- Email: [email protected]
Welwitschia mirabilis Callus Studies III. Some Effects of Morphactin
Department of Botany and Plant Tissue Culture Research Unit, University of Natal, Pietermaritzburg, South Africa
Welwitschia mirabilis Callus Studies III. Some Effects of Morphactin CHRIS H . BORNMAN With 2 figures Received December 2, 1975 . Accepted January 9, 1976
Summary Mor.phactins, fluorene-carbox ylic acid derivatives reported highly active in a number of plant morphogenic responses, retard growth and severely inhibit chlorophyll synthesis in Welwitschia mirabilis callus cultures. No organogenic response was elicited. Cell membranes appeared particularly susceptible to morphactin. ER and dictyosomes became progressively vesiculated; in the chloroplasts thylakoids were swollen and their lamellae distorted and few grana were present.
Key words: Welwitschia, Callus, Morphactin.
Introduction Callus can be proliferated quite readily from the hypocotyl-root axis of germinating Welwitschia mirabilis embryos (BORNMAN, 1976) and, at the expense of some growth, can synthesize relatively large amounts of chlorophyll when its normal growth medium (SCHENK and HILDEBRANDT, 1972) is supplemented with 4 to 8 per cent sucrose (BORNMAN and FANSHAWE, 1976). The plant itself has no apical meristem and thus far attempts at inducing adventitious bud formation in callus, have not met with success. As Welwitschia occupies an unique if somewhat controversial position in the plant kingdom (FOSTER and GIFFORD, 1959), it is of interest to learn more about its morphogenic behaviour. In this regard tissue culture seems an appropriate avenue of approach and attention therefore was focused on other substances that might induce organogenesis, as for example the morphactins, socalled morphologically active substances. According to SCHNEIDER (1972) these fluorene-carboxylic acid derivatives are highly active in a number of plant morphogenic responses such as callus formation, shoot and root growth, and flower initiation and fruit development. KATHJU and TEWARI (1971) reported that 25 mg 1-1 morphactin inhibited a-amylase activity in Cyamopsis cotyledons but enhanced that of phosphorylase. BORNMAN et a1. (1974) Z. PJlanzenphysiol. Bd. 78. S. 266-270. 1976.
Some effects of morphactin on Welwitschia callus
267
found that a concentration of 10 mg 1- 1 inhibited phosphatase and phosphodiesterase activity in Welwitschia callus. There are also some conflicting accounts regarding the action of morphactins - for example, ZIEGLER (1970) reported a shortening of pea stem internodes whereas KRISHNAMOORTHY (1971) found the opposite effect in Luffa - and their physiological role remains obscure.
Material and Methods Callus derived from the embryo of Welwitschia mirabilis was established as described earlier (BORNMAN, 1976) on SCHENK'S and HILDEBRANDT'S (1972) basal medium su pplemented with 0.3 mg 1- 1 napthaleneacetic acid, 0.1 mg 1-1 kinetin and 4 per cent sucrose. Two morphactins, f1urenol (n-butyl 9-hydroxyfluorene-(9)-carboxylate) and chlorflurenol (methyl 2-chloro-9-hydroxyfluorene-(9)-carboxylate) were incorporated in the culture medicum at concentration levels of 0.1, 1, 10, and 100 mg 1-1 • The morphactins first were dissolved in a small volume of acetone and stabilized with a droplet of Tween 20 before diluting to volume with distilled water. Fresh weights and chlorophyll content were determined 20 days after subculturing, while tissues were sampled every 10 days for electron microscopical studies. The latter were fixed, embedded and stained following the method described for Welwitschia tissue by EVERT, BORNMAN, BUTLER and GILLILAND (1973). In the case of chlorophyll content the sampling procedure adopted was that of ANSTIS and NORTHCOTE (1974) and the specific method that of ARNON (1949).
\
0,6
CONTROL
\
C>
..:J: C)
W
£'"
CHLOROPHYLL
J:
... a: ...
