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Response of Embryogenic Callus of Theobroma cacao L. to Gibberellic Acid and Inhibitors of Gibberellic Acid Synthesis
Response of Embryogenic Callus of Theobroma cacao 1. to Gibberellic Acid and Inhibitors of Gibberellic Acid Synthesis HALINA KONONOWICZ and JULES JANICK Department of Horticulture, Purdue University, West Lafayette, IN 47907, U.S.A. Received October 1, 1983 . Accepted November 7, 1983
Summary Gibberellic acid at 0.05 to 10 mg .1- 1 stimulated asexual embryogenesis from embryogeniccompetent callus of clone BC 5 but not BC 36 of Theobroma cacao. AMO 1618 stimulated embryogenesis at 0.05 to 0.1 mg .1- 1 but depressed embryogenesis above 0.1 mg .1- 1 for clone BC 5. A similar but smaller effect was observed for BC 36. Daminozide and CCC depressed embryogenesis with both clones.
Key words: Theobroma cacao, asexual embryogenesis, gibberellic acid.
Introduction
The stimulation or inhibition of in vitro somatic embryogenesis by growth regulating substances has been reported for many plant species (Steffens, 1980; Tisserat et al., 1979). Auxin and cytokinins are the growth-regulators most widely investigated (Sharp et ai., 1980; Tisserat et ai., 1979). Somatic embryogenesis via hypocotylary budding in Theobroma cacao has been described previously (Esan, 1977; Pence et ai., 1980). We have recently described the spontaneous production of somatic embryos from callus on hormone-free medium for 2 clones of cacao (Kononowicz et ai., in this issue). However, under these conditions embryogenesis occurs in relatively low frequency. Somatic embryos of Theobroma cacao initiated from callus were structurally comparable to zygotic embryos and passed through similar developmental stages, viz. globular, heart-shaped, torpedo-shaped and finally cotyledonary. Embryogenic callus was shifted toward the production of homogeneous callus with a combination of 10 % coconut water and 1.0 mg .1- 1 2,4-D or to a high frequency embryogenesis with 2,4-D at 10-3 to 10-2 mg .1- 1. Little has been reported on the role of gibberellic acid (GA3) on somatic embryogenesis. Growth retardants such as (2-chloroethyl)trimethylammonium chloride, (CCC, chlormequat), butanedioic mono-{2,2-dimethylhydrazide) (daminozide, Alar), and 2'-isopropyl-4'-dimethylamino-S'-methylphenyl-1 '-piperidine carboxylate methyl chloride (AMO 1618) inhibit GA synthesis in certain plant species (Douglas and Paleg, 1972, 1980; Kononowicz et ai., 1982; Nickell, 1979; Schneider, 1970; Wittwer, 1978). In this report, we present results of an investigation of the efZ. Pjlanzenphysiol. Bd. 113. S. 359-366. 1984.
360
HALINA KONONOWICZ and JULES JANICK
fects of GA3 and inhibitors of GA synthesis (AMO 1618, daminozide, and asexual embryogenesis via callus of Theobroma cacao.
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Materials and Methods The callus used in this study originated from cotyledons of asexual embryos of 7beobroma cacao. Culture conditions and callus characteristics have been described previously (Kononowicz et a!., in this issue). All experiments were carried out with 2 clones - BC 5 and BC 36. Pieces of friable callus about 10 to 15 mm 2 were used to initiate all experiments.
IoBc5l i
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Fig. 1: The effect of GA3 on frequency and intensity of asexual embryogenesis and growth of embryogenic competent callus of BC 5 and BC 36 of 7beobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 28.3 % for BC 5 and 39.3 % for BC 36; embryogenic intensity was 1.6 embryos per explant for BC 5 and 2.8 embryos per explant for BC36.
