- Email: [email protected]
Involvement of phytohormones in light-induced adventitious shoot formation of horseradish hairy roots
Plant Science, 86 (1992) 161-166 Elsevier Scientific Publishers Ireland Ltd.
161
Involvement of phytohormones in light-induced adventitious shoot formation of horseradish hairy roots Tsutomu Saitou, Hiroshi Kamada and Hiroshi Harada Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, lbaraki, 305 (Japan)
(Received April 6th, 1992; revision received July 9th, 1992; accepted July 10th, 1992)
Horseradish (Armoracia rusticana) hairy roots were treated with auxins or cytokinins to examine the effects on adventitious shoot formation. Naphthalene-l-acetic acid (NAA) and 5,6-dichloro-indole-3-acetic acid (5,6-C12-IAA)suppressed shoot formation under light conditions, but indole-3-acetic acid (IAA) did not inhibit it even at high concentrations. Cytokinins such as N6-benzyladenine (BA), kinetin (Kin), t-zeatin, N6-A2-isopentenyladenine (i6Ade) and N6-A2-isopentenyladenosine (i6Ado) induced shoot formation under dark conditions and enhanced the number of shoots under light conditions. Anticytokinins such as 4-substituted-2methylpyrrolo[2,3-d]pyrimidine, with p-isopropylphenyl (pPP), p-bromophenyl (pBP), p-chlorophenyl (pCP) and s-butyl (sB), partially inhibited shoot formation in light. Inhibition was overcome by co-applications of BA with the anticytokinins. These results indicate that, in horseradish hairy roots, light irradiation may result in an increase of the endogenous level of cytokinins thus leading to adventitious shoot formation. Key words: bud formation; cytokinin; hairy root; horseradish; light
Introduction Adventitious shoot f o r m a t i o n in vitro can be influenced by m a n y factors, such as physiological conditions o f m o t h e r plants [1], physical conditions of the environment [1-4] and the culture m e d i u m [5]. P h y t o h o r m o n e s are one of the most important factors a m o n g them [5,6]. In tobacco, the balance between auxin and cytokinin regulates both adventitious shoot f o r m a t i o n and adventitious root formation. Auxins and cytokinins promote adventitious root and shoot formation, respectively and an appropriate c o m b i n a t i o n promotes callus formation [7,8]. In other plants, adventitious shoot f o r m a t i o n is also p r o m o t e d by cytokinin treatments [9]. When horseradish hairy roots were cultured on p h y t o h o r m o n e - f r e e medium, adventitious shoots were vigorously formed under light conditions and Correspondence to: Tsutomu Saitou, Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan.
rarely in complete darkness [10-12]. Moreover, roots o f n o n - t r a n s f o r m e d plants of horseradish also formed shoots in light, but not in darkness [12l. In this paper, we examine the effects of p h y t o h o r m o n e s on adventitious shoot f o r m a t i o n in horseradish hairy roots cultured under light or dark conditions and discuss the possible involvement of endogenous p h y t o h o r m o n e s in lightinduced shoot f o r m a t i o n in horseradish.
Materials and Methods Plant sources Leaves and petioles of axenic horseradish (Armoracia rusticana) were infected with Agrobacterium rhizogenes strain 15 834 and hairy roots appearing on the inoculated sites were cultured in hormone-free Murashige and Skoog's m e d i u m (MS medium) [13] with Claforan (500 mg/l) for elimination of the bacterium. Several clones of hairy roots were obtained and regenerated plants from hairy roots classified into
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
162
three types, i.e. normal type, wrinkled type and rooty type according to their morphology [11]. The normal type had normal leaf morphology and normal stem length exhibiting an appearance of normal (non-transformed) plants. Normal-type hairy roots were maintained on hormone-free MS medium in darkness and used in the following experiments. One centimetre of apical tips of growing hairy roots maintained in darkness were cultured on hormone-free MS medium for more than 11 weeks in darkness. Then, 1 cm of the basal tips, that were explants from basal end (opposite end to root meristem) to 1 cm in main roots and their lateral roots were cut off at 5 mm from main roots, were cut off from pre-cultured hairy roots and transferred to MS medium with or without hormones and cultured under light or dark conditions.