21
VI
...«
~ .<:
0,4
~
Z
35
~ ~
..J ..J
>-
0,2
J:
• BUTYL ESTER METHYL
Cl.
o
0
a:
0
:e
..J
J: U
MORPHACTIN, mgl-'
Fig. 1; Effects of the morphactins flurenol (butyl ester) and chlorflurenol (methyl ester) on growth and chlorophyll production in Welwitschia callus.
z. P/lanzenphysiol. Bd. 78. S. 266-270. 1976.
268
C. H.
BORNMAN
Fig. 2: Electron micrograph of chloroplast, endoplasmic reticulum (ER), dictyosome (D) and mitochondria (M) from Welwitschia callus cultured in presence of 10 mg 1-1 flurenol. Note beading and vesiculation of ER and dictyosome, and poor development of thylakoids.
z. Pjlanzenphysiol. Bd. 78. S. 266-270. 1976.
Some effects of morphactin on Welwitschia callus
269
Results Figure 1 shows the effects of different levels of flurenol (butyl ester) and chlorflurenol (methyl ester) on growth and chlorophyll synthesis. At mg t1 chlorophyll appears unaffected although growth already is reduced by ca 28 per cent; however, 10 mg 1-1 morphactin causes a sharp reduction of 80 per cent in chlorophyll content in contrast to a decrease in growth of only 35 per cent. The relative effects of the two morphactins appear similar. At the non-physiological concentration of 100 mg 1-1 the callus was necrotic and in a state of partial collapse. Figure 2 is a composite electron micrograph of organelles from cells of callus tissue 20 days after subculturing on a medium containing 10 mg 1-1 flurenol. Invariably, the chloroplasts are very swollen and contain large deposits of starch. There are few grana per chloroplast; the lamellar organization becomes disrupted and the thylakoids, reduced in number, are distorted and swollen. The endoplasmic reticulum breaks up and, even more striking, a progressive vesiculation of the dictyosome cisternae can be observed. Mitochondria, apart from an apparent dilation and some vesiculation of the cristae, appear to be the least affected of the organelles. Not shown but observed were the following features: recessive, enlarged, weakly-granulated and electron transparent nuclei, dearth of membrane-bound coils of ribosomes as compared with the control, numerous multi vesicular bodies and vacuoles containing many vesicles.
Discussion The concentrations at which morphactins affect growth and chlorophyll synthesis in Welwitschia callus are considerably higher than those influencing growth and development of higher plants in general (ZIEGLER, 1970). It would appear that higher concentrations of morphactin are required to inhibit chlorophyll synthesis (1 mg 1-1 ) than growth (0.1 mg 1-1 ), but once thylakoid distortion has occurred the effect is final, whereas in the case of growth it appears to be progressively coupled to concentration. The increasing vesiculation of the dietyo somes suggests that the effect of morphactin on growth simply might be the inhibition of synthetic processes such as cell plate and cell wall formation, and the process of organelle turnover, III which membranous organelles are involved. Obviously, these effects would be compounded by the inhibition of chlorophyll formation. Elongated mitochondria (5 !lm and longer) were observed frequently in all treatments and their presence cannot be ascribed to morphactin per se, in contrast to ZIEGLER'S (1970) observation in pea root tips. The nature of the tissue, which consisted largely of spherical cells, precluded establishing with certainty whether morphactins act by changing the orientation of the spindle axis in dividing cells (ZIEGLER, 1970). Except for the presence of tracheary elements no tissue or organ differentiation was apparent.
z. Pflanzenphysiol. Bd. 78. S. 266-270.1976.
270
C. H. BORNMAN
Acknowledgements The financial assistance of the S. A. Council for Industrial and Scientific Research gratefully acknowledged. Celamerck, Ingelheim/Rhein, kindly supplied the morphactins.
