Cucumber cotyledon expansion as a bioassay for cytokinins

Botany Department, Gujarat University, Ahmedabad, India

Cucumber Cotyledon Expansion as a Bioassay for Cytokinins A. NARAIN and MANMOHAN M. LALORAYA With 5 figures Received July 11, 19'73

Summary A specific bioassay based on Cucumis cotyledon expansion in dark has been developed for cytokinins, with desired sensitivity. No other growth regulator was found to cause cotyledon expansion in dark. Benzimidazole, however, promotes expansion growth in dark but only at higher concentrations and is not a naturally occurring substance.

Introduction One of the major effect of cotykinins in causing seed germination of light sensitive lettuce seeds is its capacity to cause the expansion of cotyledons (IKuMA and THIMANN, 1963). The studies on cytokinin induced expansion of' isolated cotyledons, which started from the work of BANERJI and LALORAYA (1965), has since attracted a number of other workers who have shown similar effect on other cotyledon tissues e. g. Xanthium (ESASHI and LEOPOLD, 1969), flax (SVESHNIKOVA and KHOKHLOVA, 1969), mustard (LovELL and MOORE, 1970), Cucumis sativus (NARAIN and LALORAYA, 1970), Ipomea pentaphylla (SANKHLA, 1970), Sunflower (GILAD et aI., 1970), Phaleolus vulgaris (GEPsTEIN and ILAN, 1970), fenugreek (RIJVEN and PARKASH, 1970) and radish (LETHAM, 1971). The cotyledon expansion is even used as a bioassay (ESASHI and LEOPOLD, 1969; LETHAM, 1971), but these suffer from the lack of specificity. Gibberellins at relatively higher concentrations have been shown to cause expansion of various types of cotyledons in light (BANERJI and LALORAYA, 1966; RIJVEN and PARKASH, 1970; ESASHI and LEOPOLD, 1969; LETHAM, 1971) and attempts have been made to differentiate the action of the two substances. In order that cotyledon system could be accepted as a bioassay, the specificity of its response to cytokinin is to be established. As a measurable response to cytokinins is observed within a short period in cotyledon system unlike the conventional tissue culture bioassays it stands out as a potential rapid bioassay system for cytokinins. The present paper reports a bioassay system for cytokinins with desired sensitivity and specificity based on the expansion of isolated cucumber cotyledons in dark, and a comparison of the response in cotyledons of some other Cucurbitaceae plants.

Z. Pjlanzenphysiol. Bd. 71. S. 313-322. 1974.

314

A. NARAIN and M. M. LALORAYA

Materials and Methods The following plants belonging to family Cucurbitaceae were used viz. a) cucumber

(Cucumis sativus L. var. Long green), b) squash (Cucurbita maxima L. var. Golden delicious), c) muskmelon (Cucumis melo L.), d) sponge gourd (Luffa aegyptiaca L.). The seeds were

surface sterilised using 0.01 % mercuric chloride and germinated in dark at 25 ± 1 0 C. The cotyledons were excised and floated in various test solutions according to the method as earlier reported (BANER]I and LALORAYA, 1965). Since the cotyledons contain reserve food materials, no external nutrients were provided for their growth. As the age of the seedling plays an important role in kinetin induced expansion (BANER]I and LALORAYA, 1965), seedlings having developed 4-5 mm hypocotyl (about 72 hr after soaking) and which produce the best respon~e, were selected for the investigation.

