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Effect of Cyclic 3′,5′-Adenosine Monophosphate and Kinetin on Growth and Fertility in the Male Clones of the Moss Microdus brasiliensis (Dub.) Ther.
Effect of Cyclic 3',S'-Adenosine Monophosphate and Kinetin on Growth and Fertility in the Male Clones of the Moss Microdus brasiliensis (Dub.) Ther. R. N. CHOPRA and POONAM SHARMA Department of Botany, University of Delhi, Delhi 110007, India Received May 20, 1984 . Accepted July 23, 1984
Summary On basic medium the male clones of Microdus brasiliensis form normal gametophores, of which 18 % bear antheridia. Kinetin enhances the number of buds but inhibits the formation of normal gametophores. With cAMP the number of buds is reduced and the gametophores are also stunted. Co-addition of kinetin and cAMP increases the number of buds, improves the morphology of gametophores, and in some combinations bring about considerable enhancement in antheridial production.
Key words: Microdus brasiliensis, antheridial production, number of buds, cAMP, kinetin.
Introduction Considerable literature exists on the role of phytohormones in growth and development of mosses in the vegetative phase, but comparable studies on the reproductive phase are meagre (Bopp, 1983). In recent times several investigations have been carried out on mosses concerning the endogenous presence of cAMP, as well as on the morphogenetic effects of applied cAMP (Bhatia and Chopra, 1983). In the present study, attempts have been made to observe the effects of cAMP and kinetin and their interaction on bud induction, morphology of gametophores and antheridial production in the male clones of Microdus
brasiliensis.
Material and Methods Microdus brasiliensis (Dub.) Ther. is a dioecious moss of the family Dicranaceae. It was collected from Pachmarhi (M. P.) during October 1982, and aseptic cultures were raised from spores and maintained as described earlier (Chopra and Kumra, 1983). So far only male game· tophores have been observed in cultures. Kinetin was autoclaved with the basic medium, whereas cAMP was added to the autoclaved medium after filter·sterilization. Both the chemicals were tested in the range 10- 8 -10- 4 M. For a study of cAMP·kinetin interaction, 1O- 8 -10- 5 M cAMP was added together with 10- 6 and 10 - 5 M kinetin. Abbreviations: Cyclic 31,S'-adenosine monophosphate (cAMP), 6-furfurylaminopurine (kinetin; Kn), 2-isopentenyladenine (2iP).
J. Plant. Physiol. Vol.
117. pp. 293-296 (1985)
294
R. N. CHOPRA and POONAM SHARMA
The experiments were performed on 25-day-old, bud-free protonema subcultured from basic medium. Each experiment was run for 60 days. Twelve replicates were maintained for each treatment, and the experiments were repeated at least once.
Results
On basic medium buds were observed after 32 days. These developed into normal gametophores, about 18 % of which bore antheridia after 48 days. cAMP - It reduced the number of buds at all concentrations tested. Buds were moruloid and later developed into stunted gametophores. At 10- 4 M, no gametophores were observed (Table 1). Kinetin - It increased the number of buds at all concentrations tested except at 10- 4 M, at which level the protonema did not regenerate and turned brown. Buds were moruloid and these later developed into cylindrical gametophores with greatly reduced leaves (Table 1; Fig. 1 A). cAMP - kinetin interaction - Co-addition of cAMP and kinetin (10- 6 M) increased the number of buds, and these buds developed into normal gametophores. With kiTable 1: Effects of cAMP, Kn, and cAMP-Kn interaction on number of buds, gametophore morphology, and antheridial production in Microdus brasiliensis at 25±2C and 3,500-4,000 lux. Mean values and standard errors are from 12 replicates. Treatment (M)
Number of buds
Control cAMP 10-8 10-7 10-6 10-5 10-4 10- 8 Kn 10- 7 10-6 10- 5 cAMP 10- 8 -Kn 10-7 - Kn 1O-6 -Kn 1O-5 -Kn cAMP 1O-8 -Kn 1O-7 -Kn 10-6 -Kn 10-5 - Kn
10-6 10-6 10-6 10-6 10- 5 10-5 10-5 10-5
Morphology of gametophores (after 60 days)
% of fertile
(at 45 days) 148± 9 47± 4 69± 8 94± 5 79± 3 75± 5 162±11 171± 9 202± 7 318±10
Normal Abnormal (Stunted) Abnormal (Stunted) Abnormal (Stunted) Abnormal (Stunted) No gametophores Abnormal (long axis) Abnormal (long axis) Abnormal (long axis) Abnormal (long axis)
18±2
(at 30 days)*) 535± 10 587±12 485±11 421±12 491±10 580± 14 511±12 414± 9
Normal Normal Normal Normal Normal Normal Normal and Abnormal Normal and Abnormal
79±3 74±2 64±3 54±2 23±2
-) Antheridia not formed. *) After 30 days buds become too many to be counted.
