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Biochemical studies on initiation of callus in Solanum surattense
Short Communication
Biochemical studies on initiation of callus in Solanum surattense P. L. SWARNKAR,
s. P. BOHRA and N. CHANDRA
Department of Botany, University of Rajasthan, Jaipur 302004, India Received February 14, 1986 . Accepted May 18, 1986
Summary Activities of peroxidase, acid phosphatase and a-amylase and total phenols increased just before appearance of callus. Starch and reducing sugars accumulated soon after inoculation of explants to the callus forming medium and decreased subsequently. Five new isoperoxidases appeared during induction period out of which two disappeared before initiation of callus. One new isozyme of acid phosphatase also appeared prior to the appearance of callus. Key words: Solanum surattense, Biochemical changes, Callus initiation, isozymes.
Introduction The work on physiology and biochemistry of cells in cultures has largely been confined to optimising the conditions of culture and production of secondary metabolites. Very little is known about the biochemical events leading to initiation of callus. Most of the studies on this aspect were centered on respiration (Steward et aI., 1958), protein synthesis (Steward et aI., 1952) and isozymes of peroxidase (Bajaj et aI., 1973) in callus and plant parts from which the callus was derived. In the present communication, we report the changes in activities of peroxidase, acid phosphatase and examylase, isozyme pattern of peroxidase and acid phosphatase and levels of starch, reducing sugars and phenols during initiation of callus from stem segments of Solanum surattense.
Materials and Methods Plant Material
Stem internodes from in vitro grown plants of Solanum surattense Burm. f. (Syn. S. xanthocarpum Schrad and Wendl.) were transferred to Murashige and Skoog (1962) medium supplemented with 0.05 mg 1-1 of kinetin and 3.0 mg 1-1 of 2,4-D. Swelling of explants was observed on day 4 and callus appeared on day 8. Biochemical analyses were made on every alternate day.
Abbreviations: 2,4-D: 2,4-dichlorophenoxy acetic acid, IAA: Indole-3-acetic acid.
J Plant Physiol. Vol. 126. pp. 293-296 (1986)
294
P. L. SWARNKAR, S. P. BOHRA and N. CHANDRA
Enzyme assay
Plant material was homogenized in 0.1 M phosphate buffer, pH 7.0 in prechilled mortar and pestle. The homogenate was centrifuged at 5000 x g for 15 min and the supernatant was used for enzyme assay, protein estimation and electrophoresis. Peroxidase activity was measured by the guaiacol-HzO z method (Racusen and Foote, 1965). An arbitrary unit of enzyme activity was chosen as change in absorbance 0.001 sec - I. p-Nitrophenylphosphate was used as substrate for assaying the acid phosphatase activity (Zink and Veliky, 1979) and the activity was expressed in terms of mM p-nitrophenol released minute -I mg- I protein. The assay method of Bernfeld (1955) was used for estimation of a-amylase activity and the activity was expressed in terms of mg maltose liberated h -I mg - 1 protein. Electrophoresis
Isozymes were separated by disc electrophoresis on 6.5 % polyacrylamide gels following the method of Davis (1964). Gels for isozymes of peroxidase were stained by the method of Siegel and Galston (1967) and of acid phosphatase by Sako and Stahmann (1972). Metabolic constituents
Soluble proteins were estimated by the method of Lowry et a!. (1951) using bovine serum albumin as standard. Nelson's method (1944) was used for estimating reducing sugars. Phenols were assayed with Folin phenol reagent following the method of Bray and Thorpe (1954) and starch by the method of McCready et al. (1950). Results are averages (± standard error) of two experiments each with two replicates.
Results and Discussion Accumulation of starch and sugars in callus-forming explants on day 2 of inoculation and their utilization on subsequent days (Table 1) suggest that these carbohydrates serve as a source of carbon and energy for initiation of callus. The enhanced activities of a-amylase and acid phosphatase just before appearance of callus might contribute to the utilization of carbohydrates directly or indirectly through making inorganic phosphate available respectively (Murray and Collier, 1977). Increased activity of peroxidase coupled with high level of phenols on day 6 gives an indirect evidence that low level of auxin is required for cell proliferation since peroxidase and phenols both are known to be involved in oxidation of IAA (Lee and Skoog, 1965; Lee, 1980). Table 1: Changes in levels of various enzymes (specific activities) and metabolic constituents (mg g-I fresh weight) during initiation of callus in cultured stem segments of Solanum surattense. S!. No.
