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Physiological properties of inhibitory conditioning factor(s), inhibitory to somatic embryogenesis, in high-density cell cultures of carrot
Plant Science 144 (1999) 69 – 75
Physiological properties of inhibitory conditioning factor(s), inhibitory to somatic embryogenesis, in high-density cell cultures of carrot Toshihiro Kobayashi *, Katsumi Higashi, Tsutomu Saitou, Hiroshi Kamada Institute of Biological Sciences, Uni6ersity of Tsukuba, Tsukuba, Ibaraki 305 -8572, Japan Received 18 December 1998; received in revised form 29 March 1999; accepted 6 April 1999
Abstract Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells at high cell density. We showed previously that the inhibition is caused by some inhibitory factor(s) that is released into the medium of such cultures. In this study, we characterized the physiological properties of the inhibitory conditioning factor(s). Inhibition of somatic embryo formation by the inhibitory factor(s) in high-cell-density cultures was due to suppression of the rapid division of cells that is characteristic of the early stage of somatic embryogenesis. The inhibitory factor(s) did not involve suppression of somatic embryo development at certain specific stages. Suppression of the rapid cell division by the inhibitory factor(s) was reversible and did not affect embryogenic competence. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Daucus carota; High-cell-density culture; Inhibitory conditioning factor(s); Somatic embryogenesis
1. Introduction Various environmental and chemical factors influence the induction and development of carrot somatic embryos [1–8]. Auxin, in particular 2,4dichlorophenoxyacetic acid (2,4-D), is extremely important in this regard [9]. When explants are cultured on 2,4-D-containing medium for several weeks, embryogenic cells are generated and the transfer of these embryogenic cells to auxin-free medium results in formation of somatic embryos [3]. Cell density is also an important factor in carrot somatic embryogenesis [10–13]. When embryoAbbre6iations: DW, distilled water; HCM, high-cell-density conditioned medium; MS medium, Murashige and Skoog’s medium; PCV, packed cell volume at 100 × g. * Corresponding author. Gene Experiment Center, University of Tsukuba, Ten-noudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan. Tel.: +81-298-53-6588; fax: + 81-298-53-6006.
genic cells are cultured in auxin-free medium at high cell density, the formation of carrot somatic embryos is strongly reduced. Recent experiments suggested that such inhibition is due to some chemical factor(s) that is released into the culture medium [14]. Many conditioning factors that are released into the culture medium stimulate the proliferation of cells in tissue culture systems derived from various plants, including carrot [1,15–19]. A wide variety of arabinogalactan proteins are also secreted into the culture medium of carrot cell-suspension cultures. Different types of arabinogalactan proteins have stimulatory or inhibitory effect on embryogenic potential and development of somatic embryos [16,19]. However, inhibitory conditioning factors in high-celldensity cultures have not been well characterized. We showed previously that inhibition of the formation of carrot somatic embryos at high cell density was due to some inhibitory conditioning
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T. Kobayashi et al. / Plant Science 144 (1999) 69–75
factor(s) that had been released into the medium. The factor(s) had a molecular weight below 3500 and was distinct from 2,4-D that had been incorporated into embryogenic cells and introduced into the fresh culture medium via the embryogenic cells themselves [14]. In this study, we analyzed the physiological properties of the inhibitory factor(s) by using inhibitory conditioned medium from cultures of carrot cells.
2. Materials and methods
2.1. Plant materials and cell culture Details of the methods for the culture of embryogenic cells and non-embryogenic cells of Daucus carota L. cv. US-Harumakigosun have been described previously [3,20]. Embryogenic cells obtained from hypocotyls were subcultured at 2-week intervals in liquid Murashige and Skoog’s (MS) medium [21] that contained 2,4-D (1 mg/l). To induce somatic embryogenesis, small cell clusters (37 – 63 mm in diameter) were collected by successive filtering of 2-week-old suspension cultures through stainless-steel sieves of 37- and 63-mm pore size. The clusters were washed with an excess of phytohormone-free MS medium and then suspended in phytohormonefree liquid MS medium at various cell densities. Cell density was defined as the packed cell volume (PCV) in ml after centrifugation at 100 ×g of a liter culture (ml PCV/l). Non-embryogenic cells were obtained by successive subculturing, at 2-week intervals, of small clusters of cells that had been in liquid MS medium containing 2,4-D (1 mg/l) for less than 6 months. The non-embryogenic cells were maintained by subculturing in liquid MS medium containing 2,4-D (1 mg/l) at 2-week intervals. All cultures were incubated on a gyratory shaker (70 rpm) at 25°C in darkness.
2.2. Effects of cell density on the formation of somatic embryos and the proliferation of cells Embryogenic cells were cultured at 0.2, 1.0 and 5.0 ml PCV/l. Somatic embryos and cells were counted on the 3rd, 5th, 7th, 10th and 14th days of culture. Somatic embryos were counted in a counting chamber in 500 ml of culture. For
counting of cells in various cultures, cells were collected by centrifugation at 100×g for 5 min and the pelleted cells were treated with an excess of a maceration solution that consisted of 10% HNO3 and 10% CrO3 for 1 day. Then the mixture was centrifuged at 100×g and the pellet was washed once with distilled water (DW). After centrifugation, the final pellet of cells was suspended in and diluted to an appropriate cell density with DW. The cells were counted in a hemocytometer (Thoma, Erma, Tokyo).