J. Plant. Physiol. Vol.
117. pp. 293-296 (/985)
gametophores
Growth and fertility in Microdus
295
Fig.l: Microdus brasiliensis. A. Abnormal gametophore from culture on medium with lO-8M Kn. xl2S. - B. Culture on medium with 1O- 8M cAMP + lO-6M Kn showing antheridial heads. x 8. - C. Dissected antheridial head from B x 80. (All cultures are 8 weeks old.)
netin at 1O- 5 M and cAMP (10- 8 -10- 5 M) only a few gametophores grow out from the buds. With 10- 8 M cAMP + 1O- 5 M kinetin, the number of fertile gametophores increased slightly. The rest of the combinations at this level of kinetin were inhibitory. With kinetin at 10- 6 M and cAMP (10- 8 -10- 5 M) production of antheridial heads was greatly promoted and the maximum response (79%) was observed at 10- 8 M cAMP + 10- 6 M kinetin (Table 1; Fig. 1 B, C). The number of antheridia per head was also greater (10-12) as compared to the controls (8-10). However, the time of antheridial formation was not significantly affected by these treatments. Discussion Some reports are available on the effect of cAMP on bud induction in mosses. Schneider et al. (1975) observed that cAMP alone or together with 2iP does not affect bud induction in Ceratodon purpureus and Funaria hygrometrica. In Timmiella anomala cAMP inhibits bud formation and kinetin enhances this response. When added together these act synergistically (Kapur and Chopra, 1983). In Microdus brasiliensis cAMP alone inhibits bud formation, whereas kinetin has a promotory effect. Their co-addition results in an increased number of buds and production of normal gametophores. In higher plants cytokinins generally enhance femaleness, and are even known to transform sexuality (Mohan Ram, 1980; Durand and Durand, 1984). In bryophytes
J Plant. Physiol.
Vol. 117. pp. 293-296 (1985)
296
R. N. CHOPRA and POONAM SHARMA
hormones are ineffective in altering the sex, but they do influence gametangial induction. In the majority of systems investigated, cytokinins enhance archegonial production (Chopra and Bhatla, 1983). But there are some quantitative differences in the species so far investigated (Chopra and Sood, 1973; Chopra and Rawat, 1977; Bhatla and Chopra, 1981; Chopra and Kumra, 1983). The present findings on Microdus brasiliensis are interesting since in this moss kinetin in combination with cAMP markedly promotes antheridial production and also increases the number of antheridia per head. Thus, in Microdus the effects of cAMP and kinetin are dependent on each other. This is in contrast to the observations of Bhatla and Chopra (1981) who reported that in Bryum argenteum cAMP stimulates gametangial induction and has an additive effect on this response when added along with kinetin. Nothing is so far known about the internal content of these two substances and their role in regulation of sexuality in vivo. Acknowledgements Weare thankful to Prof. S. S. Kumar for determination of the moss and to the Council of Scientific and Industrial Research, New Delhi, for financial assistance to one of us (PS).