Parameters
1. 2. 3. 4. 5. 6.
Peroxidase Acid Phosphatase a-amylase Starch Reducing sugars Phenols
J Plant Physiol.
Culture period in days 0
2
4
6
8
20.2 ±2.16
1904 ±1.62 OA8±0.04
20.0 ±1.87 0.39±0.07 4.0 ±0.16 2.1 ±0.11 504 ±0.22 0.50±0.03
45.2 ±2.5 0.87±0.06 9.6 ±0.27 2.3 ±0.10 5.6 ±0.28 0.6 ±0.04
17.5 ± 1.96 0.38±0.06 3.5 ±0.21 1.8 ±0.08 4.1 ±0.22 0.36±0.03
OA9±0.04
4.8 ±0.32 1.5 ±0.08 3.8 ±0.18 OAO±O.03
6.1 ±0.24 3.5 ±0.20 8.1 ±OA2 OA4±0.05
Vol. 126. pp. 293-296 (1986)
Biochemical studies on callus initiation
295
82l: 3-
4-
56-
7-
A
8. 9-
10-
C±)
0
2
o
2
4
6
8
4
6
8
e 2 3
B
4
Days
Fig. 1: Changes in isozyme pattern of peroxidase and acid phosphatase during callus initiation in cultured stem segments of Solanum surattense. (A) Isozymes of peroxidase (B) Isozymes of acid phosphatase.
Electrophoretic studies revealed that isoperoxidases 2 and 6 appeared on day 2 and isoperoxidase 9 appeared on day 4 (Fig. 1). In addition, two transient isoperoxidases (3 and 10) were also observed. One new isozyme (2) of acid phosphatase appeared on day 4 to persist till day 8. These findings suggest that isozymes could be used as biochemical markers of the process of dedifferentiation. Acknowledgements One of us (PLS) is thankful to UGC, India for award of teacher research fellowship.
References BAJAJ, Y. P. 5., M. Bopp, and S. BAJAJ: Phytomorphology 23,43-52 (1973). BERNFELD, P.: Methods in Enzymol. 1, 149-158 (1955). BRAY, H. G. and W. V. THORPE: In: GLICK, D. (ed.): Methods of biochemical analysis 1, 27-57, Interscience Publishers Inc., New York (1954).
J Plant Physiol. Vol. 126. pp. 293-296 (1986)
296
P. L. SWARNKAR, S. P. BOHRA and N. CHANDRA
DAVIS, B. J.: Ann. N. Y. Acad. Sci. 121, 404-427 (1964). LEE, T. T.: Physio!' Plant. 50, 107 -117 (1980). LEE, T. T. and F. SKOOG: Physio!. Plant. 18, 577-585 (1965). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: J. Bio!. Chern. 193,265-275 (1951). McCREADY, R. M., J. GUGGOLZ, V. SILVIERA, and H. S. OWENS: Ana!. Chern. 22, 1156-1158 (1950). MURASHIGE, T. and F. SKOOG: Physio!. Plant. 15,473-497 (1962). MURRAY, D. R. and M. D. COLLIER: Aust. J. Plant Physio!. 4,843-848 (1977). NELSON, N.: J. Bio!. Chern. 153, 375-380 (1944). RACUSEN, D. and M. FOOTE: Can. J. Bot. 43, 817-824 (1965). SAKO, N. and M. A. STAHMANN: Physio!. Plant Path. 2, 217 -226 (1972). SIEGEL, B. Z. and A. W. GALSTON: Plant Physio!. 42, 221-226 (1967). STEWARD, F. c., R. G. S. BIDWELL, and E. W. YEMM: J. Exp. Bot. 9,11-51 (1958). STEWARD, F. c., S. M. CAPLIN, and F. K. MILLAR: Ann. Bot. 16,58-77 (1952). ZINK, M. W. and L A. VELIKY: Can. J. Bot. 57, 739-753 (1979).
J Plant Physiol. Vol.