Z. Pjlanzenphysiol. Bd. 113. S. 359-366. 1984.
Response of cacao callus to GA and GA-inhibitors
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Fig. 2: The effect of AMO 1618 on frequency and intensity of asexual embryogenesis and growth of embryogenic callus of BC 5 and BC 36 of 7beobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 25 % for BC 5 and 42.7 % for BC 36; embryogenic intensity was 1.8 embryos per explant for BC 5 and 2.2 embryos per explant for BC36.
The basal medium contained the following constituents per liter: Murashige and Skoog (MS) salts (10), thiamine· HCI (0.1 mg), pyridoxine· HCI (0.5 mg), i-inositol (100 mg), nicotinic acid (0.5 mg), glycine (2.0 mg), casein hydrolysate (1 g), sucrose (30 g), Bacto-agar (8 g). Growth regulators (GAl, CCC, AMO 1618, daminozide) were sterilized by filtration through a Milipore filter (0.45/-1m) and added to the partially cooled medium after autoclaving to obtain concentrations of 0.05 to 10 mg .1- 1•
Z. Pjlanzenphysiol. Ed. 113. S. 359-366. 1984.
362
HALINA KONONOWICZ and JULES JANICK
The data presented are based on growth and development of the cultures after 6 weeks. Callus was measured and area calculated as described previously (Kononowicz et aI., in this issue). Frequency of embryogenesis are presented as a percentage of callus cultures producing somatic embryos. Intensity of embryogenesis is expressed as the average number of embryos per culture. Results were expressed relative to a percentage of the control. Control cultures were grown on basal medium lacking hormones. Experiments were repeated 2 or 3 times and the results averaged. Results
GA3 The effect of GA3 on callus growth and embryogenesis shown in Fig. 1A-C represent the averages of 3 experiments. GA3 stimulated embryogenesis for BC 5 (Fig. 1 A). Maximal response was observed at 1mg·l- 1• GA3 at 10mg·l- 1 increased embryogenesis over that of the controls, but embryos showed many abnormal features with finger-like structures formed instead of cotyledons. The intensity of embryogenesis responded in the same manner as frequency for BC 5 (Fig. 1 B). The maximum embryogenic response was at 1 mg .1- 1 at which intensity of embryo production increased 3.5 times that of the control. Embryogenic frequency or intensity from BC 36 callus was unaffected by GA3. Callus growth of both clones increased with GA3 from 0.05 to 1 mg .1- 1 and decreased at concentration above 1 mg .1- 1 (Fig. 1 C). AMO 1618
Results presented in Fig. 2 represent the averages of 3 experiments. AMO 1618 increased the frequency of embryo formation of BC 5 at concentrations of 0.05 to 1 mg .1- 1, and depressed embryogenesis completely above 1 mg .1- 1 (Fig. 2 A). Intensity of embryogenesis responded similarly to frequency for BC 5 (Fig. 2 B). The embryogenic effect of AMO 1618 on BC 36 callus was considerably less than that ofBC 5 (Fig. 2 A, B). AMO 1618 at 0.05 mg .1- 1 increased slightly the frequency and intensity of embryo production but above this concentration embryogenesis decreased. Callus growth of BC 5 was increased slightly by 0.05 mg .1- 1 AMO 1618, but there was no effect at higher concentrations (Fig. 2 C). AMO 1618 reduced callus growth of BC 36 at concentrations as low as 0.05 mg . 1-1, but no additional inhibition was observed as AMO 1618 concentrations increased.
Daminozide Results presented in Fig. 3 represent the averages of 2 experiments. Daminozide decreased embryogenesis at concentrations of 0.05 mg .1- 1 and above in both clones. For BC 5 complete inhibition of embryo formation occurred above 0.05 mg .1- 1 (Fig. 3 A). Intensity of embryo production responded similarly to frequency (Fig. 3 B). Daminozide suppressed growth of callus for both clones, but this inhibitory effect was considerably greater for BC 5 than BC 36 (Fig. 3 C). Z. Pjlanzenphysiol. Bd. 113. S.359-366. 1984.