References BHAl'LA, S. C. and R. N. CHOPRA: Hormonal regulation of gametangial formation in the moss Bryum argenteum Hedw. J. Exp. Bot. 32, 1243-1256 (1981). - - Biological Significance of cyclic adenosine 3',5'-monophosphate in mosses. Physiol. Plant. 57, 383-389 (1983). Bopp, M.: Developmental physiology of bryophytes. In: SCHUSTER, R. M. (ed.), New Manual of Bryology 1, 276-324. The Hattori Botanical Laboratory, Japan, 1983. CHOPRA, R. N. and S. C. BHATLA: Regulation of gametangial formation in bryophytes. Bot. Rev. 49, 29-63 (1983). CHOPRA, R. N. and P. K. KUMRA: Hormonal regulation of growth and antheridial production in three mosses grown tn vitro. J. Bryol. 12, 491- 502 (1983). CHOPRA, R. N. and M. S. RAWAT: Studies on the initiation of sexual phase in the moss Leptobryum pyriforme. Beitr. BioI. Pflanzen 53, 353-357 (1977). CHOPRA, R. N. and S. SOOD: In vitro studies in Marchantiales. I. Effects of some carbohydrates, agar, pH, light and growth regulators on growth and sexuality in Rima crystallina. Phytomorphology 23, 230-244 (1973). DURAND, R. and B. DURAND: Sexual differentiation in higher plants. Physiol. Plant. 60, 267274 (1984). KApUR, A. and R. N. CHOPRA: Effect of kinetin and cyclic 3',5'-adenosine monophosphate on bud induction in the moss Timmiella anomala. New Phytol. 94, 393-399 (1983). MOHAN RAM, H. Y.: Hormones and flower sex. PI. Biochem. J. S. M. Sircar Memorial Volume, 77 -88 (1980). SCHNEIDER, J., SZWEYKOWSKA, A., and M. SPYCHALA: Evidence against mediation of adenosine3',5'-cyclic monophosphate in bud-inducing effect of cytokinins in moss protonema. Acta Soc. Bot. Pol. 44, 607 -614 (1975).
J Plant. Physiol. Vol.
117. pp. 293-296 (1985)
Summary On basic medium the male clones of Microdus brasiliensis form normal gametophores, of which 18 % bear antheridia. Kinetin enhances the number of buds but inhibits the formation of normal gametophores. With cAMP the number of buds is reduced and the gametophores are also stunted. Co-addition of kinetin and cAMP increases the number of buds, improves the morphology of gametophores, and in some combinations bring about considerable enhancement in antheridial production.
Key words: Microdus brasiliensis, antheridial production, number of buds, cAMP, kinetin.
Introduction Considerable literature exists on the role of phytohormones in growth and development of mosses in the vegetative phase, but comparable studies on the reproductive phase are meagre (Bopp, 1983). In recent times several investigations have been carried out on mosses concerning the endogenous presence of cAMP, as well as on the morphogenetic effects of applied cAMP (Bhatia and Chopra, 1983). In the present study, attempts have been made to observe the effects of cAMP and kinetin and their interaction on bud induction, morphology of gametophores and antheridial production in the male clones of Microdus
brasiliensis.
Material and Methods Microdus brasiliensis (Dub.) Ther. is a dioecious moss of the family Dicranaceae. It was collected from Pachmarhi (M. P.) during October 1982, and aseptic cultures were raised from spores and maintained as described earlier (Chopra and Kumra, 1983). So far only male game· tophores have been observed in cultures. Kinetin was autoclaved with the basic medium, whereas cAMP was added to the autoclaved medium after filter·sterilization. Both the chemicals were tested in the range 10- 8 -10- 4 M. For a study of cAMP·kinetin interaction, 1O- 8 -10- 5 M cAMP was added together with 10- 6 and 10 - 5 M kinetin. Abbreviations: Cyclic 31,S'-adenosine monophosphate (cAMP), 6-furfurylaminopurine (kinetin; Kn), 2-isopentenyladenine (2iP).