2.3. Effects of high-cell-density conditioned medium on the formation of somatic embryos and the proliferation of cells Details of the preparation of conditioned cellfree medium have been described previously [14]. Conditioned medium was prepared by passing 2week-old high-cell-density cultures (5.0 ml PCV/l) through a filter (GF/F, Whatman, Maidstone, UK). The conditioned medium was designated HCM (high-cell-density conditioned medium). HCM was diluted 2:1, 1:1, 1:2 and 1:4 (v/v) with DW and added to an equal volume of 2,4-D-free double-strength MS medium. In control experiments, DW or MS medium was added to double-strength MS medium. The pH of the medium was adjusted to 5.7 and the medium was sterilized by filtration through a cellulose acetate membrane (DISMIC-25cs, 0.45 mm; ADVANTEC, Tokyo). Small clusters (37–63 mm) of embryogenic cells, collected as described above, were cultured at a low cell density (0.2 ml PCV/l) in medium that contained HCM. The somatic embryos and cells in each medium were counted on the 3rd, 5th, 10th and 14th days of culture.
2.4. Effects of HCM on the proliferation of embryogenic and non-embryogenic cells with or without 2,4 -D Embryogenic cells (37–63 mm) and non-embryogenic cells were cultured in medium that contained HCM with and without 2,4-D (1 mg/l) at 0.2 ml PCV/l. After 14 days, cells were counted. In control experiments, medium that consisted of equal volume of double-strength MS medium and DW or MS medium was used.
T. Kobayashi et al. / Plant Science 144 (1999) 69–75
2.5. Stage specificity of inhibition by HCM of somatic embryogenesis After cells had been cultured for 14 days at low cell density (0.2 ml PCV/l), we collected globular, heart-shaped and torpedo-shaped somatic embryos manually from cultures. The embryos at each stage of development were suspended in the medium composed of equal volumes of HCM and double strength MS medium and cultured for 2 weeks. Then, the somatic embryos at each stage of development were counted and numbers of cells were determined. In control experiments, DW or MS medium was added to an equal volume of double-strength MS medium.
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cell density (0.2 ml PCV/l), the total number of cells increased rapidly for 20 days and was already 100-times the initial number after 14 days of culture. In contrast, the increase in cell number in cultures initiated at 5.0 ml PCV/l occurred more slowly than at 0.2 ml PCV/l (Fig. 1d). When embryogenic cells were cultured in HCMcontaining medium at a low cell density (0.2 ml PCV/l), the formation of somatic embryos was inhibited as the concentration of HCM increased (Fig. 2). Throughout the culture period, the increase in number of both somatic embryos and cells was strongly inhibited in medium that contained HCM as compared to control media without HCM (Fig. 3a–d).
2.6. Effects of the duration of culture in HCM-containing medium on somatic embryogenesis Embryogenic cells were cultured at 0.2 ml PCV/l in MS medium that contained HCM for 3, 5, 10, and 14 days and then cells were collected by centrifugation at 100× g. The cells were resuspended in fresh MS medium without HCM and cultured for a further 14 days after the transfer to fresh MS medium without HCM. In all experiments, cultures were incubated in 50-ml flasks that contained 15 ml of test medium. All experiments were repeated at least twice and the average values with S.D. (n= 4) are shown.
3. Results
3.1. Effects of cell density and HCM on the de6elopment of somatic embryos and the proliferation of cells At a low cell density (0.2 ml PCV/l), the total number of somatic embryos increased rapidly during the culture (Fig. 1a) and many embryos had developed into heart- and torpedo-shaped embryos by the late stage (14 days) of culture. In contrast, at high cell densities (1.0 and 5.0 ml PCV/l), the total number of somatic embryos that formed was significantly lower than at 0.2 ml PCV/l. Both heart- and torpedo-shaped embryos were observed at the late stage (10th and 14th days) of culture for cells cultured at 0.2 and 1.0 ml PCV/l but not 5.0 ml PCV/l (Fig. 1b,c). At the low
Fig. 1. Effects of cell density on the formation of somatic embryos and proliferation of cells. Embryogenic cells were cultured in phytohormone-free MS medium at various cell densities (0.2, 1.0 and 5.0 ml PCV/l). Somatic embryos and cells were counted after various times, as indicated. (a – c) The number of somatic embryos at 0.2 (a), 1.0 (b) and 5.0 (c) ml PCV/l. Closed boxes, globular embryos; stripped boxes, heart-shaped embryos; open boxes, torpedo-shaped embryos. (d) Number of cells at 0.2 (), 1.0 () and 5.0 () ml PCV/l. Results represent means with S.D. (n = 4).