IS
References ANSTIS, P. J. P., and D. H. NORTHCOTE: Chlorophyll accumulation by callus tissues of Glycine max. Planta (Berl.) 116, 105-108 (1974). ARNON, D. 1.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physio!. (Lancaster) 24, 1-15 (1949). BORNMAN, C. H.: Welwitschia mirabilis callus studies 1. Initiation and growth. Z. Pflanzenphysio!. 78, 182-186 (1976). BORNMAN, C. H., R. BURCHARDT, and M. G. GILLILAND: Effects of morphactins on Welwitschia callus. Proc. Electron. Microsc. Soc. South. Afr. 4, 43-44 (1974). BORNMAN, C. H., and N. C. FANSHAWE: Welwitschia mirabilis callus studies II. Some effects of sucrose. Z ..Pflanzenphysio!. 78, 217-221 (1976). EVERT, R. F., C. H. BORNMAN, V. BUTLER, and M. G. GILLILAND: Structure and Development of the Sieve-Cell Protoplast in Leaf Veins of Welwitschia. Protoplasm a 76, 1-21 (1973). FOSTER, A. S., and E. M. GIFFORD Jr.: Comparative Morphology of Vascular Plants. Freeman and Company, San Francisco, 1959. KATHJU, S., and M. N. TEWARI: Physiological and enzymological effects of morphactin. Abst: Int. Seminar on Physio!. of Differentiation, 17 (1971) . KRISHNAMOORTHY, H. N.: Effect of morphactin on growth, flowering and sex expression of Luffa acutangula. Abst: Int. Seminar on Physio!. of Differentiation, 26 (1971). SCHENK, R. U., and A. C. HILDEBRANDT: Medium and techniques for induction of and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199-204 (1972). SCHNEIDER, G.: Morphactins and plant growth regulation. In: H. KALDEWEY and Y. VARDAR (Eds.), Hormonal Regulation in Plant Growth and Development. pp 317-331. Verlag Chemie, Weinheim, 1972. ZIEGLER, H.: Morphactins. Endeavour XXIX, 112-116 (1970).
Prof. Dr. CHRIS H. BORNMAN, Department of Botany, University of Pretoria. Pretoria, 0002 South Africa.
Z. PJlanzenphysiol. Bd. 78. S. 266-270. 1976.
Welwitschia mirabilis Callus Studies III. Some Effects of Morphactin CHRIS H . BORNMAN With 2 figures Received December 2, 1975 . Accepted January 9, 1976
Summary Mor.phactins, fluorene-carbox ylic acid derivatives reported highly active in a number of plant morphogenic responses, retard growth and severely inhibit chlorophyll synthesis in Welwitschia mirabilis callus cultures. No organogenic response was elicited. Cell membranes appeared particularly susceptible to morphactin. ER and dictyosomes became progressively vesiculated; in the chloroplasts thylakoids were swollen and their lamellae distorted and few grana were present.
Key words: Welwitschia, Callus, Morphactin.
Introduction Callus can be proliferated quite readily from the hypocotyl-root axis of germinating Welwitschia mirabilis embryos (BORNMAN, 1976) and, at the expense of some growth, can synthesize relatively large amounts of chlorophyll when its normal growth medium (SCHENK and HILDEBRANDT, 1972) is supplemented with 4 to 8 per cent sucrose (BORNMAN and FANSHAWE, 1976). The plant itself has no apical meristem and thus far attempts at inducing adventitious bud formation in callus, have not met with success. As Welwitschia occupies an unique if somewhat controversial position in the plant kingdom (FOSTER and GIFFORD, 1959), it is of interest to learn more about its morphogenic behaviour. In this regard tissue culture seems an appropriate avenue of approach and attention therefore was focused on other substances that might induce organogenesis, as for example the morphactins, socalled morphologically active substances. According to SCHNEIDER (1972) these fluorene-carboxylic acid derivatives are highly active in a number of plant morphogenic responses such as callus formation, shoot and root growth, and flower initiation and fruit development. KATHJU and TEWARI (1971) reported that 25 mg 1-1 morphactin inhibited a-amylase activity in Cyamopsis cotyledons but enhanced that of phosphorylase. BORNMAN et a1. (1974) Z. PJlanzenphysiol. Bd. 78. S. 266-270. 1976.