J. Plant. Physiol. Vol.
117. pp. 293-296 (1985)
294
R. N. CHOPRA and POONAM SHARMA
The experiments were performed on 25-day-old, bud-free protonema subcultured from basic medium. Each experiment was run for 60 days. Twelve replicates were maintained for each treatment, and the experiments were repeated at least once.
Results
On basic medium buds were observed after 32 days. These developed into normal gametophores, about 18 % of which bore antheridia after 48 days. cAMP - It reduced the number of buds at all concentrations tested. Buds were moruloid and later developed into stunted gametophores. At 10- 4 M, no gametophores were observed (Table 1). Kinetin - It increased the number of buds at all concentrations tested except at 10- 4 M, at which level the protonema did not regenerate and turned brown. Buds were moruloid and these later developed into cylindrical gametophores with greatly reduced leaves (Table 1; Fig. 1 A). cAMP - kinetin interaction - Co-addition of cAMP and kinetin (10- 6 M) increased the number of buds, and these buds developed into normal gametophores. With kiTable 1: Effects of cAMP, Kn, and cAMP-Kn interaction on number of buds, gametophore morphology, and antheridial production in Microdus brasiliensis at 25±2C and 3,500-4,000 lux. Mean values and standard errors are from 12 replicates. Treatment (M)
Number of buds
Control cAMP 10-8 10-7 10-6 10-5 10-4 10- 8 Kn 10- 7 10-6 10- 5 cAMP 10- 8 -Kn 10-7 - Kn 1O-6 -Kn 1O-5 -Kn cAMP 1O-8 -Kn 1O-7 -Kn 10-6 -Kn 10-5 - Kn
10-6 10-6 10-6 10-6 10- 5 10-5 10-5 10-5
Morphology of gametophores (after 60 days)
% of fertile
(at 45 days) 148± 9 47± 4 69± 8 94± 5 79± 3 75± 5 162±11 171± 9 202± 7 318±10
Normal Abnormal (Stunted) Abnormal (Stunted) Abnormal (Stunted) Abnormal (Stunted) No gametophores Abnormal (long axis) Abnormal (long axis) Abnormal (long axis) Abnormal (long axis)
18±2
(at 30 days)*) 535± 10 587±12 485±11 421±12 491±10 580± 14 511±12 414± 9
Normal Normal Normal Normal Normal Normal Normal and Abnormal Normal and Abnormal
79±3 74±2 64±3 54±2 23±2
-) Antheridia not formed. *) After 30 days buds become too many to be counted.
J. Plant. Physiol. Vol.
117. pp. 293-296 (/985)
gametophores
Growth and fertility in Microdus
295
Fig.l: Microdus brasiliensis. A. Abnormal gametophore from culture on medium with lO-8M Kn. xl2S. - B. Culture on medium with 1O- 8M cAMP + lO-6M Kn showing antheridial heads. x 8. - C. Dissected antheridial head from B x 80. (All cultures are 8 weeks old.)
netin at 1O- 5 M and cAMP (10- 8 -10- 5 M) only a few gametophores grow out from the buds. With 10- 8 M cAMP + 1O- 5 M kinetin, the number of fertile gametophores increased slightly. The rest of the combinations at this level of kinetin were inhibitory. With kinetin at 10- 6 M and cAMP (10- 8 -10- 5 M) production of antheridial heads was greatly promoted and the maximum response (79%) was observed at 10- 8 M cAMP + 10- 6 M kinetin (Table 1; Fig. 1 B, C). The number of antheridia per head was also greater (10-12) as compared to the controls (8-10). However, the time of antheridial formation was not significantly affected by these treatments. Discussion Some reports are available on the effect of cAMP on bud induction in mosses. Schneider et al. (1975) observed that cAMP alone or together with 2iP does not affect bud induction in Ceratodon purpureus and Funaria hygrometrica. In Timmiella anomala cAMP inhibits bud formation and kinetin enhances this response. When added together these act synergistically (Kapur and Chopra, 1983). In Microdus brasiliensis cAMP alone inhibits bud formation, whereas kinetin has a promotory effect. Their co-addition results in an increased number of buds and production of normal gametophores. In higher plants cytokinins generally enhance femaleness, and are even known to transform sexuality (Mohan Ram, 1980; Durand and Durand, 1984). In bryophytes
J Plant. Physiol.