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3.4. Effects of the duration of culture in HCM-containing medium on somatic embryogenesis
Fig. 2. Effects of the concentration of HCM on the formation of somatic embryos. HCM was diluted with DW and added to an equal volume of phytohormone-free double-strength MS medium, and embryogenic cells were cultured in the medium at 0.2 ml PCV/l. In control experiments, DW or phytohormone-free MS medium instead of HCM was added. Somatic embryos were counted at 14 days of culture. Closed boxes, globular embryos; stripped boxes, heart-shaped embryos; open boxes, torpedo-shaped embryos. Results represent means with S.D. (n =4).
The number of somatic embryos formed on the 14th day after the start of the culture decreased with increasing duration of culture in HCM-containing medium (Fig. 6a). However, 2 weeks after the transfer of cells from HCM-containing medium to fresh MS medium without HCM, the number of somatic embryos that formed in all experiments was similar to that in controls (no treatment with HCM) irrespective of the duration of culture in HCM-containing medium (Fig. 6b).
3.2. Effects of HCM on the proliferation of embryogenic and non-embryogenic cells In medium without 2,4-D (in which formation of somatic embryos was normally observed), proliferation of cells was strongly inhibited in the presence of HCM (Fig. 4b). The proliferation of embryogenic cells in medium that contained 2,4-D was not inhibited by the presence of HCM (Fig. 4a). The proliferation of non-embryogenic cells in medium with and without 2,4-D was also unaffected by the presence of HCM (Fig. 4c,d).
3.3. Stage specificity of the inhibition by HCM of the formation of somatic embryos When globular somatic embryos were cultured in HCM-containing medium, the number of heartand torpedo-shaped embryos that developed was significantly lower than that in control media without HCM (Fig. 5a). However, when heart- or torpedo-shaped embryos were cultured in HCMcontaining medium, numbers of developed embryos were similar to those in control media without HCM (Fig. 5b,c). We observed similar effects when we monitored cell proliferation (Fig. 5d–f).
Fig. 3. Effects of HCM on the formation of somatic embryos and the proliferation of cells. HCM was added to an equal volume of phytohormone-free double-strength MS medium, and embryogenic cells were cultured in the medium at 0.2 ml PCV/l. In control experiments, distilled water (DW) or phytohormone-free MS medium (MS) was added to double-strength MS medium. Somatic embryos and cells were counted at various times as indicated. (a – c) Number of somatic embryos in medium prepared with DW (a), MS (b) and HCM (c). Closed boxes, globular embryos; stripped boxes, heartshaped embryos; open boxes, torpedo-shaped embryos. (d) Number of cells in cultures prepared with DW (), MS () and HCM (). Results represent means with S.D. (n = 4).
T. Kobayashi et al. / Plant Science 144 (1999) 69–75
Fig. 4. Effects of HCM on the proliferation of embryogenic and non-embryogenic cells. Embryogenic and non-embryogenic cells at 0.2 ml PCV/l were cultured for 2 weeks in medium composed of double-strength MS medium and HCM with and without 2,4-D (1 mg/l), and then cells were counted. In control experiments, distilled water (DW) or MS medium (MS) was added to double-strength MS medium. (a and b) Embryogenic cells were cultured in medium with (a) and without 2,4-D (b). (c and d) Non-embryogenic cells were cultured in medium with (c) and without 2,4-D (d). Initial indicates the number of embryogenic or non-embryogenic cells at start of the culture. Results represent means with S.D. (n= 4).
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the medium prepared with HCM was inhibited much more strongly than that in the medium prepared with MS medium or DW (Fig. 2). In contrast, the number of somatic embryos in control culture was different in each experiment. These differences might have been caused by a gradual decline in embryogenic competence with increasing duration of subculture [22,23]. In our previous study, we showed that inhibition was caused by some inhibitory factor(s) in the conditioned medium and that the factor(s) had molecular weight below 3500 and was not 2,4-D that had been incorporated into embryogenic cells and introduced into the fresh medium by the embryogenic cells themselves [14]. It has been proposed that the inhibition of somatic embryogenesis in high-cell-density cultures of carrot cells is due to suppression of the transition from globular stage to the heart-shaped stage [11–13]. We found that when embryogenic cells were cultured at high-cell-density or in medium that contained HCM at low cell density, the total number of somatic embryos formed decreased dra-
4. Discussion Formation of carrot somatic embryos is strongly inhibited when embryogenic cells are cultured at high cell density [10–13]. In this study, we confirmed this inhibition (Fig. 1) and demonstrated that such inhibition could be achieved by the addition of conditioned medium in which embryogenic cells had been cultured at high cell density. Moreover, the extent of inhibition depended on the concentration of conditioned medium (Figs. 2 and 3). We observed slight inhibition of the formation of somatic embryos in control medium prepared by adding MS medium instead of HCM, as compared to that in medium to which DW had been added (Figs. 2–6). This weak inhibition might have been caused by the high concentration of nutrients in the medium prepared with MS medium instead of HCM (1.5-fold higher than in medium to which DW had been added) because high concentration of MS nutrients resulted in inhibition of somatic embryogenesis (data not shown). However, the formation of somatic embryos in
Fig. 5. Stage specificity of the inhibition by HCM of the formation of somatic embryos. Globular (a, d), heart-shaped (b, e) and torpedoshaped (c, f) embryos were separately cultured in HCM-containing medium (HCM) for 2 weeks. In control experiments, distilled water (DW) or MS medium (MS) was added to double-strength MS medium. (a – c) The number of somatic embryos at each stage of development. Closed boxes, globular embryos; stripped boxes, heartshaped embryos; open boxes, torpedo-shaped embryos. (d–f) The number of cells. Results represent means with S.D. (n = 4).