IAA) and naphthalene-l-acetic acid (NAA). Cytokinins were N6-benzyladenine (BA), kinetin (Kin), t-zeatin, N6-A2-isopentenyladenine (i6Ade) and N6-za2-isopentenyladenosine (i6Ado). Anticytokinins were 2-methylpyrrolo[2,3-dlpyrimidines, with p-isopropylphenyl (pPP), p-bromophenyl (pBP), p-chlorophenyl (pCP) and s-butyl (sB) substituents in the 4 position [14]. BA, Kin, IAA and NAA were added to MS medium before autoclaving. Other cytokinins except for BA and Kin, 5,6-C12-IAA and anticytokinins were dissolved in 100% dimethyl sulfoxide (DMSO) and added to MS medium after autoclaving. The final concentration of DMSO was 0.1% in MS medium. Auxins, cytokinins and anticytokinins were applied at 0-57, 0-40 and 0-40/zmol/1, respectively. One centimetre basal tips of hairy roots were cultured on MS medium containing the indicated phytohormones and placed in continuous darkness or light conditions (16 light / 8 dark per day, 78 #mol/m2s light intensity). White light was obtained from a Toshiba lamp (FL40SS. W/37).
Hormone treatments
Auxins used in this study were indole-3-acetic acid (IAA), 5,6-Cl2-indole-3-acetic acid (5,6-C12--°~- 150~
Light
Dark
lOolO
-
"L
o
250 -
Light
Dark
200 6~ =v150.
~ 100 o
5O 0
0
0.057
0.57
5.7
~ 57
0
concentration ( p.M )
0.057
0.57
5.7
57
Fig. 1. Effects of exogenously applied auxins on adventitious shoot formation under light conditions in horseradish hairy roots. Basal ends (1 cm) of dark cultured hairy roots of horseradish were cut off and transferred to MS medium containing the indicated auxins and cultured under light or dark conditions. The relative frequency of shoot formation (no. of explants forming shoots/total explants) and relative no. of shoots (no. of shoots/total explants) were recorded after 4 weeks of the culture. Both values (relative frequency and relative number) on control (hormone-free) MS medium under light conditions ([2]) were taken as 100 and values in each treatment were calculated by comparing to the control value. IAA: 1"3 5, 6-CI2-1AA: ~, N A A : B. Experiments were repeated more than four times with 20 replications. Bars indicate S.E.
163
Light intensity was measured with a Toshiba Photocell Illuminometer SPI-5. After 4 weeks of culture, the frequency of adventitious shoot formation (frequency of shoot formation and number of shoots) was recorded. The frequency (%) of shoot formation is expressed as (number of explants forming shoots/total explants) × 100, and the relative number of shoots means the (number of shoots / total explants) x 100. All experiments were repeated at least twice with more than 15 replications. Results
Effects of auxins on adventitious shoot formation IAA showed no effect on shoot formation at concentrations lower than 57 /~mol/1 under dark and light conditions (Fig. 1). 5, 6-CI2-IAA and NAA decreased both the frequency of shoot formation and the number of shoots when they were applied at 57 #mold under light conditions and did not enhance them under dark conditions at the concentrations tested. When 5,6-CIz-IAA and NAA were applied at 0.57/~mol/1 under light conditions, the number of shoots increased compared
to those on hormone-free MS medium. IAA also enhanced the number of shoots under light conditions when it was applied at concentrations higher than 0.57/~mol/1.
Effects of cytokinins and~or anticytokinins on adventitious shoot formation All cytokinins tested at 4.5/~mol/1 strongly increased the frequency of shoot formation and the number of shoots under dark conditions (Fig. 2). However, only BA showed stimulative effects on the number of shoots at a concentration of 0.45 #mol/1 under dark conditions. On the other hand, under light conditions, addition of cytokinins increased the number of shoots per explant as compared to that on hormone-free MS medium and did not enhance the frequency of shoot formation. Under dark conditions, cytokinins induced shoot formation (Fig. 2), so that anticytokinins were added to MS medium under light conditions to clarify whether they inhibit adventitious shoot formation induced by light, sB and pPP scarcely inhibited shoot formation at concentrations lower than 40/~mol/1 (Fig. 3). pBP and pCP decreased both the frequency of shoot formation and the
o~- 1 5 0 ~
"~g
loo
~E
.~m ~~ 5o1
200-
Light
100
~ .
Dark
6~ c~ > o ~-, o "6
.
.
.
.
.
.
.
.
.
5000
0.45
4.5
0
0.45
4.5
concentration ( ~M )
Fig. 2. Effects of exogenously applied cytokinins on adventitious shoot formation under dark conditions. The cedure was the same as that in Fig. 1, except that basal ends (1 cm) of dark cultured hairy roots were cut off and medium containing cytokinins and cultured under light or dark conditions. The basal ends were treated with medium under light or dark conditions ( ) , BA (O), Kin ( ~ ) , t-zeatin (N!IIIE), i6Ade ( I ) and i6Ado (B). repeated more than three times with 20 replications. Bars indicate S.E.
experimental protransferred to MS hormone-free MS Experiments were
164
+++i+ ........ ~ sol o
150 °c °'2+100 ~
.