Some effects of morphactin on Welwitschia callus
267
found that a concentration of 10 mg 1- 1 inhibited phosphatase and phosphodiesterase activity in Welwitschia callus. There are also some conflicting accounts regarding the action of morphactins - for example, ZIEGLER (1970) reported a shortening of pea stem internodes whereas KRISHNAMOORTHY (1971) found the opposite effect in Luffa - and their physiological role remains obscure.
Material and Methods Callus derived from the embryo of Welwitschia mirabilis was established as described earlier (BORNMAN, 1976) on SCHENK'S and HILDEBRANDT'S (1972) basal medium su pplemented with 0.3 mg 1- 1 napthaleneacetic acid, 0.1 mg 1-1 kinetin and 4 per cent sucrose. Two morphactins, f1urenol (n-butyl 9-hydroxyfluorene-(9)-carboxylate) and chlorflurenol (methyl 2-chloro-9-hydroxyfluorene-(9)-carboxylate) were incorporated in the culture medicum at concentration levels of 0.1, 1, 10, and 100 mg 1-1 • The morphactins first were dissolved in a small volume of acetone and stabilized with a droplet of Tween 20 before diluting to volume with distilled water. Fresh weights and chlorophyll content were determined 20 days after subculturing, while tissues were sampled every 10 days for electron microscopical studies. The latter were fixed, embedded and stained following the method described for Welwitschia tissue by EVERT, BORNMAN, BUTLER and GILLILAND (1973). In the case of chlorophyll content the sampling procedure adopted was that of ANSTIS and NORTHCOTE (1974) and the specific method that of ARNON (1949).
\
0,6
CONTROL
\
C>
..:J: C)
W
£'"
CHLOROPHYLL
J:
... a: ...
21
VI
...«
~ .<:
0,4
~
Z
35
~ ~
..J ..J
>-
0,2
J:
• BUTYL ESTER METHYL
Cl.
o
0
a:
0
:e
..J
J: U
MORPHACTIN, mgl-'
Fig. 1; Effects of the morphactins flurenol (butyl ester) and chlorflurenol (methyl ester) on growth and chlorophyll production in Welwitschia callus.
z. P/lanzenphysiol. Bd. 78. S. 266-270. 1976.
268
C. H.
BORNMAN
Fig. 2: Electron micrograph of chloroplast, endoplasmic reticulum (ER), dictyosome (D) and mitochondria (M) from Welwitschia callus cultured in presence of 10 mg 1-1 flurenol. Note beading and vesiculation of ER and dictyosome, and poor development of thylakoids.
z. Pjlanzenphysiol. Bd. 78. S. 266-270. 1976.
Some effects of morphactin on Welwitschia callus
269
Results Figure 1 shows the effects of different levels of flurenol (butyl ester) and chlorflurenol (methyl ester) on growth and chlorophyll synthesis. At mg t1 chlorophyll appears unaffected although growth already is reduced by ca 28 per cent; however, 10 mg 1-1 morphactin causes a sharp reduction of 80 per cent in chlorophyll content in contrast to a decrease in growth of only 35 per cent. The relative effects of the two morphactins appear similar. At the non-physiological concentration of 100 mg 1-1 the callus was necrotic and in a state of partial collapse. Figure 2 is a composite electron micrograph of organelles from cells of callus tissue 20 days after subculturing on a medium containing 10 mg 1-1 flurenol. Invariably, the chloroplasts are very swollen and contain large deposits of starch. There are few grana per chloroplast; the lamellar organization becomes disrupted and the thylakoids, reduced in number, are distorted and swollen. The endoplasmic reticulum breaks up and, even more striking, a progressive vesiculation of the dictyosome cisternae can be observed. Mitochondria, apart from an apparent dilation and some vesiculation of the cristae, appear to be the least affected of the organelles. Not shown but observed were the following features: recessive, enlarged, weakly-granulated and electron transparent nuclei, dearth of membrane-bound coils of ribosomes as compared with the control, numerous multi vesicular bodies and vacuoles containing many vesicles.