Response of cacao callus to GA and GA-inhibitors
363
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Fig. 3: Effect of daminozide on frequency and intensity of asexual embryogenesis and growth of embryogenic callus of Be 5 and Be 36 of Theobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 37.5% for Be 5 and 40% for Be 36; embryogenic intensity was 4.2 embryos per explant for Be 5 and 4.8 embryos per explant for Be 36.
CCC Results presented in Fig. 4 are the averages of 2 experiments. The effect of CCC on embryogenesis was similar to the effect of daminozide. CCC at 0.05 mg .1- 1 inhibited embryo formation in BC 5, and above this concentration embryogenesis was completely depressed (Fig. 4 A). Although BC 36 responded similarly to BC 5, the inhibitory effect was not as extreme. Intensity of embryogenesis with both clones responded similarly to frequency (Fig. 4 B). Z. Pjlanzenphysiol. Bd. 113. S. 359-366. 1984.
364
HALINA KONONOWICZ
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eee (mq/liter) Fig. 4: Effect of CCC on frequency and intensity of asexual embryogenesis and growth of embryogenic callus of BC 5 and BC 36 of Theobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 50 % for BC 5 and 50 % for BC 36; embryogenic intensity was 3.1 embryos per explant for BC 5 and 5.3 embryos per explant for BC 36.
CCC reduced callus growth by both clones at concentrations as low 0.05 mg .1- 1 (Fig. 4 C). No additional inhibition was observed up to 10 mg '1- 1, Although the growth of BC 5 callus was twice as fast as that of BC 36 in the control, growth was reduced approximately to the same level for both clones in the presence ofCCC. Z. Pf/anzenphysiol. Ed. 113. S.359--366. 1984.
Response of cacao callus to GA and GA-inhibitors
365
Discussion
Our results indicate that the effect of GA on embryogenesis is clone-specific. For BC 5, the continuous application of GA3 for six weeks stimulated embryogenesis. However, there was no effect of GA3 with BC 36. Three inhibitors of GA synthesis, AMO 1618, daminozide, and CCC, did not respond identically. AMO 1618 stimulated embryogenesis at very low concentrations (0.05 and 0.1 mg .1- 1) with promotion considerably greater for BC 5 than BC 36. CCC and daminozide affected embryogenesis similarly, with inhibition proportional to concentration. Both retardants were more inhibitory with BC 5 than BC 36. The observation of GA-stimulated embryogenesis is in contrast to the inhibition of embryogenesis by GA reported for citrus ovular callus (Kochba et ai., 1978), carrot cells (Fujimura and Komomine, 1975; Kamada and Harada, 1979), caraway cells (Ammirato, 1977), and soybean callus (Phillips and Collins, 1981). This disagreement suggests that the effect of GA on embryogenesis differs by species or clone. Our observation of GA-induced abnormalities at high concentration also was observed by Ammirato (1977) for caraway somatic embryos. AMO 1618, which has been classified as a growth inhibitor, promoted embryogenesis at very low concentrations but inhibited embryogenesis at high concentration especially for BC 5. The inhibitory effect of AMO 1618 on somatic embryogenesis has been reported for carrot (Kamada and Harada, 1979). Phillips and Collins (1981) reported that AMO 1618 improved development of soybean somatic embryos but with no increase in number of embryos produced from callus. The plant growth retardants CCC and daminozide depressed somatic embryogenesis and callus growth of Theobroma cacao. In contrast, Kochba et ai. (1978) reported that these two growth retardants significantly stimulated embryogenesis in citrus callus in the range of 0.01 to 10 mg .1- 1• The conflicting results of the effects of GA and GA synthesis inhibitors on embryogenesis is difficult to interpret. There may be a major interaction of these growth substances with species and, in our study, differences due to clone. For example, we observed increased embryogenic response to GA for only one clone of cacao. With this fact in mind, it is not surprising that well-known inhibitors of GA synthesis would decrease or completely depress embryogenesis of this clone. The stimulatory effect of AMO 1618 at very low concentrations would suggest that this growth substance also has effects other than the inhibition of GA synthesis. Our results support the concept that asexual embryogenesis is a complex phenomenon influenced by many endogenous and/or exogenous growth substances.