+++
.
.
.
5
~o m J= 5n~
0~
0
1
4 10 concenlration ( ~M )
40
Fig. 3. Effects of anticytokinins on adventitious shoot formation under light conditions. The experimental procedure was the same as that in Fig+ 1, except that basal ends (1 cm) of dark cultured hairy roots were cut off and transferred to MS medium containing anticytokinins and cultured under light conditions. The basal ends were treated with hormone-free MS medium ( ~ ) , pPP (D), pBP (1~1~),pCP (I) and sB (B). Experiments were repeated more than twice with 20 replications. Bars indicate S.E.
1501
"62 lOO ~E ._> 3
~ -~
s° l o 200 T
•~
150
~> oo
100
~
50
0 0.4 4 BA concentration ( ~tM )
40
Fig. 4. Effects of simultaneous application of BA and anticytokinins on adventitious shoot formation under light conditions. The experimental procedure was the same as that in Fig. 1, except that basal ends (1 cm) of dark cultured hairy roots were cut off and transferred to MS medium containing both BA and each of anticytokinins indicated and cultured under light conditions. The basal ends were treated with BA alone (-'~ and BA with 40/~M pBP (El) and BA with 40/~mpCP (II). Experiments were repeated more than 3 times with 15 replications. Bars indicate S.E.
number of shoots at concentrations higher than 10 /~mol/l. Other types of anticytokinins which were s-triazine types [15] and carbamate types [16] were also tested and they partially inhibited shoot formation at 100 izmol/1 under light conditions (data not shown). In the next experiment, both anticytokinins and BA were simultaneously added to MS medium under light conditions to clarify whether inhibition of light-induced shoot formation by anticytokinins is overcome by co-application of BA with anticytokinins. To MS medium containing pBP or pCP at 40/~mol/l, up to 40/~mol/l BA was added. When BA was added at 4 or 40 ~mol/l with anticytokinins, the frequency of shoot formation and the number of shoots were the same values as those obtained in hormone-free MS medium (Fig. 4).
Discussion Morphogenesis in higher plants is known to be affected by a balance between auxin and cytokinin [7-9]. In tobacco, it was shown that adventitious roots emerged when the level of endogenous auxin was higher than that of cytokinin and that adventitious shoots were induced when the auxin level was lower than that of cytokinin [8]. In horseradish hairy roots, adventitious shoots can be formed without exogenous application o f phytohormones, when they are cultured under light conditions [10-12]. It was thought that light irradiation induced phytohormonal change which caused a change in balance between endogenous levels of auxin and cytokinin and thus, shoot formation in horseradish hairy roots was induced. At first, we thought that an endogenous auxin, IAA, might be metabolized or inactivated when hairy roots were cultured under light conditions. If this is true, exogenous application of auxins in MS medium under light conditions would suppress shoot formation. However, shoot formation was not suppressed at a concentration lower than 57 /~mol/1 of I A A (Fig. 1). The synthetic auxins N A A and 5,6-C12-IAA, strongly inhibited shoot formation at the high concentration (57 ~mol/l) (Fig. 1). Auxins at this excessively high concentration must induce numerous physiological changes in plants
165 in addition to inhibition of light-induced shoot formation. In fact, at this concentration, hairy roots formed calli without shoots or died. When NAA or 5,6-C12-IAA were applied at 0.57/~mol/l, the number of shoots increased as compared to those untreated with phytohormones. This phenomenon might be induced by the increase of the rate of cell division and cell elongation in hairy roots. With the application of IAA, shoots differentiated on hairy roots, but did not develop further at the high concentrations. The differences in morphogenetic activities depending on the types of auxin might be caused by the differences of catabolism and/or inactivation of the auxins in plant cells and by their stability in MS medium under light conditions. These results suggest that endogenous auxin is not the main factor controlling light-induced shoot formation in horseradish hairy roots. Secondly, to clarify the possibility that the endogenous level of cytokinin might increase when hairy roots were cultured under light conditions, we examined effects of exogenously applied cytokinins such as BA and Kin, t-zeatin, i6Ade and i6Ado under dark conditions. As shown in Fig. 2, applications of cytokinins induced shoots in hairy roots cultured under dark conditions. These results indicate the possibility that light irradiation resulted in an increase of endogenous cytokinin level and the increment of endogenous cytokinin induced shoot formation. When cytokinins were applied under light conditions, the number of shoots increased. These results could be explained in two ways. One was that adventitious shoot formation induced by light is independent of cytokinin-induced shoot formation. Another was that light irradiation induces an increase of endogenous cytokinin level and that the amounts of exogenously applied cytokinins were much higher than the increased level of endogenous cytokinin and this promoted shoot formation in,addition to light-induced shoot formation. In order to study these two possibilities, we examined the effects of anticytokinins under light conditions. Anticytokinins which were used in this study are thought to competitively inhibit the effects of cytokinins on the receptor(s) [14]. Anticytokinins partially inhibited the shoot forma-
tion induced by light (Fig. 3). Inhibition by anticytokinins was reversed by simultaneous application of BA (Fig. 4). This result indicates that the inhibitory effects of anticytokinins on shoot formation must be specific for cytokinins. Inhibition of light-induced shoot formation by s-triazine (no. 12) [15] and carbamate (no. 55) [16] types of anticytokinins was also reversed by simultaneous application of BA at 10 /~mol/l (data not shown here). These results suggest the involvement of endogenous cytokinins in light-induced shoot formation and supported the latter possibility described above. Inhibitory effects of anticytokinins on shoot formation described in this study were different from those previously reported in a tobacco tissue culture system [14]. This might be caused by the different types of exogenous cytokinins and plant species used in the bio-assay. These differences would affect the sensitivity to anticytokinins. In this report, we showed that cytokinin might be involved in adventitious shoot formation in horseradish hairy roots induced by light. We are now trying to measure the endogenous level of cytokinin in horseradish hairy roots cultured under light and dark conditions.
