4.10-7 M) to the proembryonic culture (+2,4-D) yielded an increase of the cell density, in contrast to a proembryonic culture grown without exogenous application of cytokinins. Embryogenesis was induced by transferring the cells to a hormone-free medium. Embryo development was promoted by isopentenyladenine and isopentenyladenosine (5.10 -8 to 5" 10- 7 M), higher concentrations (5" 10- 6 M) inhibited embryogenesis. The effect of cytokinins on embryogenesis was only promotive until the third day of culture, i.e. coincident with cell growth rather than differentiation. Key words: Cell culture - Cell growth - Cytokinin and embryogenesis - Embryogenesis in cell culture - Pimpinella.
These results indicate a possible relationship between embryo development and cytokinin level. However, i) endogenous cytokinins in the carrot culture, which might show an influence on cell development together with the externally applied cytokinins, are not known, and ii) a direct comparison of embryonic and proembryonic cultures has not been reported up to now. This prompted us to investigate the influence of an exogenous application of cytokinins on a proembryonic cell suspension culture, as well as during embryogenesis in the same culture. Two cytokinins, isopentenyladenine (i6Ade) and isopentenyladenosine (i6Ado), were tested because they were found to be present in high concentrations in the cell culture of Pimpinella anisum (Ernst et al. 1984). The general question to be answered here is whether cytokinins merely promote growth or whether they stimulate embryogenesis as well. Material and methods
Introduction
Chemicals. 2,4-Diehlorophenoxyacetic acid (2,4-D), isopentenyladenosine (i6Ado) and isopentenyladenine (i6Ade) were from Sigma (Munich, FRG), all other chemicals were obtained from Merck (Darmstadt, FRG).
Cytokinins are known to occur in plant tissue cultures, and to exhibit a change in concentration during cell growth (Mackenzie and Street 1972; Short and Torrey 1972; Nishinari and Syono 1980; Ernst et al. 1984). Recently we have demonstrated a change in cytokinin content during somatic embryogenesis in an anise cell culture (Ernst et al. 1984). Bennici and Cionini (1979) demonstrated an increase of in-vitro growth in Phaseolus coccineus embryos by the addition of zeatin or zeatin riboside. Fujiumura and Komamine (1975) reported a promotive effect on embryo development in a carrot cell culture by the application of zeatin.
Plant material and culture methods. The isolation and tiae general growth conditions of the anise culture were as described (Ernst et al. 1983, 1984). The culture was grown at 27 ~ C under continuous illumination (fluorescent lamps, 1000 lx). The proembryonic culture ( + 2,4-D) was incubated in 250-ml Delong flasks with 50 ml B5-medium (Gamborg 1975) on a gyratory shaker (140 rpm). The embryonic culture was incubated in Petri dishes (60 mm diameter, 15 rnm deep) with 10 ml of the same medium ommitting 2,4-D, and maintained at 60 rpm under the same conditions. The cytokinins, i6Ado or i6Ade, were added by sterile injection (0.2 gm Acrodisc-filter, Gelman, Ann Arbor, USA) to the culture. The final concentrations of the cytokinins ranged from 4.10 -6 to 5"10 -8 M.
Abbreviations: 2,4-D = 2,4-dichlorophenoxyacetic acid; i6Ade = isopentenyladenine; i6Ado - isopentenyladenosine
Cell and embryo counting, i) Proembryos: to 5-ml samples of cell suspension 1 ml of 90% trichloroacetic acid (w/v) was add-
D. Ernst and D. Oesterhelt: Cytokinins, growth and somatic embryogenesis in Pimpinella cells
247
ed and the cells were counted as described by Huber et al. (1978). ii) Embryos: the number of embryos formed was counted under a light microscope (2540 x ), according to the morphological embryo stages: globular, heart-shaped and torpedo-shaped.
adenosine (i6Ado) on cell growth in a proembryonic anise culture (50 ml). The initial cell density was 8.104 cells ml-L The results are the average of two replicate experiments, n.d. =not determined
Results and discussion
Cytokinin (x 10-s M)
lsopentenyladenosine and i6Ade (4.10-s to 4.10 -6 M) were added to the anise cell culture containing 2,4-D. We used this range of hormone concentration, as the endogenous pool of i6Ado was about 1.5' 10 -7 mol kg-1 fresh weight during logarithmic growth (Ernst et al. 1984). Concentrations of 4.10-6 M showed no remarkable enhancement of the cell density after an incubation time of 48h or 90h, whereas concentrations of 4.10-7 M and 4.10-s M both increased the cell density by 46% (for i6Ade) and by 24% (for i6Ado) after 48 h of growth (Table 1). An enhancement of about 10% was still evident after 90 h of incubation. A similar effect was reported by Mackenzie et al. (1972) for the growth of a sycamore cell culture with cytokinin concentrations ranging from 10- 8 to 10- 6 M. Addition of i6Ado or i6Ade in concentrations from 5.10 -8 to 5.10 -7 M to the embryonic anise cell culture without 2,4-D caused an increased number of embryos after 6 d, whereas a concentration of 5.10- 6 M had no effect (Table 2). Similar results were obtained by Fujiumura and Komamine (1975, 1980) when embryo formation was measured in a carrot cell culture. However, these authors investigated only embryo formation and not the influence of cytokinins on the proembryonic stage. We further investigated the possibility of cytokinin sensitivity during the various stages of cell growth. Isopentenyladenosine or i6Ade (5"10 -s M) was added to the cultures at different times after the induction of embryogenesis. The maximum enhancement of embryo formation was obtained by adding i6Ado or i6Ade at day two of the culture medium (Table 3). At this time no embryos were visible in the culture; the cells are still in the logarithmic phase of cell growth (Ernst et al. 1984). From Table 3, it is obvious that embryo formation is increased only by a cytokinin application during the first 3 d (up to 46%). Addition of i6Ado or i6Ade after day three of culture produced no enhancement in the number of embryos formed. Furthermore, we found no difference in the percentage of individual embryo stages. Irrespective of the time of application, about 78-82% globular and 18-22% heart- and torpedoshaped embryos were present after a culture period of 6d.