Vol. 117. pp. 293-296 (1985)
296
R. N. CHOPRA and POONAM SHARMA
hormones are ineffective in altering the sex, but they do influence gametangial induction. In the majority of systems investigated, cytokinins enhance archegonial production (Chopra and Bhatla, 1983). But there are some quantitative differences in the species so far investigated (Chopra and Sood, 1973; Chopra and Rawat, 1977; Bhatla and Chopra, 1981; Chopra and Kumra, 1983). The present findings on Microdus brasiliensis are interesting since in this moss kinetin in combination with cAMP markedly promotes antheridial production and also increases the number of antheridia per head. Thus, in Microdus the effects of cAMP and kinetin are dependent on each other. This is in contrast to the observations of Bhatla and Chopra (1981) who reported that in Bryum argenteum cAMP stimulates gametangial induction and has an additive effect on this response when added along with kinetin. Nothing is so far known about the internal content of these two substances and their role in regulation of sexuality in vivo. Acknowledgements Weare thankful to Prof. S. S. Kumar for determination of the moss and to the Council of Scientific and Industrial Research, New Delhi, for financial assistance to one of us (PS).
References BHAl'LA, S. C. and R. N. CHOPRA: Hormonal regulation of gametangial formation in the moss Bryum argenteum Hedw. J. Exp. Bot. 32, 1243-1256 (1981). - - Biological Significance of cyclic adenosine 3',5'-monophosphate in mosses. Physiol. Plant. 57, 383-389 (1983). Bopp, M.: Developmental physiology of bryophytes. In: SCHUSTER, R. M. (ed.), New Manual of Bryology 1, 276-324. The Hattori Botanical Laboratory, Japan, 1983. CHOPRA, R. N. and S. C. BHATLA: Regulation of gametangial formation in bryophytes. Bot. Rev. 49, 29-63 (1983). CHOPRA, R. N. and P. K. KUMRA: Hormonal regulation of growth and antheridial production in three mosses grown tn vitro. J. Bryol. 12, 491- 502 (1983). CHOPRA, R. N. and M. S. RAWAT: Studies on the initiation of sexual phase in the moss Leptobryum pyriforme. Beitr. BioI. Pflanzen 53, 353-357 (1977). CHOPRA, R. N. and S. SOOD: In vitro studies in Marchantiales. I. Effects of some carbohydrates, agar, pH, light and growth regulators on growth and sexuality in Rima crystallina. Phytomorphology 23, 230-244 (1973). DURAND, R. and B. DURAND: Sexual differentiation in higher plants. Physiol. Plant. 60, 267274 (1984). KApUR, A. and R. N. CHOPRA: Effect of kinetin and cyclic 3',5'-adenosine monophosphate on bud induction in the moss Timmiella anomala. New Phytol. 94, 393-399 (1983). MOHAN RAM, H. Y.: Hormones and flower sex. PI. Biochem. J. S. M. Sircar Memorial Volume, 77 -88 (1980). SCHNEIDER, J., SZWEYKOWSKA, A., and M. SPYCHALA: Evidence against mediation of adenosine3',5'-cyclic monophosphate in bud-inducing effect of cytokinins in moss protonema. Acta Soc. Bot. Pol. 44, 607 -614 (1975).
J Plant. Physiol. Vol.
117. pp. 293-296 (1985)