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Fig. 6. Effects of the duration of culture in HCM-containing medium on the formation of somatic embryos. Embryogenic cells at low cell density (0.2 ml PCV/l) were cultured in phytohormone-free doublestrength MS medium supplemented with distilled water (DW), MS medium (MS) or HCM for 0, 3, 5, 10 and 14 days. Then they were transferred to phytohormone-free MS medium without HCM and cultured for 14 days. In each experiment, somatic embryos were counted after the start of the culture (a) and 14 days after transfer to MS medium without HCM (b). Closed boxes, globular embryos; stripped boxes, heart-shaped embryos; open boxes, torpedo-shaped embryos. Results represent means with S.D. (n= 4).
matically, but some somatic embryos still developed to the heart- and torpedo-shaped stages (Fig. 1c and Fig. 3c). Thus, it is possible that inhibition is not caused by interruption of the development of embryos (from the globular stage to the heartand torpedo-shaped stages) but by some other mechanism. We showed clearly, however, that cell proliferation was inhibited in high-cell-density cultures and in medium that contained HCM at low cell density (Fig. 1d and Fig. 3d). Thus, inhibition of the formation of somatic embryos at high cell density (or by HCM) appeared to be caused by suppression of cell proliferation, and those embryos that occasionally formed developed to maturity. Exceptionally rapid division of cells is observed during the formation of globular embryos at the early stages of culture [24,25]. We examined whether the inhibitory factor(s) in HCM specifically suppressed this rapid division of cells or whether it suppressed the division of cells in general. HCM strongly inhibited the proliferation of embryogenic cells in 2,4-D-free medium but not that of non-embryogenic cells in medium with and without 2,4-D nor that of embryogenic cells in the medium with 2,4-D (Fig. 4). Thus, it appeared that the inhibitory factor(s) specifically suppressed the rapid division of cells that is characteristic of
somatic embryogenesis. This possibility was supported by other results. When somatic embryos at the early globular, heart- and torpedo-shaped stages were separately cultured in HCM-containing medium, increases in number of developed somatic embryos were suppressed only in cultures of early globular embryos (Fig. 5). These results suggest that the inhibitory factor(s) in HCM suppressed the formation of somatic embryos by inhibiting the rapid division of cells that is characteristic at early stage of somatic embryogenesis. As a consequence, the total number of somatic embryos decreased. To examine the reversibility of the effect of the inhibitory factor(s), we cultured embryogenic cells in medium without HCM after several days of culture in HCM-containing medium. During the culture in HCM-containing medium, small clusters of embryogenic cells developed into small aggregates of cells, resembling those at the early stage of formation of globular embryos. Those aggregates did not develop to somatic embryos in HCM-containing medium but they did develop to heart- and torpedo-shaped embryos after transfer to medium without HCM (Fig. 6b). Thus, the inhibitory factor(s) did not affect embryogenic competence and its effects were reversible. Several phytohormones and chemically synthesized compounds inhibit carrot embryogenesis [5,10,26,27]. The existence and concentration of the inhibitors in conditioned medium have not previously been examined, except in the case of p-hydroxybenzoic acid. Fridborg et al. [10] reported that p-hydroxybenzoic acid accumulates in the medium during carrot somatic embryogenesis and inhibits the formation of somatic embryos. However, formation of somatic embryos was not inhibited in MS medium after addition of p-hydroxybenzoic acid at a concentration equal to that in our HCM (our unpublished data). Some factor(s) that stimulates formation of somatic embryos is also present in conditioned medium [14] (Matsubayashi et al., personal communication). In fact, the formation of somatic embryos was stimulated by addition of conditioned medium prepared from low-cell-density cultures (our unpublished data). Thus, somatic embryogenesis from carrot cells is influenced by the balance of levels of the stimulatory and inhibitory conditioning factors in the medium. However, inhibition appears to be stronger than
T. Kobayashi et al. / Plant Science 144 (1999) 69–75
stimulation since strong inhibition was observed in the presence of HCM. In an attempt to clarify the chemical characteristics of the inhibitory conditioning factor(s) and to analyze in further detail the mechanism of inhibition of somatic embryogenesis in high-cell-density cultures, we are now isolating and purifying the inhibitory factor(s) so that we can determine the chemical structure(s).