Discussion The concentrations at which morphactins affect growth and chlorophyll synthesis in Welwitschia callus are considerably higher than those influencing growth and development of higher plants in general (ZIEGLER, 1970). It would appear that higher concentrations of morphactin are required to inhibit chlorophyll synthesis (1 mg 1-1 ) than growth (0.1 mg 1-1 ), but once thylakoid distortion has occurred the effect is final, whereas in the case of growth it appears to be progressively coupled to concentration. The increasing vesiculation of the dietyo somes suggests that the effect of morphactin on growth simply might be the inhibition of synthetic processes such as cell plate and cell wall formation, and the process of organelle turnover, III which membranous organelles are involved. Obviously, these effects would be compounded by the inhibition of chlorophyll formation. Elongated mitochondria (5 !lm and longer) were observed frequently in all treatments and their presence cannot be ascribed to morphactin per se, in contrast to ZIEGLER'S (1970) observation in pea root tips. The nature of the tissue, which consisted largely of spherical cells, precluded establishing with certainty whether morphactins act by changing the orientation of the spindle axis in dividing cells (ZIEGLER, 1970). Except for the presence of tracheary elements no tissue or organ differentiation was apparent.
z. Pflanzenphysiol. Bd. 78. S. 266-270.1976.
270
C. H. BORNMAN
Acknowledgements The financial assistance of the S. A. Council for Industrial and Scientific Research gratefully acknowledged. Celamerck, Ingelheim/Rhein, kindly supplied the morphactins.
IS
References ANSTIS, P. J. P., and D. H. NORTHCOTE: Chlorophyll accumulation by callus tissues of Glycine max. Planta (Berl.) 116, 105-108 (1974). ARNON, D. 1.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physio!. (Lancaster) 24, 1-15 (1949). BORNMAN, C. H.: Welwitschia mirabilis callus studies 1. Initiation and growth. Z. Pflanzenphysio!. 78, 182-186 (1976). BORNMAN, C. H., R. BURCHARDT, and M. G. GILLILAND: Effects of morphactins on Welwitschia callus. Proc. Electron. Microsc. Soc. South. Afr. 4, 43-44 (1974). BORNMAN, C. H., and N. C. FANSHAWE: Welwitschia mirabilis callus studies II. Some effects of sucrose. Z ..Pflanzenphysio!. 78, 217-221 (1976). EVERT, R. F., C. H. BORNMAN, V. BUTLER, and M. G. GILLILAND: Structure and Development of the Sieve-Cell Protoplast in Leaf Veins of Welwitschia. Protoplasm a 76, 1-21 (1973). FOSTER, A. S., and E. M. GIFFORD Jr.: Comparative Morphology of Vascular Plants. Freeman and Company, San Francisco, 1959. KATHJU, S., and M. N. TEWARI: Physiological and enzymological effects of morphactin. Abst: Int. Seminar on Physio!. of Differentiation, 17 (1971) . KRISHNAMOORTHY, H. N.: Effect of morphactin on growth, flowering and sex expression of Luffa acutangula. Abst: Int. Seminar on Physio!. of Differentiation, 26 (1971). SCHENK, R. U., and A. C. HILDEBRANDT: Medium and techniques for induction of and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199-204 (1972). SCHNEIDER, G.: Morphactins and plant growth regulation. In: H. KALDEWEY and Y. VARDAR (Eds.), Hormonal Regulation in Plant Growth and Development. pp 317-331. Verlag Chemie, Weinheim, 1972. ZIEGLER, H.: Morphactins. Endeavour XXIX, 112-116 (1970).
Prof. Dr. CHRIS H. BORNMAN, Department of Botany, University of Pretoria. Pretoria, 0002 South Africa.
Z. PJlanzenphysiol. Bd. 78. S. 266-270. 1976.