References AMMlRATO, P. U.: Plant Physiol. 59, 579-586 (1977). DOUGLAS, T. J. and L. G. PALEG: Plant Physiol. 49, 417-420 (1972). DOUGLAs, T. J. and L. G. PALEG: Phytochemistry 17, 713-718 (1980).
Z. Pjlanzenphysiol. Bd. 113. S.359-366. 1984.
366
HALINA KONONOWICZ and JULES JANICK
ESAN, E. G.: Tissue culture studies on cacao (Theobroma cacao L.). A supplementation of current research. Proceedings V International Cacao Research Conference, 1975, pp. 116-125. Cacao Research Institute of Nigeria, Ibadan, Nigeria, 1977. FUJIMURA, T. and A. KOMOMINE: Plant Sci. Lett. 5, 359-364 (1975). KAMADA, H. and H. HARADA: Z. Pflanzenphysiol. 91, 255-266 (1979). KOCHBA, Y., P. SPIEGEL-Roy, H. NEWMANN, and S. SAAD: Z. Pflanzenphysiol. 89, 427-432 (1978).
KONONOWICZ, H., A. K. KONONOWICZ, L. WASILEWSKA-DABROWSKA, and K. KLECZKOWSKI: Int. J. Biochem. 14, 421-428 (1982). MURASHIGE, T. and F. SKOOG: Can. J. Bot. 48, 277-285 (1962). NICKELL, L. G.: Controlling biological behavior of plants with synthetic plant growth regulating chemicals. In: MADAVA, N. B. (ed.): Plant growth substances. American Chemical Society Symposium Series III, American Chemical Society, Washington D.C., pp. 263-279 (1979).
PENCE, V. c., P. M. HASEGAWA, andJ. JANICK: Z. Pflanzenphysiol. 98,1-14 (1980). PHILLIPS, G. C. and G. B. COLLINS: Plant Cell Tissue Organ Culture 1, 123-129 (1981). SCHNEIDER, G.: Annu. Rev. Plant Physiol. 21, 449-536 (1970). SHARP, W. R., M. R. SONDAHL, L. S. CALDUS, and S. B. MARAFFA: Hort. Rev. 2, 268-310 (1980). STEFFENS, G. L.: Applied uses of growth substances - growth inhibitors. In: SKOOG, F. (ed.): Plant growth substances 1979. Springer-Verlag, Berlin, pp. 397-408 (1980). TISSERAT, B., E. B. ESAN, and T. MURASHIGE: Hort. Rev. 1, 1-79 (1979). WITTWER, S. H.: Phytohormones and chemical regulators in agriculture. In: LETHAM, E. D., GOODWIN, P. G., HIGGINS, T. Y. O. (ed.): Phytohormones and related compounds: A comprehensive treatise, Vol. 2. Elsevier, Amsterdam, pp. 599-615 (1978).
Z. Pjlanzenphysiol. Ed. 113. S. 359-366.1984.
Summary Gibberellic acid at 0.05 to 10 mg .1- 1 stimulated asexual embryogenesis from embryogeniccompetent callus of clone BC 5 but not BC 36 of Theobroma cacao. AMO 1618 stimulated embryogenesis at 0.05 to 0.1 mg .1- 1 but depressed embryogenesis above 0.1 mg .1- 1 for clone BC 5. A similar but smaller effect was observed for BC 36. Daminozide and CCC depressed embryogenesis with both clones.
Key words: Theobroma cacao, asexual embryogenesis, gibberellic acid.