Acknowledgements
[11]
[12]
[13]
[14]
This work was supported in part by grants-inAid for Special Research on Priority Areas from the Ministry of Education, Science, Culture and Sports, Japan, and by the Program for Promotion of Basic Research Activities for Innovative Biosciences.
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Physiological properties of inhibitory conditioning factor(s), inhibitory to somatic embryogenesis, in high-density cell cultures of carrot Toshihiro Kobayashi *, Katsumi Higashi, Tsutomu Saitou, Hiroshi Kamada Institute of Biological Sciences, Uni6ersity of Tsukuba, Tsukuba, Ibaraki 305 -8572, Japan Received 18 December 1998; received in revised form 29 March 1999; accepted 6 April 1999
Abstract Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells at high cell density. We showed previously that the inhibition is caused by some inhibitory factor(s) that is released into the medium of such cultures. In this study, we characterized the physiological properties of the inhibitory conditioning factor(s). Inhibition of somatic embryo formation by the inhibitory factor(s) in high-cell-density cultures was due to suppression of the rapid division of cells that is characteristic of the early stage of somatic embryogenesis. The inhibitory factor(s) did not involve suppression of somatic embryo development at certain specific stages. Suppression of the rapid cell division by the inhibitory factor(s) was reversible and did not affect embryogenic competence. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Daucus carota; High-cell-density culture; Inhibitory conditioning factor(s); Somatic embryogenesis
1. Introduction Various environmental and chemical factors influence the induction and development of carrot somatic embryos [1–8]. Auxin, in particular 2,4dichlorophenoxyacetic acid (2,4-D), is extremely important in this regard [9]. When explants are cultured on 2,4-D-containing medium for several weeks, embryogenic cells are generated and the transfer of these embryogenic cells to auxin-free medium results in formation of somatic embryos [3]. Cell density is also an important factor in carrot somatic embryogenesis [10–13]. When embryoAbbre6iations: DW, distilled water; HCM, high-cell-density conditioned medium; MS medium, Murashige and Skoog’s medium; PCV, packed cell volume at 100 × g. * Corresponding author. Gene Experiment Center, University of Tsukuba, Ten-noudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan. Tel.: +81-298-53-6588; fax: + 81-298-53-6006.
genic cells are cultured in auxin-free medium at high cell density, the formation of carrot somatic embryos is strongly reduced. Recent experiments suggested that such inhibition is due to some chemical factor(s) that is released into the culture medium [14]. Many conditioning factors that are released into the culture medium stimulate the proliferation of cells in tissue culture systems derived from various plants, including carrot [1,15–19]. A wide variety of arabinogalactan proteins are also secreted into the culture medium of carrot cell-suspension cultures. Different types of arabinogalactan proteins have stimulatory or inhibitory effect on embryogenic potential and development of somatic embryos [16,19]. However, inhibitory conditioning factors in high-celldensity cultures have not been well characterized. We showed previously that inhibition of the formation of carrot somatic embryos at high cell density was due to some inhibitory conditioning
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factor(s) that had been released into the medium. The factor(s) had a molecular weight below 3500 and was distinct from 2,4-D that had been incorporated into embryogenic cells and introduced into the fresh culture medium via the embryogenic cells themselves [14]. In this study, we analyzed the physiological properties of the inhibitory factor(s) by using inhibitory conditioned medium from cultures of carrot cells.
2. Materials and methods
2.1. Plant materials and cell culture Details of the methods for the culture of embryogenic cells and non-embryogenic cells of Daucus carota L. cv. US-Harumakigosun have been described previously [3,20]. Embryogenic cells obtained from hypocotyls were subcultured at 2-week intervals in liquid Murashige and Skoog’s (MS) medium [21] that contained 2,4-D (1 mg/l). To induce somatic embryogenesis, small cell clusters (37 – 63 mm in diameter) were collected by successive filtering of 2-week-old suspension cultures through stainless-steel sieves of 37- and 63-mm pore size. The clusters were washed with an excess of phytohormone-free MS medium and then suspended in phytohormonefree liquid MS medium at various cell densities. Cell density was defined as the packed cell volume (PCV) in ml after centrifugation at 100 ×g of a liter culture (ml PCV/l). Non-embryogenic cells were obtained by successive subculturing, at 2-week intervals, of small clusters of cells that had been in liquid MS medium containing 2,4-D (1 mg/l) for less than 6 months. The non-embryogenic cells were maintained by subculturing in liquid MS medium containing 2,4-D (1 mg/l) at 2-week intervals. All cultures were incubated on a gyratory shaker (70 rpm) at 25°C in darkness.
2.2. Effects of cell density on the formation of somatic embryos and the proliferation of cells Embryogenic cells were cultured at 0.2, 1.0 and 5.0 ml PCV/l. Somatic embryos and cells were counted on the 3rd, 5th, 7th, 10th and 14th days of culture. Somatic embryos were counted in a counting chamber in 500 ml of culture. For
counting of cells in various cultures, cells were collected by centrifugation at 100×g for 5 min and the pelleted cells were treated with an excess of a maceration solution that consisted of 10% HNO3 and 10% CrO3 for 1 day. Then the mixture was centrifuged at 100×g and the pellet was washed once with distilled water (DW). After centrifugation, the final pellet of cells was suspended in and diluted to an appropriate cell density with DW. The cells were counted in a hemocytometer (Thoma, Erma, Tokyo).
