EKP would reflect changes in fine structure of the plasmalemma (Haydon, 1961;. Senn and .... Sinnott. Î. W. (1960) Plant morphogenesis (New York: McGraw Hill).
J. Biosci., Vol. 12, Number 4, December 1987, pp. 393–397. © Printed in India.
Relation between electrokinetic potentials and growth in callus cultures of Trigonella foenum-graecum S. S. ΑΡΤΕ, C. K. KOKATE and D. RAMBHAU University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506 009, India MS received 24 March 1986: revised 27 July 1987 Abstract. Callus cultures of Trigonella foenum-graecum were initiated from radicle or cotyledon portions of seedlings and young leaves and maintained on modified 1-B5 medium. The callus mass was disaggregated by mechanical agitation and the discrete cells thus obtained were used to measure their electrokinetic potential. Studies pertaining to the effects of ageing on electrokinetic potential and growth index revealed a relationship between these two parameters. Thus, the rate of change of electrokinotie potential with age could be employed as a parameter to study the growth kinetics of cells in callus cultures. Keywords. Electrokinetic potential; Trigonella foenum graecum callus cultures; growth index.
Introduction Cell growth in tissue cultures is accompanied by changes in the structure of the cell walls (Amino and Komamine, 1982). Since differentiation is accompanied by changes in the composition of the cell wall, the wall serves as a key to study cell differentiation (Sinnott, 1960). Any attempt to study the changes in cell wall may help in understanding the behaviour of cells during growth. Recent studies on the structure of plant cell walls suggest that changes in the composition of primary cell wall occur in association with cell growth (Asamizu et al., 1977; Nishi and Asamizu, 1982). However such studies involving chemical analysis appear to be of limited value since they employ drastic treatments to isolate cell wall fractions. Moreover, isolation results in disruption of the natural integrity of the cell wall. Thus studies of the integral cell wall employing non-invasive techniques appears to be necessary and such studies should be extended to interpret major cellular activity. The electrokinetic potential (EKP) is a sensitive measure of cell surface charge and changes in EKP would reflect changes in fine structure of the plasmalemma (Haydon, 1961; Senn and Pilet, 1981), the measurement made without disrupting the cells. In this paper EKP has been employed as a parameter to study the growth kinetics of cells in callus cultures obtained from radicle and cotyledon portions of seedlings and from young leaves of Trigonella foenum-graecum. Materials and methods Initiation and maintenance of callus cultures The callus cultures were initiated from the radicle or cotyledon portions of seedlings Abbreviations used: EKP, Electrokinetic potential; GI, growth index: Ml, mitotic index; SCP, surface charge parameter; GP, growth parameter.
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and from the young leaves of Τ. foenum-graecum on 1-B5 medium (Gamborg and Wetter, 1975), modified by adding sucrose (20 g/1), yeast extract (2 g/1) and casein hydrolysate (3 g/1). The seeds were surface sterilized by dipping in ethanol (70% v/v) for 30 s followed by immersion in bromine water (1% v/v) for 2 to 3 min. Following surface sterilization, the seeds were washed 4 times with sterile distilled water and transferred aseptically to petri dishes containing moistened filter papers. The Petri dishes were incubated in the dark at 27 ± 1°C for 48 h. Following germination the radicle or cotyledon portions were separated with a sterilized knife and aseptically transferred on to modified 1-B5 medium in culture flasks. Young leaves were washed thoroughly with distilled water and surface-sterilized by immersing in ethanol (70% v/v) for 30 s followed by treatment with 10% (w/v) calcium hypochlorite solution for 10 min. Finally the leaves were washed repeatedly with sterile distilled water before transferring into flasks containing modified 1-B5 medium. The flasks were in000000000cubated in the dark at 27 ± 1°C for 4 weeks. The age of 4-week old callus was considered as ‘zero week’. The callus cultures were maintained by subculturing onto the same medium after interval of 3 weeks for a total period of 9 weeks. At the end of each interval, growth index (GI) was determined on dry weight basis. Mitotic index (MI) was also determined at each time. Preparation of cell suspension and measurement of EKP At the end of each interval a portion of callus mass was shaken with about 20 ml of distilled water on a vibratory shaker for 15 min. The suspension was filtered through a 64 mesh sieve. The cells in the filtrate were separated by centrifugation at a low speed and washed 5 times with distilled water to remove all traces of medium. A packed cell volume of 0·5 ml was always made up to 10 ml with distilled water for the EKP measurements. The EKP of cells was measured on Zetameter of Zetameter Inc., USA. Fifty cells were tracked and the average EKP was calculated. The measurements were made at the time of subculturing until 12 weeks of age. Results and discussion The cells of zero week old callus from radicle, cotyledon and leaf shown average EKP of –33·5, –34·52 and – 31·72mV, respectively. Though the cells were from calli initiated from different tissues of the plant, no significant difference could be noticed in their average EKP values. During ageing, the EKP of cells from all the cultures showed a definite decline. Thus, for cells of radicle cultures, the initial EKP declined to –32·18 , –31·62 and –29·89 mV at the end of 3, 6 and 9 weeks, respectively. The cells of cotyledon and leaf calli had EKP of –28·15 and –30·07 mV, respectively at 9 weeks. GI also showed a pattern of changes similar to that of EKP (figure 1). There appears to be a broad qualitative correlation between Gl and EKP. The initial higher GI indicates high rate of cellular proliferation. This is also clear from the comparatively higher MI’s at zero and 3 weeks. The corresponding higher average EKP values probably indicate the same. Higher EKP of rapidly proliferating cells has been reported for animal cells (Ben-Or and Pethica, 1960; Eisenberg et al.,
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Figure 1. Changes in average EKP, GI and MI of callus cultures derived from T-.foenumgraecum during ageing.