Introduction
The stimulation or inhibition of in vitro somatic embryogenesis by growth regulating substances has been reported for many plant species (Steffens, 1980; Tisserat et al., 1979). Auxin and cytokinins are the growth-regulators most widely investigated (Sharp et ai., 1980; Tisserat et ai., 1979). Somatic embryogenesis via hypocotylary budding in Theobroma cacao has been described previously (Esan, 1977; Pence et ai., 1980). We have recently described the spontaneous production of somatic embryos from callus on hormone-free medium for 2 clones of cacao (Kononowicz et ai., in this issue). However, under these conditions embryogenesis occurs in relatively low frequency. Somatic embryos of Theobroma cacao initiated from callus were structurally comparable to zygotic embryos and passed through similar developmental stages, viz. globular, heart-shaped, torpedo-shaped and finally cotyledonary. Embryogenic callus was shifted toward the production of homogeneous callus with a combination of 10 % coconut water and 1.0 mg .1- 1 2,4-D or to a high frequency embryogenesis with 2,4-D at 10-3 to 10-2 mg .1- 1. Little has been reported on the role of gibberellic acid (GA3) on somatic embryogenesis. Growth retardants such as (2-chloroethyl)trimethylammonium chloride, (CCC, chlormequat), butanedioic mono-{2,2-dimethylhydrazide) (daminozide, Alar), and 2'-isopropyl-4'-dimethylamino-S'-methylphenyl-1 '-piperidine carboxylate methyl chloride (AMO 1618) inhibit GA synthesis in certain plant species (Douglas and Paleg, 1972, 1980; Kononowicz et ai., 1982; Nickell, 1979; Schneider, 1970; Wittwer, 1978). In this report, we present results of an investigation of the efZ. Pjlanzenphysiol. Bd. 113. S. 359-366. 1984.
360
HALINA KONONOWICZ and JULES JANICK
fects of GA3 and inhibitors of GA synthesis (AMO 1618, daminozide, and asexual embryogenesis via callus of Theobroma cacao.
ccq on
Materials and Methods The callus used in this study originated from cotyledons of asexual embryos of 7beobroma cacao. Culture conditions and callus characteristics have been described previously (Kononowicz et a!., in this issue). All experiments were carried out with 2 clones - BC 5 and BC 36. Pieces of friable callus about 10 to 15 mm 2 were used to initiate all experiments.
IoBc5l i
L~~
800
c
700
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~ 600
~
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IS
400
g
300 200 100 0r------+--4-----~--~------~~--_1
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~
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Fig. 1: The effect of GA3 on frequency and intensity of asexual embryogenesis and growth of embryogenic competent callus of BC 5 and BC 36 of 7beobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 28.3 % for BC 5 and 39.3 % for BC 36; embryogenic intensity was 1.6 embryos per explant for BC 5 and 2.8 embryos per explant for BC36.
Z. Pjlanzenphysiol. Bd. 113. S. 359-366. 1984.
Response of cacao callus to GA and GA-inhibitors
361
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Fig. 2: The effect of AMO 1618 on frequency and intensity of asexual embryogenesis and growth of embryogenic callus of BC 5 and BC 36 of 7beobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 25 % for BC 5 and 42.7 % for BC 36; embryogenic intensity was 1.8 embryos per explant for BC 5 and 2.2 embryos per explant for BC36.
The basal medium contained the following constituents per liter: Murashige and Skoog (MS) salts (10), thiamine· HCI (0.1 mg), pyridoxine· HCI (0.5 mg), i-inositol (100 mg), nicotinic acid (0.5 mg), glycine (2.0 mg), casein hydrolysate (1 g), sucrose (30 g), Bacto-agar (8 g). Growth regulators (GAl, CCC, AMO 1618, daminozide) were sterilized by filtration through a Milipore filter (0.45/-1m) and added to the partially cooled medium after autoclaving to obtain concentrations of 0.05 to 10 mg .1- 1•