2.3. Effects of high-cell-density conditioned medium on the formation of somatic embryos and the proliferation of cells Details of the preparation of conditioned cellfree medium have been described previously [14]. Conditioned medium was prepared by passing 2week-old high-cell-density cultures (5.0 ml PCV/l) through a filter (GF/F, Whatman, Maidstone, UK). The conditioned medium was designated HCM (high-cell-density conditioned medium). HCM was diluted 2:1, 1:1, 1:2 and 1:4 (v/v) with DW and added to an equal volume of 2,4-D-free double-strength MS medium. In control experiments, DW or MS medium was added to double-strength MS medium. The pH of the medium was adjusted to 5.7 and the medium was sterilized by filtration through a cellulose acetate membrane (DISMIC-25cs, 0.45 mm; ADVANTEC, Tokyo). Small clusters (37–63 mm) of embryogenic cells, collected as described above, were cultured at a low cell density (0.2 ml PCV/l) in medium that contained HCM. The somatic embryos and cells in each medium were counted on the 3rd, 5th, 10th and 14th days of culture.
2.4. Effects of HCM on the proliferation of embryogenic and non-embryogenic cells with or without 2,4 -D Embryogenic cells (37–63 mm) and non-embryogenic cells were cultured in medium that contained HCM with and without 2,4-D (1 mg/l) at 0.2 ml PCV/l. After 14 days, cells were counted. In control experiments, medium that consisted of equal volume of double-strength MS medium and DW or MS medium was used.
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2.5. Stage specificity of inhibition by HCM of somatic embryogenesis After cells had been cultured for 14 days at low cell density (0.2 ml PCV/l), we collected globular, heart-shaped and torpedo-shaped somatic embryos manually from cultures. The embryos at each stage of development were suspended in the medium composed of equal volumes of HCM and double strength MS medium and cultured for 2 weeks. Then, the somatic embryos at each stage of development were counted and numbers of cells were determined. In control experiments, DW or MS medium was added to an equal volume of double-strength MS medium.
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cell density (0.2 ml PCV/l), the total number of cells increased rapidly for 20 days and was already 100-times the initial number after 14 days of culture. In contrast, the increase in cell number in cultures initiated at 5.0 ml PCV/l occurred more slowly than at 0.2 ml PCV/l (Fig. 1d). When embryogenic cells were cultured in HCMcontaining medium at a low cell density (0.2 ml PCV/l), the formation of somatic embryos was inhibited as the concentration of HCM increased (Fig. 2). Throughout the culture period, the increase in number of both somatic embryos and cells was strongly inhibited in medium that contained HCM as compared to control media without HCM (Fig. 3a–d).
2.6. Effects of the duration of culture in HCM-containing medium on somatic embryogenesis Embryogenic cells were cultured at 0.2 ml PCV/l in MS medium that contained HCM for 3, 5, 10, and 14 days and then cells were collected by centrifugation at 100× g. The cells were resuspended in fresh MS medium without HCM and cultured for a further 14 days after the transfer to fresh MS medium without HCM. In all experiments, cultures were incubated in 50-ml flasks that contained 15 ml of test medium. All experiments were repeated at least twice and the average values with S.D. (n= 4) are shown.
3. Results
3.1. Effects of cell density and HCM on the de6elopment of somatic embryos and the proliferation of cells At a low cell density (0.2 ml PCV/l), the total number of somatic embryos increased rapidly during the culture (Fig. 1a) and many embryos had developed into heart- and torpedo-shaped embryos by the late stage (14 days) of culture. In contrast, at high cell densities (1.0 and 5.0 ml PCV/l), the total number of somatic embryos that formed was significantly lower than at 0.2 ml PCV/l. Both heart- and torpedo-shaped embryos were observed at the late stage (10th and 14th days) of culture for cells cultured at 0.2 and 1.0 ml PCV/l but not 5.0 ml PCV/l (Fig. 1b,c). At the low
Fig. 1. Effects of cell density on the formation of somatic embryos and proliferation of cells. Embryogenic cells were cultured in phytohormone-free MS medium at various cell densities (0.2, 1.0 and 5.0 ml PCV/l). Somatic embryos and cells were counted after various times, as indicated. (a – c) The number of somatic embryos at 0.2 (a), 1.0 (b) and 5.0 (c) ml PCV/l. Closed boxes, globular embryos; stripped boxes, heart-shaped embryos; open boxes, torpedo-shaped embryos. (d) Number of cells at 0.2 (), 1.0 () and 5.0 () ml PCV/l. Results represent means with S.D. (n = 4).