Acknowledgments The authors thank Dr. Iwamura of Kyoto University for kind supply of anticytokinins used in this experiment. This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H.H. and H.K. We wish to thank Dr. Macer for his critical reading of the manuscript.
References 1 S. Tanimotoand H. Harada, Influencesof environmental and physiologicalconditions on floral bud formation of Torenia stem segments cultured in vitro. Z. Pflanzenphysiol., 95 (1979) 33-41. 2 A.S. Economouand P.E. Read, Light quality control of shoot and root formationin vitro in petuniacultures (No. 750). HortScience,21 (1986) S-761. 3 P.G. Kadkade and H. Jopson, Influenceof light quality on organogenesis from the embryo-derived callus of
166
4
5
6
7
8
9
douglas fir (Pseudotsuga menziesii). Plant Sci. Lett., 13 (1978) 67-73. S. Predieri and F.F.F. Malavasi, High frequency shoot regeneration from leaves of the apple rootstock M26 (Malus pumila Mill.). Plant Cell Tissue Organ Cult., 17 (1989) 133-142. H. Kamada and H. Harada, Influence of several growth regulators and amino acids on in vitro organogenesis of Toreniafournieri Lind. J. Exp. Bot., 30 (1979) 27-36. S. Tanimoto and H. Harada, Hormonal control of morphogenesis in leaf explants of Perilla frutescens Britton var. crispa Decaisne f. viridi-crispa Makino. Ann. Bot., 45 (1980) 321-327. F. Skoog and C.O. Miller, Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol., I1 (1957) 118-131. K. Ishikawa, H. Kamada, I. Yamaguchi, N. Takahashi and H. Harada, Morphology and hormone levels of tobacco and carrot tissues transformed by Agrobacterium tumefaciens I. auxin and cytokinin contents of cultured tissues transformed with wild-type and mutant Ti plasmids. Plant Cell Physiol., 29 (1988) 461-466. S. Tanimoto and H. Harada, Roles of auxin and cytokinin in organogenesis in Torenia stem segments cultured in vitro. J. Plant Physiol., 115 (1984) 11-18.
10
11
12
13
14
15
16
T. Noda, N. Tanaka, Y. Mano, S. Nabeshima, H. Ohkawa and C. Matsui, Regeneration of horseradish hairy roots incited Agrobacterium rhizogenes infection. Plant Cell Rep., 6 (1987) 283-286. T. Saitou, H. Kamada and H. Harada, Isoperoxidases in hairy roots and regenerated plants of horseradish (Armoracia lapathifolia). Plant Sci., 75 (1991) 195-201. T. Saitou, H. Kamada and H. Harada, Light requirement for shoot regeneration in horseradish hairy roots. Plant Physiol., in press. T. Murashige and F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15 (1962) 473-497. H. Iwamura, N. Masuda, K. Koshimizu and S. Matsubara, Cytokinin - - agonistic and antagonistic activities of 4-substituted-2-methylpyrrolo[2,3-d]pyrimidines, 7deaza analogs of cytokinin-active adenine derivatives. Phytochemistry, 18 (1979) 217-222. R. Shimizu, H. lwamura, S. Matsubara and T. Fujita, Development of s-triazine anticytokinins and their quantitative structure-activity relationship. J. Agric. Food Chem., 37 (1989) 236-240. R. Shimizu, H. Iwamura and T. Fujita, Anticytokinin activity of N-phenyl-and N-pyridylcarbamates. Z. Naturforsch., 45c (1990) 89-95.