Table 1. Effect of isopentenyladenine (i6Ade) and isopentenyl-
Cell density (x 105 cells m1-1) 48 h
90 h
400 (i6Ade) 40 (i6Ade) 4 (i6Ade) 400 (i6Ado) 40 (i6Ado) 4 (i6Ado)
1.20 1.71 1.62 1.26 1.46 1.49
n.d. 2.09 2.09 n.d. 2.05 2.04
Control
1.17
1.86
Table 2. Effect of isopentenyladenine (i6Ade) and isopentenyl-
adenosine (i6Ado) on somatic embryogenesis in an anise cell culture. The cytokinins were added at the beginning of the incubation time and embryos were counted after 6 d. The initial cell density was 1.2.105 cells m1-1 in a 10-ml culture volume. The results are the average of three independent experiments. Standard deviation: between 10% and 20% Cytokinin ( x 10-a M)
Embryos per Petri-dish
500 (i6Ade) 50 (i6Ade) 5 (i6Ade)
3675 4388
500 (i6Ado) 50 (i6Ado) 5 (i6Ade)
3 964 4572 4635
Control
3838
4631
Table 3. Effect of isopentenyladenine (i6Ade) and isopentenyl-
adenosine (i6Ado) on anise embryo formation. The initial cell density was 1.2" 105 cells m1-1 in a 10-ml culture volume. The cytokinins (5.10 -8 M) were added at different times to the culture and the embryos counted 6 d after the induction of embryogenesis. The results represent the average of three independent experiments. Standard deviation: between 10% and 20%. (Control: 3838 embryos per Petri-dish) Time (d) of cytokinin addition
0 l 2 3 4
Embryos per Petri-dish i6Ade
i6Ado
4631 4803 5 640 4762 3509
4635 4725 5 499 4617 3936
The results indicate that the addition of cytokinins enhances cell division during this period of growth; more cells are yielded and there is, therefore, the possibility of the formation of more embryos at a later stage. This assumption is supported
248
D. Ernst and D. Oesterhelt: Cytokinins, growth and somatic embryogenesis in Pimp&ella cells
by a number of other facts: i) addition of i6Ado or i6Ade to the embryo culture after day three yielded about the same number of embryos as a control culture (Table 3); ii) addition of i6Ado or i6Ade to a proembryonic culture increased the cell density (Table 1); iii) the endogenous maxima of cytokinins are correlated with logarithmic growth and not with a morphologically visible embryo formation (Ernst et al. 1984); iv) Huber et al. (1978) demonstrated a direct correlation of the number of anise embryos formed in a cell culture with the initial cell density: higher initial cell densities yielded more embryos; v) application of zeatin or zeatin riboside to Phaseolus coccineus embryos increased the embryo length, indicating a cytokinin influence on cell growth (Bennici and Cionini 1979). Our observations and the results from the literature lead to the following conclusion: cytokinins are not involved in embryo development directly, rather they show a stimulating effect on cell division, resulting in an increased number of cells and, therefore, more embryos in the culture.
References
Ernst, D., Sch/ifer, W., Oesterhelt, D. (1983) Isolation and quantitation of isopentenyladenosine in an anise cell culture by single ion monitoring, radioimmunoassay and bioassay. Planta 159, 216-221 Ernst, D., Oesterhelt, D., Schiller, W. (1984) Endogenous cytokinins during embryogenesis in an anise cell culture (Pirnpinella an&urn L.). Planta 161,240-245 Fujiumura, T., Komamine, A. (1975) Effects of various growth regulators on the embryogenesis in a carrot cell suspension culture. Plant Sci. Lett. 5, 359-364 Fujiumura, T., Komamine, A. (1980) Mode of action of 2,4-D and zeatin on somatic embryogenesis in a carrot cell suspension culture. Z. Pflanzenphysiol. 99, 1-8 Gamborg, O.L. (1975) Callus and cell culture. In: Plant tissue culture methods, pp. 1-11, Gamborg, O.L., Wetter, L.R., eds. National Research Council of Canada, Saskatoon, Saskatchewan Huber, J., Constabel, F., Gamborg, O.L. (1978) A cell counting procedure applied to embryogenesis in cell suspension cultures of anise (Pirnpinella an&urn L.). Plant Sci. Lett. 12, 209-215 Mackenzie, I.A., Street, H.E. (1972) The cytokinins of cultured sycamore cells. New Phytol. 71,621-631 Mackenzie, I.A., Konar, A., Street, H.E. (1972) Cytokinins and the growth of cultured sycamore cells. New Phytol. 71, 633-638 Nishinari, N., Syono, K. (1980) Changes in endogenous cytokinin levels in partially synchronized cultured tobacco cells. Plant Physiol. 65, 437-441 Short, C.K., Torrey, J.G. (1972) Cytokinin production in relation to the growth of pea-root callus tissue. J. Exp. Bot. 23, 1099-1105
Bennici, A., Cionini, P.G. (1979) Cytokinins and in vitro development of Phaseolus coccineus embryos. Planta 147, 27-29
Received 12 November 1983 ; accepted 7 February 1984