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3.4. Effects of the duration of culture in HCM-containing medium on somatic embryogenesis
Fig. 2. Effects of the concentration of HCM on the formation of somatic embryos. HCM was diluted with DW and added to an equal volume of phytohormone-free double-strength MS medium, and embryogenic cells were cultured in the medium at 0.2 ml PCV/l. In control experiments, DW or phytohormone-free MS medium instead of HCM was added. Somatic embryos were counted at 14 days of culture. Closed boxes, globular embryos; stripped boxes, heart-shaped embryos; open boxes, torpedo-shaped embryos. Results represent means with S.D. (n =4).
The number of somatic embryos formed on the 14th day after the start of the culture decreased with increasing duration of culture in HCM-containing medium (Fig. 6a). However, 2 weeks after the transfer of cells from HCM-containing medium to fresh MS medium without HCM, the number of somatic embryos that formed in all experiments was similar to that in controls (no treatment with HCM) irrespective of the duration of culture in HCM-containing medium (Fig. 6b).
3.2. Effects of HCM on the proliferation of embryogenic and non-embryogenic cells In medium without 2,4-D (in which formation of somatic embryos was normally observed), proliferation of cells was strongly inhibited in the presence of HCM (Fig. 4b). The proliferation of embryogenic cells in medium that contained 2,4-D was not inhibited by the presence of HCM (Fig. 4a). The proliferation of non-embryogenic cells in medium with and without 2,4-D was also unaffected by the presence of HCM (Fig. 4c,d).
3.3. Stage specificity of the inhibition by HCM of the formation of somatic embryos When globular somatic embryos were cultured in HCM-containing medium, the number of heartand torpedo-shaped embryos that developed was significantly lower than that in control media without HCM (Fig. 5a). However, when heart- or torpedo-shaped embryos were cultured in HCMcontaining medium, numbers of developed embryos were similar to those in control media without HCM (Fig. 5b,c). We observed similar effects when we monitored cell proliferation (Fig. 5d–f).
Fig. 3. Effects of HCM on the formation of somatic embryos and the proliferation of cells. HCM was added to an equal volume of phytohormone-free double-strength MS medium, and embryogenic cells were cultured in the medium at 0.2 ml PCV/l. In control experiments, distilled water (DW) or phytohormone-free MS medium (MS) was added to double-strength MS medium. Somatic embryos and cells were counted at various times as indicated. (a – c) Number of somatic embryos in medium prepared with DW (a), MS (b) and HCM (c). Closed boxes, globular embryos; stripped boxes, heartshaped embryos; open boxes, torpedo-shaped embryos. (d) Number of cells in cultures prepared with DW (), MS () and HCM (). Results represent means with S.D. (n = 4).
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Fig. 4. Effects of HCM on the proliferation of embryogenic and non-embryogenic cells. Embryogenic and non-embryogenic cells at 0.2 ml PCV/l were cultured for 2 weeks in medium composed of double-strength MS medium and HCM with and without 2,4-D (1 mg/l), and then cells were counted. In control experiments, distilled water (DW) or MS medium (MS) was added to double-strength MS medium. (a and b) Embryogenic cells were cultured in medium with (a) and without 2,4-D (b). (c and d) Non-embryogenic cells were cultured in medium with (c) and without 2,4-D (d). Initial indicates the number of embryogenic or non-embryogenic cells at start of the culture. Results represent means with S.D. (n= 4).
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the medium prepared with HCM was inhibited much more strongly than that in the medium prepared with MS medium or DW (Fig. 2). In contrast, the number of somatic embryos in control culture was different in each experiment. These differences might have been caused by a gradual decline in embryogenic competence with increasing duration of subculture [22,23]. In our previous study, we showed that inhibition was caused by some inhibitory factor(s) in the conditioned medium and that the factor(s) had molecular weight below 3500 and was not 2,4-D that had been incorporated into embryogenic cells and introduced into the fresh medium by the embryogenic cells themselves [14]. It has been proposed that the inhibition of somatic embryogenesis in high-cell-density cultures of carrot cells is due to suppression of the transition from globular stage to the heart-shaped stage [11–13]. We found that when embryogenic cells were cultured at high-cell-density or in medium that contained HCM at low cell density, the total number of somatic embryos formed decreased dra-
4. Discussion Formation of carrot somatic embryos is strongly inhibited when embryogenic cells are cultured at high cell density [10–13]. In this study, we confirmed this inhibition (Fig. 1) and demonstrated that such inhibition could be achieved by the addition of conditioned medium in which embryogenic cells had been cultured at high cell density. Moreover, the extent of inhibition depended on the concentration of conditioned medium (Figs. 2 and 3). We observed slight inhibition of the formation of somatic embryos in control medium prepared by adding MS medium instead of HCM, as compared to that in medium to which DW had been added (Figs. 2–6). This weak inhibition might have been caused by the high concentration of nutrients in the medium prepared with MS medium instead of HCM (1.5-fold higher than in medium to which DW had been added) because high concentration of MS nutrients resulted in inhibition of somatic embryogenesis (data not shown). However, the formation of somatic embryos in
Fig. 5. Stage specificity of the inhibition by HCM of the formation of somatic embryos. Globular (a, d), heart-shaped (b, e) and torpedoshaped (c, f) embryos were separately cultured in HCM-containing medium (HCM) for 2 weeks. In control experiments, distilled water (DW) or MS medium (MS) was added to double-strength MS medium. (a – c) The number of somatic embryos at each stage of development. Closed boxes, globular embryos; stripped boxes, heartshaped embryos; open boxes, torpedo-shaped embryos. (d–f) The number of cells. Results represent means with S.D. (n = 4).