Z. Pjlanzenphysiol. Ed. 113. S. 359-366. 1984.
362
HALINA KONONOWICZ and JULES JANICK
The data presented are based on growth and development of the cultures after 6 weeks. Callus was measured and area calculated as described previously (Kononowicz et aI., in this issue). Frequency of embryogenesis are presented as a percentage of callus cultures producing somatic embryos. Intensity of embryogenesis is expressed as the average number of embryos per culture. Results were expressed relative to a percentage of the control. Control cultures were grown on basal medium lacking hormones. Experiments were repeated 2 or 3 times and the results averaged. Results
GA3 The effect of GA3 on callus growth and embryogenesis shown in Fig. 1A-C represent the averages of 3 experiments. GA3 stimulated embryogenesis for BC 5 (Fig. 1 A). Maximal response was observed at 1mg·l- 1• GA3 at 10mg·l- 1 increased embryogenesis over that of the controls, but embryos showed many abnormal features with finger-like structures formed instead of cotyledons. The intensity of embryogenesis responded in the same manner as frequency for BC 5 (Fig. 1 B). The maximum embryogenic response was at 1 mg .1- 1 at which intensity of embryo production increased 3.5 times that of the control. Embryogenic frequency or intensity from BC 36 callus was unaffected by GA3. Callus growth of both clones increased with GA3 from 0.05 to 1 mg .1- 1 and decreased at concentration above 1 mg .1- 1 (Fig. 1 C). AMO 1618
Results presented in Fig. 2 represent the averages of 3 experiments. AMO 1618 increased the frequency of embryo formation of BC 5 at concentrations of 0.05 to 1 mg .1- 1, and depressed embryogenesis completely above 1 mg .1- 1 (Fig. 2 A). Intensity of embryogenesis responded similarly to frequency for BC 5 (Fig. 2 B). The embryogenic effect of AMO 1618 on BC 36 callus was considerably less than that ofBC 5 (Fig. 2 A, B). AMO 1618 at 0.05 mg .1- 1 increased slightly the frequency and intensity of embryo production but above this concentration embryogenesis decreased. Callus growth of BC 5 was increased slightly by 0.05 mg .1- 1 AMO 1618, but there was no effect at higher concentrations (Fig. 2 C). AMO 1618 reduced callus growth of BC 36 at concentrations as low as 0.05 mg . 1-1, but no additional inhibition was observed as AMO 1618 concentrations increased.
Daminozide Results presented in Fig. 3 represent the averages of 2 experiments. Daminozide decreased embryogenesis at concentrations of 0.05 mg .1- 1 and above in both clones. For BC 5 complete inhibition of embryo formation occurred above 0.05 mg .1- 1 (Fig. 3 A). Intensity of embryo production responded similarly to frequency (Fig. 3 B). Daminozide suppressed growth of callus for both clones, but this inhibitory effect was considerably greater for BC 5 than BC 36 (Fig. 3 C). Z. Pjlanzenphysiol. Bd. 113. S.359-366. 1984.
Response of cacao callus to GA and GA-inhibitors
363
c 800
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o A
• 20
•
•
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Fig. 3: Effect of daminozide on frequency and intensity of asexual embryogenesis and growth of embryogenic callus of Be 5 and Be 36 of Theobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 37.5% for Be 5 and 40% for Be 36; embryogenic intensity was 4.2 embryos per explant for Be 5 and 4.8 embryos per explant for Be 36.
CCC Results presented in Fig. 4 are the averages of 2 experiments. The effect of CCC on embryogenesis was similar to the effect of daminozide. CCC at 0.05 mg .1- 1 inhibited embryo formation in BC 5, and above this concentration embryogenesis was completely depressed (Fig. 4 A). Although BC 36 responded similarly to BC 5, the inhibitory effect was not as extreme. Intensity of embryogenesis with both clones responded similarly to frequency (Fig. 4 B). Z. Pjlanzenphysiol. Bd. 113. S. 359-366. 1984.