Results A. Survey of kinetin effects on cotyledon expansion in light Results are presented in table 1. All the plant materials responded to Although Luffa cotyledons gave the best response, the germination of seeds uniform and took a longer time. Cucumber seeds gave high percentage of germination and response towards kinetin, and was therefore chosen for investigation.

kinetin. was not uniform detailed

B. Effect of different growth regulators I t has been shown earlier that both kinetin and gibberellic acid bring about a marked expansion of light grown pumpkin cotyledons, whereas indole-3-acetic acid has no effect (BANERJI and LALORAYA, 1966). Similar effect of gibberellic acid has been reported for Xanthium and radish cotyledons (ESASHI and LEOPOLD, 1969; LETHAM, 1971). A similar effect is observed in cucumber cotyledons grown in light (Table 2 and Fig. 1). However the sensitivity towards kinetin is more than in gibberellic acid. The morphological features of cotyledons are also different in the two group of growth substances as also observed in earlier studies (BANERJI and LALORAYA, 1966). Kinetin and gibberellic acid together bring about a synergistic effect on the growth of the cotyledons (Table 3). Thus in light both gibberellic acid and kinetin bring about the expansion of cotyledons.

Kinetin has been shown to stimulate cotyledon expansion even in dark (BANERJI, 1966). Cucumber cotyledons when grown in complete darkness in water do not show any expansion growth. From the results presented in table 4 and fig. 2, it is clear that kinetin (10 mg/l) stimulates the expansion of cotyledons whereas indole-3-acetic acid (100 mgll) and gibberellic acid (100 mg/l) were found to be ineffective. Benzimidazole was found to affect the growth only at a high concentration (100 mg/l) but was ineffective at lower concentrations (Table 5). As compared to kinetin it is ten times less sensitive. Benzimidazole is not a naturally occurring compound but is known to produce cytokinin like effects in a number of systems (HILLMAN, 1955;

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P/lanzenphysiol. Bd. 71. S. 313-322. 1974.

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standard deviation

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0.56

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± SD

Table 1: Effect of kinetin (10 mg/l) on the growth of excised cotyledons in light at 72 hours.

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Fig. 1: Effect of gibberellic acid and kinetin on the growth of cucumber cotyledons at 72 hours. Constructed on the basis of data shown in Table 2. PERSON et aI., 1957; WANG and WAYGOOD, 1959). Cucumber cotyledon expanSIon in dark can therefore serve as a bioassay system for cytokinins. A comparison of the activity of different concentrations of kinetin and benzylaminopurine on cotyledon expansion in dark is shown in table 6 and figs. 3 to 5. It will be seen that benzylaminopurine is more active than kinetin in cucumber cotyledons as has been reported for Xanthium. The lower concentrations of benzylaminopurine were more effective than kinetin on cotyledon growth. No significant difference in response could be obtained with increasing time of cotyledon culture (Table 7), and the best effect was observed at 3-day growth.

Discussion In the present study a specific bioassay for cytokinins based on cotyledon expansion has been developed. The other bioassays proposed uptodate based on the cotyledon expansion by cytokinins have one or other discrepencies. ESASHI and LEOPOLD (1969) used Xanthium cotyledons, which responds to gibberellic acid as well as kinetin. In order to eliminate the interference from gibberellic acid they used mannitol (0.25 M) but this considerably reduced sensitivity towards cytokinins. Similarly LETHAM (1971) using radish cotyledons showed that mannitol minimised the response to gibberellic acid, without much affecting the response of cytokinin.

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Pjlanzenphysiol. Bd. 71. S. 313-322. 1974.

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Table 2: Effect of different concentrations of gibberellic acid (GA 3 ) and kinetin (KN) on the growth of cucumber cotyledons in light at 72 hours after treatment.

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Table 3: Effect of kinetin (10 mg/l), GA 3 (100 mg/l) and kinetin cocumber cotyledons in light at 72 hours.

::. Control

KN ::. Control GA 3 ::. Control I(N + GA 3 ::. Control

=

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158.0

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268.0

water control

Table 4: Effect of kinetin (10 mg/l), GA 3 (100 mg/l) and IAA (100 mg/l) on the growth of cucumber cotyledons in dark at 72 hours. Growth Fresh Wt. mg/cotyledon Area sq. cm

Treatment

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225.3

118.5

117.5

108.5

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Fig. 2: Effect of gibberellic acid (100 mg/l), kinetin (10 mg/l) and indole-3-acetic acid (100 mg/l) on the growth of cotyledons at 72 hours in dark, C = water control.