161
Involvement of phytohormones in light-induced adventitious shoot formation of horseradish hairy roots Tsutomu Saitou, Hiroshi Kamada and Hiroshi Harada Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, lbaraki, 305 (Japan)
(Received April 6th, 1992; revision received July 9th, 1992; accepted July 10th, 1992)
Horseradish (Armoracia rusticana) hairy roots were treated with auxins or cytokinins to examine the effects on adventitious shoot formation. Naphthalene-l-acetic acid (NAA) and 5,6-dichloro-indole-3-acetic acid (5,6-C12-IAA)suppressed shoot formation under light conditions, but indole-3-acetic acid (IAA) did not inhibit it even at high concentrations. Cytokinins such as N6-benzyladenine (BA), kinetin (Kin), t-zeatin, N6-A2-isopentenyladenine (i6Ade) and N6-A2-isopentenyladenosine (i6Ado) induced shoot formation under dark conditions and enhanced the number of shoots under light conditions. Anticytokinins such as 4-substituted-2methylpyrrolo[2,3-d]pyrimidine, with p-isopropylphenyl (pPP), p-bromophenyl (pBP), p-chlorophenyl (pCP) and s-butyl (sB), partially inhibited shoot formation in light. Inhibition was overcome by co-applications of BA with the anticytokinins. These results indicate that, in horseradish hairy roots, light irradiation may result in an increase of the endogenous level of cytokinins thus leading to adventitious shoot formation. Key words: bud formation; cytokinin; hairy root; horseradish; light
Introduction Adventitious shoot f o r m a t i o n in vitro can be influenced by m a n y factors, such as physiological conditions o f m o t h e r plants [1], physical conditions of the environment [1-4] and the culture m e d i u m [5]. P h y t o h o r m o n e s are one of the most important factors a m o n g them [5,6]. In tobacco, the balance between auxin and cytokinin regulates both adventitious shoot f o r m a t i o n and adventitious root formation. Auxins and cytokinins promote adventitious root and shoot formation, respectively and an appropriate c o m b i n a t i o n promotes callus formation [7,8]. In other plants, adventitious shoot f o r m a t i o n is also p r o m o t e d by cytokinin treatments [9]. When horseradish hairy roots were cultured on p h y t o h o r m o n e - f r e e medium, adventitious shoots were vigorously formed under light conditions and Correspondence to: Tsutomu Saitou, Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan.
rarely in complete darkness [10-12]. Moreover, roots o f n o n - t r a n s f o r m e d plants of horseradish also formed shoots in light, but not in darkness [12l. In this paper, we examine the effects of p h y t o h o r m o n e s on adventitious shoot f o r m a t i o n in horseradish hairy roots cultured under light or dark conditions and discuss the possible involvement of endogenous p h y t o h o r m o n e s in lightinduced shoot f o r m a t i o n in horseradish.
Materials and Methods Plant sources Leaves and petioles of axenic horseradish (Armoracia rusticana) were infected with Agrobacterium rhizogenes strain 15 834 and hairy roots appearing on the inoculated sites were cultured in hormone-free Murashige and Skoog's m e d i u m (MS medium) [13] with Claforan (500 mg/l) for elimination of the bacterium. Several clones of hairy roots were obtained and regenerated plants from hairy roots classified into
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
162
three types, i.e. normal type, wrinkled type and rooty type according to their morphology [11]. The normal type had normal leaf morphology and normal stem length exhibiting an appearance of normal (non-transformed) plants. Normal-type hairy roots were maintained on hormone-free MS medium in darkness and used in the following experiments. One centimetre of apical tips of growing hairy roots maintained in darkness were cultured on hormone-free MS medium for more than 11 weeks in darkness. Then, 1 cm of the basal tips, that were explants from basal end (opposite end to root meristem) to 1 cm in main roots and their lateral roots were cut off at 5 mm from main roots, were cut off from pre-cultured hairy roots and transferred to MS medium with or without hormones and cultured under light or dark conditions.
IAA) and naphthalene-l-acetic acid (NAA). Cytokinins were N6-benzyladenine (BA), kinetin (Kin), t-zeatin, N6-A2-isopentenyladenine (i6Ade) and N6-za2-isopentenyladenosine (i6Ado). Anticytokinins were 2-methylpyrrolo[2,3-dlpyrimidines, with p-isopropylphenyl (pPP), p-bromophenyl (pBP), p-chlorophenyl (pCP) and s-butyl (sB) substituents in the 4 position [14]. BA, Kin, IAA and NAA were added to MS medium before autoclaving. Other cytokinins except for BA and Kin, 5,6-C12-IAA and anticytokinins were dissolved in 100% dimethyl sulfoxide (DMSO) and added to MS medium after autoclaving. The final concentration of DMSO was 0.1% in MS medium. Auxins, cytokinins and anticytokinins were applied at 0-57, 0-40 and 0-40/zmol/1, respectively. One centimetre basal tips of hairy roots were cultured on MS medium containing the indicated phytohormones and placed in continuous darkness or light conditions (16 light / 8 dark per day, 78 #mol/m2s light intensity). White light was obtained from a Toshiba lamp (FL40SS. W/37).