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Fig. 6. Effects of the duration of culture in HCM-containing medium on the formation of somatic embryos. Embryogenic cells at low cell density (0.2 ml PCV/l) were cultured in phytohormone-free doublestrength MS medium supplemented with distilled water (DW), MS medium (MS) or HCM for 0, 3, 5, 10 and 14 days. Then they were transferred to phytohormone-free MS medium without HCM and cultured for 14 days. In each experiment, somatic embryos were counted after the start of the culture (a) and 14 days after transfer to MS medium without HCM (b). Closed boxes, globular embryos; stripped boxes, heart-shaped embryos; open boxes, torpedo-shaped embryos. Results represent means with S.D. (n= 4).
matically, but some somatic embryos still developed to the heart- and torpedo-shaped stages (Fig. 1c and Fig. 3c). Thus, it is possible that inhibition is not caused by interruption of the development of embryos (from the globular stage to the heartand torpedo-shaped stages) but by some other mechanism. We showed clearly, however, that cell proliferation was inhibited in high-cell-density cultures and in medium that contained HCM at low cell density (Fig. 1d and Fig. 3d). Thus, inhibition of the formation of somatic embryos at high cell density (or by HCM) appeared to be caused by suppression of cell proliferation, and those embryos that occasionally formed developed to maturity. Exceptionally rapid division of cells is observed during the formation of globular embryos at the early stages of culture [24,25]. We examined whether the inhibitory factor(s) in HCM specifically suppressed this rapid division of cells or whether it suppressed the division of cells in general. HCM strongly inhibited the proliferation of embryogenic cells in 2,4-D-free medium but not that of non-embryogenic cells in medium with and without 2,4-D nor that of embryogenic cells in the medium with 2,4-D (Fig. 4). Thus, it appeared that the inhibitory factor(s) specifically suppressed the rapid division of cells that is characteristic of
somatic embryogenesis. This possibility was supported by other results. When somatic embryos at the early globular, heart- and torpedo-shaped stages were separately cultured in HCM-containing medium, increases in number of developed somatic embryos were suppressed only in cultures of early globular embryos (Fig. 5). These results suggest that the inhibitory factor(s) in HCM suppressed the formation of somatic embryos by inhibiting the rapid division of cells that is characteristic at early stage of somatic embryogenesis. As a consequence, the total number of somatic embryos decreased. To examine the reversibility of the effect of the inhibitory factor(s), we cultured embryogenic cells in medium without HCM after several days of culture in HCM-containing medium. During the culture in HCM-containing medium, small clusters of embryogenic cells developed into small aggregates of cells, resembling those at the early stage of formation of globular embryos. Those aggregates did not develop to somatic embryos in HCM-containing medium but they did develop to heart- and torpedo-shaped embryos after transfer to medium without HCM (Fig. 6b). Thus, the inhibitory factor(s) did not affect embryogenic competence and its effects were reversible. Several phytohormones and chemically synthesized compounds inhibit carrot embryogenesis [5,10,26,27]. The existence and concentration of the inhibitors in conditioned medium have not previously been examined, except in the case of p-hydroxybenzoic acid. Fridborg et al. [10] reported that p-hydroxybenzoic acid accumulates in the medium during carrot somatic embryogenesis and inhibits the formation of somatic embryos. However, formation of somatic embryos was not inhibited in MS medium after addition of p-hydroxybenzoic acid at a concentration equal to that in our HCM (our unpublished data). Some factor(s) that stimulates formation of somatic embryos is also present in conditioned medium [14] (Matsubayashi et al., personal communication). In fact, the formation of somatic embryos was stimulated by addition of conditioned medium prepared from low-cell-density cultures (our unpublished data). Thus, somatic embryogenesis from carrot cells is influenced by the balance of levels of the stimulatory and inhibitory conditioning factors in the medium. However, inhibition appears to be stronger than
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stimulation since strong inhibition was observed in the presence of HCM. In an attempt to clarify the chemical characteristics of the inhibitory conditioning factor(s) and to analyze in further detail the mechanism of inhibition of somatic embryogenesis in high-cell-density cultures, we are now isolating and purifying the inhibitory factor(s) so that we can determine the chemical structure(s).
Acknowledgements
[11]
[12]
[13]
[14]
This work was supported in part by grants-inAid for Special Research on Priority Areas from the Ministry of Education, Science, Culture and Sports, Japan, and by the Program for Promotion of Basic Research Activities for Innovative Biosciences.
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