126. pp. 293-296 (1986)
Biochemical studies on initiation of callus in Solanum surattense P. L. SWARNKAR,
s. P. BOHRA and N. CHANDRA
Department of Botany, University of Rajasthan, Jaipur 302004, India Received February 14, 1986 . Accepted May 18, 1986
Summary Activities of peroxidase, acid phosphatase and a-amylase and total phenols increased just before appearance of callus. Starch and reducing sugars accumulated soon after inoculation of explants to the callus forming medium and decreased subsequently. Five new isoperoxidases appeared during induction period out of which two disappeared before initiation of callus. One new isozyme of acid phosphatase also appeared prior to the appearance of callus. Key words: Solanum surattense, Biochemical changes, Callus initiation, isozymes.
Introduction The work on physiology and biochemistry of cells in cultures has largely been confined to optimising the conditions of culture and production of secondary metabolites. Very little is known about the biochemical events leading to initiation of callus. Most of the studies on this aspect were centered on respiration (Steward et aI., 1958), protein synthesis (Steward et aI., 1952) and isozymes of peroxidase (Bajaj et aI., 1973) in callus and plant parts from which the callus was derived. In the present communication, we report the changes in activities of peroxidase, acid phosphatase and examylase, isozyme pattern of peroxidase and acid phosphatase and levels of starch, reducing sugars and phenols during initiation of callus from stem segments of Solanum surattense.
Materials and Methods Plant Material
Stem internodes from in vitro grown plants of Solanum surattense Burm. f. (Syn. S. xanthocarpum Schrad and Wendl.) were transferred to Murashige and Skoog (1962) medium supplemented with 0.05 mg 1-1 of kinetin and 3.0 mg 1-1 of 2,4-D. Swelling of explants was observed on day 4 and callus appeared on day 8. Biochemical analyses were made on every alternate day.
Abbreviations: 2,4-D: 2,4-dichlorophenoxy acetic acid, IAA: Indole-3-acetic acid.
J Plant Physiol. Vol. 126. pp. 293-296 (1986)
294
P. L. SWARNKAR, S. P. BOHRA and N. CHANDRA
Enzyme assay
Plant material was homogenized in 0.1 M phosphate buffer, pH 7.0 in prechilled mortar and pestle. The homogenate was centrifuged at 5000 x g for 15 min and the supernatant was used for enzyme assay, protein estimation and electrophoresis. Peroxidase activity was measured by the guaiacol-HzO z method (Racusen and Foote, 1965). An arbitrary unit of enzyme activity was chosen as change in absorbance 0.001 sec - I. p-Nitrophenylphosphate was used as substrate for assaying the acid phosphatase activity (Zink and Veliky, 1979) and the activity was expressed in terms of mM p-nitrophenol released minute -I mg- I protein. The assay method of Bernfeld (1955) was used for estimation of a-amylase activity and the activity was expressed in terms of mg maltose liberated h -I mg - 1 protein. Electrophoresis
Isozymes were separated by disc electrophoresis on 6.5 % polyacrylamide gels following the method of Davis (1964). Gels for isozymes of peroxidase were stained by the method of Siegel and Galston (1967) and of acid phosphatase by Sako and Stahmann (1972). Metabolic constituents
Soluble proteins were estimated by the method of Lowry et a!. (1951) using bovine serum albumin as standard. Nelson's method (1944) was used for estimating reducing sugars. Phenols were assayed with Folin phenol reagent following the method of Bray and Thorpe (1954) and starch by the method of McCready et al. (1950). Results are averages (± standard error) of two experiments each with two replicates.
Results and Discussion Accumulation of starch and sugars in callus-forming explants on day 2 of inoculation and their utilization on subsequent days (Table 1) suggest that these carbohydrates serve as a source of carbon and energy for initiation of callus. The enhanced activities of a-amylase and acid phosphatase just before appearance of callus might contribute to the utilization of carbohydrates directly or indirectly through making inorganic phosphate available respectively (Murray and Collier, 1977). Increased activity of peroxidase coupled with high level of phenols on day 6 gives an indirect evidence that low level of auxin is required for cell proliferation since peroxidase and phenols both are known to be involved in oxidation of IAA (Lee and Skoog, 1965; Lee, 1980). Table 1: Changes in levels of various enzymes (specific activities) and metabolic constituents (mg g-I fresh weight) during initiation of callus in cultured stem segments of Solanum surattense. S!. No.