Hormone treatments
Auxins used in this study were indole-3-acetic acid (IAA), 5,6-Cl2-indole-3-acetic acid (5,6-C12--°~- 150~
Light
Dark
lOolO
-
"L
o
250 -
Light
Dark
200 6~ =v150.
~ 100 o
5O 0
0
0.057
0.57
5.7
~ 57
0
concentration ( p.M )
0.057
0.57
5.7
57
Fig. 1. Effects of exogenously applied auxins on adventitious shoot formation under light conditions in horseradish hairy roots. Basal ends (1 cm) of dark cultured hairy roots of horseradish were cut off and transferred to MS medium containing the indicated auxins and cultured under light or dark conditions. The relative frequency of shoot formation (no. of explants forming shoots/total explants) and relative no. of shoots (no. of shoots/total explants) were recorded after 4 weeks of the culture. Both values (relative frequency and relative number) on control (hormone-free) MS medium under light conditions ([2]) were taken as 100 and values in each treatment were calculated by comparing to the control value. IAA: 1"3 5, 6-CI2-1AA: ~, N A A : B. Experiments were repeated more than four times with 20 replications. Bars indicate S.E.
163
Light intensity was measured with a Toshiba Photocell Illuminometer SPI-5. After 4 weeks of culture, the frequency of adventitious shoot formation (frequency of shoot formation and number of shoots) was recorded. The frequency (%) of shoot formation is expressed as (number of explants forming shoots/total explants) × 100, and the relative number of shoots means the (number of shoots / total explants) x 100. All experiments were repeated at least twice with more than 15 replications. Results
Effects of auxins on adventitious shoot formation IAA showed no effect on shoot formation at concentrations lower than 57 /~mol/1 under dark and light conditions (Fig. 1). 5, 6-CI2-IAA and NAA decreased both the frequency of shoot formation and the number of shoots when they were applied at 57 #mold under light conditions and did not enhance them under dark conditions at the concentrations tested. When 5,6-CIz-IAA and NAA were applied at 0.57/~mol/1 under light conditions, the number of shoots increased compared
to those on hormone-free MS medium. IAA also enhanced the number of shoots under light conditions when it was applied at concentrations higher than 0.57/~mol/1.
Effects of cytokinins and~or anticytokinins on adventitious shoot formation All cytokinins tested at 4.5/~mol/1 strongly increased the frequency of shoot formation and the number of shoots under dark conditions (Fig. 2). However, only BA showed stimulative effects on the number of shoots at a concentration of 0.45 #mol/1 under dark conditions. On the other hand, under light conditions, addition of cytokinins increased the number of shoots per explant as compared to that on hormone-free MS medium and did not enhance the frequency of shoot formation. Under dark conditions, cytokinins induced shoot formation (Fig. 2), so that anticytokinins were added to MS medium under light conditions to clarify whether they inhibit adventitious shoot formation induced by light, sB and pPP scarcely inhibited shoot formation at concentrations lower than 40/~mol/1 (Fig. 3). pBP and pCP decreased both the frequency of shoot formation and the
o~- 1 5 0 ~
"~g
loo
~E
.~m ~~ 5o1
200-
Light
100
~ .
Dark
6~ c~ > o ~-, o "6
.
.
.
.
.
.
.
.
.
5000
0.45
4.5
0
0.45
4.5
concentration ( ~M )
Fig. 2. Effects of exogenously applied cytokinins on adventitious shoot formation under dark conditions. The cedure was the same as that in Fig. 1, except that basal ends (1 cm) of dark cultured hairy roots were cut off and medium containing cytokinins and cultured under light or dark conditions. The basal ends were treated with medium under light or dark conditions ( ) , BA (O), Kin ( ~ ) , t-zeatin (N!IIIE), i6Ade ( I ) and i6Ado (B). repeated more than three times with 20 replications. Bars indicate S.E.
experimental protransferred to MS hormone-free MS Experiments were
164
+++i+ ........ ~ sol o
150 °c °'2+100 ~
.
+++
.
.
.