364
HALINA KONONOWICZ
~
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1200
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and JULES JANICK
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eee (mq/liter) Fig. 4: Effect of CCC on frequency and intensity of asexual embryogenesis and growth of embryogenic callus of BC 5 and BC 36 of Theobroma cacao. Mean values of the control were as follows: Embryogenic frequency was 50 % for BC 5 and 50 % for BC 36; embryogenic intensity was 3.1 embryos per explant for BC 5 and 5.3 embryos per explant for BC 36.
CCC reduced callus growth by both clones at concentrations as low 0.05 mg .1- 1 (Fig. 4 C). No additional inhibition was observed up to 10 mg '1- 1, Although the growth of BC 5 callus was twice as fast as that of BC 36 in the control, growth was reduced approximately to the same level for both clones in the presence ofCCC. Z. Pf/anzenphysiol. Ed. 113. S.359--366. 1984.
Response of cacao callus to GA and GA-inhibitors
365
Discussion
Our results indicate that the effect of GA on embryogenesis is clone-specific. For BC 5, the continuous application of GA3 for six weeks stimulated embryogenesis. However, there was no effect of GA3 with BC 36. Three inhibitors of GA synthesis, AMO 1618, daminozide, and CCC, did not respond identically. AMO 1618 stimulated embryogenesis at very low concentrations (0.05 and 0.1 mg .1- 1) with promotion considerably greater for BC 5 than BC 36. CCC and daminozide affected embryogenesis similarly, with inhibition proportional to concentration. Both retardants were more inhibitory with BC 5 than BC 36. The observation of GA-stimulated embryogenesis is in contrast to the inhibition of embryogenesis by GA reported for citrus ovular callus (Kochba et ai., 1978), carrot cells (Fujimura and Komomine, 1975; Kamada and Harada, 1979), caraway cells (Ammirato, 1977), and soybean callus (Phillips and Collins, 1981). This disagreement suggests that the effect of GA on embryogenesis differs by species or clone. Our observation of GA-induced abnormalities at high concentration also was observed by Ammirato (1977) for caraway somatic embryos. AMO 1618, which has been classified as a growth inhibitor, promoted embryogenesis at very low concentrations but inhibited embryogenesis at high concentration especially for BC 5. The inhibitory effect of AMO 1618 on somatic embryogenesis has been reported for carrot (Kamada and Harada, 1979). Phillips and Collins (1981) reported that AMO 1618 improved development of soybean somatic embryos but with no increase in number of embryos produced from callus. The plant growth retardants CCC and daminozide depressed somatic embryogenesis and callus growth of Theobroma cacao. In contrast, Kochba et ai. (1978) reported that these two growth retardants significantly stimulated embryogenesis in citrus callus in the range of 0.01 to 10 mg .1- 1• The conflicting results of the effects of GA and GA synthesis inhibitors on embryogenesis is difficult to interpret. There may be a major interaction of these growth substances with species and, in our study, differences due to clone. For example, we observed increased embryogenic response to GA for only one clone of cacao. With this fact in mind, it is not surprising that well-known inhibitors of GA synthesis would decrease or completely depress embryogenesis of this clone. The stimulatory effect of AMO 1618 at very low concentrations would suggest that this growth substance also has effects other than the inhibition of GA synthesis. Our results support the concept that asexual embryogenesis is a complex phenomenon influenced by many endogenous and/or exogenous growth substances.
References AMMlRATO, P. U.: Plant Physiol. 59, 579-586 (1977). DOUGLAS, T. J. and L. G. PALEG: Plant Physiol. 49, 417-420 (1972). DOUGLAs, T. J. and L. G. PALEG: Phytochemistry 17, 713-718 (1980).
Z. Pjlanzenphysiol. Bd. 113. S.359-366. 1984.
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HALINA KONONOWICZ and JULES JANICK
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