Parameters
1. 2. 3. 4. 5. 6.
Peroxidase Acid Phosphatase a-amylase Starch Reducing sugars Phenols
J Plant Physiol.
Culture period in days 0
2
4
6
8
20.2 ±2.16
1904 ±1.62 OA8±0.04
20.0 ±1.87 0.39±0.07 4.0 ±0.16 2.1 ±0.11 504 ±0.22 0.50±0.03
45.2 ±2.5 0.87±0.06 9.6 ±0.27 2.3 ±0.10 5.6 ±0.28 0.6 ±0.04
17.5 ± 1.96 0.38±0.06 3.5 ±0.21 1.8 ±0.08 4.1 ±0.22 0.36±0.03
OA9±0.04
4.8 ±0.32 1.5 ±0.08 3.8 ±0.18 OAO±O.03
6.1 ±0.24 3.5 ±0.20 8.1 ±OA2 OA4±0.05
Vol. 126. pp. 293-296 (1986)
Biochemical studies on callus initiation
295
82l: 3-
4-
56-
7-
A
8. 9-
10-
C±)
0
2
o
2
4
6
8
4
6
8
e 2 3
B
4
Days
Fig. 1: Changes in isozyme pattern of peroxidase and acid phosphatase during callus initiation in cultured stem segments of Solanum surattense. (A) Isozymes of peroxidase (B) Isozymes of acid phosphatase.
Electrophoretic studies revealed that isoperoxidases 2 and 6 appeared on day 2 and isoperoxidase 9 appeared on day 4 (Fig. 1). In addition, two transient isoperoxidases (3 and 10) were also observed. One new isozyme (2) of acid phosphatase appeared on day 4 to persist till day 8. These findings suggest that isozymes could be used as biochemical markers of the process of dedifferentiation. Acknowledgements One of us (PLS) is thankful to UGC, India for award of teacher research fellowship.
References BAJAJ, Y. P. 5., M. Bopp, and S. BAJAJ: Phytomorphology 23,43-52 (1973). BERNFELD, P.: Methods in Enzymol. 1, 149-158 (1955). BRAY, H. G. and W. V. THORPE: In: GLICK, D. (ed.): Methods of biochemical analysis 1, 27-57, Interscience Publishers Inc., New York (1954).
J Plant Physiol. Vol. 126. pp. 293-296 (1986)
296
P. L. SWARNKAR, S. P. BOHRA and N. CHANDRA
DAVIS, B. J.: Ann. N. Y. Acad. Sci. 121, 404-427 (1964). LEE, T. T.: Physio!' Plant. 50, 107 -117 (1980). LEE, T. T. and F. SKOOG: Physio!. Plant. 18, 577-585 (1965). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: J. Bio!. Chern. 193,265-275 (1951). McCREADY, R. M., J. GUGGOLZ, V. SILVIERA, and H. S. OWENS: Ana!. Chern. 22, 1156-1158 (1950). MURASHIGE, T. and F. SKOOG: Physio!. Plant. 15,473-497 (1962). MURRAY, D. R. and M. D. COLLIER: Aust. J. Plant Physio!. 4,843-848 (1977). NELSON, N.: J. Bio!. Chern. 153, 375-380 (1944). RACUSEN, D. and M. FOOTE: Can. J. Bot. 43, 817-824 (1965). SAKO, N. and M. A. STAHMANN: Physio!. Plant Path. 2, 217 -226 (1972). SIEGEL, B. Z. and A. W. GALSTON: Plant Physio!. 42, 221-226 (1967). STEWARD, F. c., R. G. S. BIDWELL, and E. W. YEMM: J. Exp. Bot. 9,11-51 (1958). STEWARD, F. c., S. M. CAPLIN, and F. K. MILLAR: Ann. Bot. 16,58-77 (1952). ZINK, M. W. and L A. VELIKY: Can. J. Bot. 57, 739-753 (1979).
J Plant Physiol. Vol.
126. pp. 293-296 (1986)