5
~o m J= 5n~
0~
0
1
4 10 concenlration ( ~M )
40
Fig. 3. Effects of anticytokinins on adventitious shoot formation under light conditions. The experimental procedure was the same as that in Fig+ 1, except that basal ends (1 cm) of dark cultured hairy roots were cut off and transferred to MS medium containing anticytokinins and cultured under light conditions. The basal ends were treated with hormone-free MS medium ( ~ ) , pPP (D), pBP (1~1~),pCP (I) and sB (B). Experiments were repeated more than twice with 20 replications. Bars indicate S.E.
1501
"62 lOO ~E ._> 3
~ -~
s° l o 200 T
•~
150
~> oo
100
~
50
0 0.4 4 BA concentration ( ~tM )
40
Fig. 4. Effects of simultaneous application of BA and anticytokinins on adventitious shoot formation under light conditions. The experimental procedure was the same as that in Fig. 1, except that basal ends (1 cm) of dark cultured hairy roots were cut off and transferred to MS medium containing both BA and each of anticytokinins indicated and cultured under light conditions. The basal ends were treated with BA alone (-'~ and BA with 40/~M pBP (El) and BA with 40/~mpCP (II). Experiments were repeated more than 3 times with 15 replications. Bars indicate S.E.
number of shoots at concentrations higher than 10 /~mol/l. Other types of anticytokinins which were s-triazine types [15] and carbamate types [16] were also tested and they partially inhibited shoot formation at 100 izmol/1 under light conditions (data not shown). In the next experiment, both anticytokinins and BA were simultaneously added to MS medium under light conditions to clarify whether inhibition of light-induced shoot formation by anticytokinins is overcome by co-application of BA with anticytokinins. To MS medium containing pBP or pCP at 40/~mol/l, up to 40/~mol/l BA was added. When BA was added at 4 or 40 ~mol/l with anticytokinins, the frequency of shoot formation and the number of shoots were the same values as those obtained in hormone-free MS medium (Fig. 4).
Discussion Morphogenesis in higher plants is known to be affected by a balance between auxin and cytokinin [7-9]. In tobacco, it was shown that adventitious roots emerged when the level of endogenous auxin was higher than that of cytokinin and that adventitious shoots were induced when the auxin level was lower than that of cytokinin [8]. In horseradish hairy roots, adventitious shoots can be formed without exogenous application o f phytohormones, when they are cultured under light conditions [10-12]. It was thought that light irradiation induced phytohormonal change which caused a change in balance between endogenous levels of auxin and cytokinin and thus, shoot formation in horseradish hairy roots was induced. At first, we thought that an endogenous auxin, IAA, might be metabolized or inactivated when hairy roots were cultured under light conditions. If this is true, exogenous application of auxins in MS medium under light conditions would suppress shoot formation. However, shoot formation was not suppressed at a concentration lower than 57 /~mol/1 of I A A (Fig. 1). The synthetic auxins N A A and 5,6-C12-IAA, strongly inhibited shoot formation at the high concentration (57 ~mol/l) (Fig. 1). Auxins at this excessively high concentration must induce numerous physiological changes in plants
165 in addition to inhibition of light-induced shoot formation. In fact, at this concentration, hairy roots formed calli without shoots or died. When NAA or 5,6-C12-IAA were applied at 0.57/~mol/l, the number of shoots increased as compared to those untreated with phytohormones. This phenomenon might be induced by the increase of the rate of cell division and cell elongation in hairy roots. With the application of IAA, shoots differentiated on hairy roots, but did not develop further at the high concentrations. The differences in morphogenetic activities depending on the types of auxin might be caused by the differences of catabolism and/or inactivation of the auxins in plant cells and by their stability in MS medium under light conditions. These results suggest that endogenous auxin is not the main factor controlling light-induced shoot formation in horseradish hairy roots. Secondly, to clarify the possibility that the endogenous level of cytokinin might increase when hairy roots were cultured under light conditions, we examined effects of exogenously applied cytokinins such as BA and Kin, t-zeatin, i6Ade and i6Ado under dark conditions. As shown in Fig. 2, applications of cytokinins induced shoots in hairy roots cultured under dark conditions. These results indicate the possibility that light irradiation resulted in an increase of endogenous cytokinin level and the increment of endogenous cytokinin induced shoot formation. When cytokinins were applied under light conditions, the number of shoots increased. These results could be explained in two ways. One was that adventitious shoot formation induced by light is independent of cytokinin-induced shoot formation. Another was that light irradiation induces an increase of endogenous cytokinin level and that the amounts of exogenously applied cytokinins were much higher than the increased level of endogenous cytokinin and this promoted shoot formation in,addition to light-induced shoot formation. In order to study these two possibilities, we examined the effects of anticytokinins under light conditions. Anticytokinins which were used in this study are thought to competitively inhibit the effects of cytokinins on the receptor(s) [14]. Anticytokinins partially inhibited the shoot forma-
tion induced by light (Fig. 3). Inhibition by anticytokinins was reversed by simultaneous application of BA (Fig. 4). This result indicates that the inhibitory effects of anticytokinins on shoot formation must be specific for cytokinins. Inhibition of light-induced shoot formation by s-triazine (no. 12) [15] and carbamate (no. 55) [16] types of anticytokinins was also reversed by simultaneous application of BA at 10 /~mol/l (data not shown here). These results suggest the involvement of endogenous cytokinins in light-induced shoot formation and supported the latter possibility described above. Inhibitory effects of anticytokinins on shoot formation described in this study were different from those previously reported in a tobacco tissue culture system [14]. This might be caused by the different types of exogenous cytokinins and plant species used in the bio-assay. These differences would affect the sensitivity to anticytokinins. In this report, we showed that cytokinin might be involved in adventitious shoot formation in horseradish hairy roots induced by light. We are now trying to measure the endogenous level of cytokinin in horseradish hairy roots cultured under light and dark conditions.