1962; Ambrose et al., 1956), bacteria (Sato et al., 1981) and Chlorella cells (Lukiewicz and Korohoda, 1965). Pilet et al. (1984) observed higher EKP values for protoplasts isolated from rapidly elongating cells of the upper part of gravireacting maize root compared to those isolated from the slow growing lower portion. Our observations on plant cells in tissue culture are in agreement with these reports. It is important to note that none of these reports suggest any quantitative relation between growth and EKP. When EKP was plotted against GI no statistically significant relation could be established. The possibility of a quantitative relation between EKP and cell proliferation was explored by introducing rate of change of EKP and of growth as parameters. Rate of change of EKP was expressed as (EKPt/EKPto)/t, where EKPt is EKP at time t, EKPto is EKP of cells of zero-weekold culture and t is time in weeks. Similarly, the rate of change of growth with age was expressed as GI/t. These parameters were designated as surface charge parameter (SCP) and growth parameter (GP), respectively. When SCP was plotted against GP a straight line was obtained for all the 3 cultures (figure 2). On regression analysis, a statistically significant correlation between these two parameters was revealed (for cotyledon cultures r= 0·9999, for radicle cultures r= 0·9995 and for leaf cultures r= 0·9974 at P< 0·01).
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Figure 2. Relation between SCP and GP values of callus cultures of T, foenum-graecum during ageing.
These results indicate that the changes in average EKP values of cells correspond to the rate of proliferation in tissue cultures. As EKP is a cell surface property, variation in EKP suggests alteration in cell surface composition. The occurrence of changes in surface composition has been reported for cells of suspension cultures of carrot and for some other cultures (Nishi and Asamizu, 1982; Amino and Komamine, 1982). Senn and Pilet (1981) reported that such changes in composition could be reflected in surface charge density. Accordingly they believe that surface charge of cells strongly depend on their cytological origin as well as on their physiological and inherent growth properties. Thus, EKP could be employed as a parameter with which changes in surface composition of cells could be followed without disturbing their integrity. Further, by extending the studies using specific chemical treatment made on animal cells (Cook et αl., 1961; Bangham and Pethica, 1961) to plant cells, it would be possible to identify the cell surface groups contributing to surface charge. This non-invasive technique is much simpler and less time consuming and seems promising for studies of plant cell surfaces. References Ambrose, E. J., James, A. M. and Lowick, J. Η. Β. (1956) Nature (London), 177, 576. Amino, S. and Komamine, A. (1982) in Plant tissue culture (ed. A. Fujiwara) (Tokyo: Jap. Assoc. Plant Tissue Culture) p. 65. Asamizu, Τ., Tanaka, Κ.. Takebe, I. and Nishi, A. (1977) Physiol. Plant., 40, 215. Bangham, A. D. and Pethica, Β. Α. (1961) Proc. R. Soc. Edinburgh, B28, 43. Ben-Or, S., Eisenberg, S. and Doljanski, F. (1961) Nature (London), 188, 1200. Cook, G. M. W., Heard, D. H. and Seaman, G. V. F. (1961) Nature (London), 191, 44. Eisenberg, S., Ben-Or, S. and Doljanski, F. (1962) Exp. Cell Res., 26, 451. Gamborg, O. L. and Wetter, L. R. (1975) Plant tissue culture methods (Saskatoon, Saskatchewan, Canada: National Research Council of Canada). Haydon, D. A. (1961) Biochim. Biophys, Acta, 50, 450.
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Lukiewicz, S. and Korohoda, W. (1965) in Cell Electrophoresis (ed. Ε. J. Ambrose) (London: J. and Α. Churchill Ltd.,) p. 171. Nishi, A. and Asamizu, T. (1982) in Plant tissue culture (ed. A. Fujiwara) (Tokyo: Jpn. Assoc. Plant Tissue Culture) p. 37. Pilet, P. E., Herve, R. and Senn, A. (1984) Planta, 162, 17. Sato, C, Kojima, K., Nishizawa, K. and Hirota, Y. (1981) Radiat. Res., 87, 646. Senn, A. and Pilet, P. E. (1981) Z Plazenphysiol., 102, 19. Sinnott. Ε. W. (1960) Plant morphogenesis (New York: McGraw Hill).