Acknowledgments The authors thank Dr. Iwamura of Kyoto University for kind supply of anticytokinins used in this experiment. This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan to H.H. and H.K. We wish to thank Dr. Macer for his critical reading of the manuscript.
References 1 S. Tanimotoand H. Harada, Influencesof environmental and physiologicalconditions on floral bud formation of Torenia stem segments cultured in vitro. Z. Pflanzenphysiol., 95 (1979) 33-41. 2 A.S. Economouand P.E. Read, Light quality control of shoot and root formationin vitro in petuniacultures (No. 750). HortScience,21 (1986) S-761. 3 P.G. Kadkade and H. Jopson, Influenceof light quality on organogenesis from the embryo-derived callus of
166
4
5
6
7
8
9
douglas fir (Pseudotsuga menziesii). Plant Sci. Lett., 13 (1978) 67-73. S. Predieri and F.F.F. Malavasi, High frequency shoot regeneration from leaves of the apple rootstock M26 (Malus pumila Mill.). Plant Cell Tissue Organ Cult., 17 (1989) 133-142. H. Kamada and H. Harada, Influence of several growth regulators and amino acids on in vitro organogenesis of Toreniafournieri Lind. J. Exp. Bot., 30 (1979) 27-36. S. Tanimoto and H. Harada, Hormonal control of morphogenesis in leaf explants of Perilla frutescens Britton var. crispa Decaisne f. viridi-crispa Makino. Ann. Bot., 45 (1980) 321-327. F. Skoog and C.O. Miller, Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol., I1 (1957) 118-131. K. Ishikawa, H. Kamada, I. Yamaguchi, N. Takahashi and H. Harada, Morphology and hormone levels of tobacco and carrot tissues transformed by Agrobacterium tumefaciens I. auxin and cytokinin contents of cultured tissues transformed with wild-type and mutant Ti plasmids. Plant Cell Physiol., 29 (1988) 461-466. S. Tanimoto and H. Harada, Roles of auxin and cytokinin in organogenesis in Torenia stem segments cultured in vitro. J. Plant Physiol., 115 (1984) 11-18.
10
11
12
13
14
15
16
T. Noda, N. Tanaka, Y. Mano, S. Nabeshima, H. Ohkawa and C. Matsui, Regeneration of horseradish hairy roots incited Agrobacterium rhizogenes infection. Plant Cell Rep., 6 (1987) 283-286. T. Saitou, H. Kamada and H. Harada, Isoperoxidases in hairy roots and regenerated plants of horseradish (Armoracia lapathifolia). Plant Sci., 75 (1991) 195-201. T. Saitou, H. Kamada and H. Harada, Light requirement for shoot regeneration in horseradish hairy roots. Plant Physiol., in press. T. Murashige and F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15 (1962) 473-497. H. Iwamura, N. Masuda, K. Koshimizu and S. Matsubara, Cytokinin - - agonistic and antagonistic activities of 4-substituted-2-methylpyrrolo[2,3-d]pyrimidines, 7deaza analogs of cytokinin-active adenine derivatives. Phytochemistry, 18 (1979) 217-222. R. Shimizu, H. lwamura, S. Matsubara and T. Fujita, Development of s-triazine anticytokinins and their quantitative structure-activity relationship. J. Agric. Food Chem., 37 (1989) 236-240. R. Shimizu, H. Iwamura and T. Fujita, Anticytokinin activity of N-phenyl-and N-pyridylcarbamates. Z. Naturforsch., 45c (1990) 89-95.