Z. Pflanzenphysiol. Ed. 71. S. 313-322. 1974.

Cucumber Cotyledon Expansion as a Bioassay for Cytokinins

319

In order to distinguish the response of the two growth regulators, he recommended placing of hypocotyl sections along with the cotyledons, which elongate in presence of gibberellic acid while cytokinin suppressed the elongation growth. This makes a bioassay more complicated and it is like performing two bioassays. The cucumber cotyledon expansion in dark on the other hand is a simple and sensitive bioassay for cytokinins. RIJVEN and PARKASH (1970) have shown that fenugreek cotyledons also

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Fig. 4: Effect of kinetin (K) on the expansion of cucumber cotyledons at 72 hours in dark.

Z. Pflanzenphysiol. Bd. 71. S. 313-322. 1974.

320

A.

NARAIN

and M. M.

LALORAYA

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Fig. 5: Effect of 6-benzylaminopurine (6-BAP) on the expansion of cucumber cotyledons at 72 hours in dark.

Table 5: Effect of benzimidazole on cotyledon growth in dark at 72 hours after treatment. Concn. 1.0 10.0 100.0

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%

104.0 111.0 185.3

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%

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respond to kinetin only in dark. The sensitivity to kinetin in cucumber is comparable to that reported for Xanthium cotyledons in dark (ESASHI and LEOPOLD, 1969). In Xanthium benzylaminopurine is shown to be much more effective than kinetin. Similar is the case with cucumber. Benzimidazole however, also brings about expansion growth in dark but its sensitivity as compared to kinetin and benzylaminopurine is very low. RIJVEN and PARKASH (1970) and LETHAM (1971) claim to have obtained no response with benzimidazole in fenugreek and radish cotyledons. The concentrations used by them were probably low and therefore ineffective.

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Pflanienphysiol. Bd. 71. S. 313-322. 1974.

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%

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1.40

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0.66

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± 0.02 ± 0.04

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Fresh Wt. mg/cotyledon Control Treated

Table 7: Effect of 6-benzylaminopurine (BAP) on the growth of cucumber cotyledons in dark at 144 hours after treatment.

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Table 6: Effect of kinetin (K) and 6-benzylaminopurine (BAP) on the growth of cucumber cotyledons in dark at 72 hours after treatment.

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References BANERJI, D.: Studies on protein metabolism in plants with particular reference to kinetin effects. Ph. D. Thesis, Allahabad University, Allahabad, India (1966). BANERJI, D., and M. M. LALORAYA: Expansion of isolated pumpkin cotyledons with kinetin: Effect of seedlings age. Naturwiss. 52, 349 (1965). - - Comparative effects of indole-3-acetic acid, kinetin and gibberellic acid on the growth of isolated Cucurbita pepo cotyledons. Curr. Sci. 35, 601 (1966). ESASHI, Y., and A. C. LEOPOLD: Cotyledon expansion as a bioassy for cytokinins. Plant Physiol. 44, 618 (1969). GEPSTEIN, S., and 1. ILAN: A promotive action of kinetin on amylase activity in cotyledons of Phaseolus vulgaris. Plant and Cell Physiol. 11, 819 (1970). GILAD, T., 1. ILAN, and L. REINHOLD: The effect of kinetin and of the embryo axis on the level of reducing sugars in sunflower cotyledons. Israel ]. Bot. 19, 447 (1970). HILLMAN, W. S.: The action of benzimidazole on Lemna minor. Plant Physiol. 30,535 (1955). IKUMA, H., and K. V. THIMANN: Action of kinetin on photosensitive germination of lettuce seed as compared with that of gibberellic acid. Plant and Cell Physiol. 4, 113 (1963). LETHAM, D. S.: Regulators of cell division in plant tissues XII. A cytokinin bioassay using excised radish cotyledons. Physiol. Plant 25, 391 (1971). LOVELL, P. H., and K. G. MOORE: The effects of benzylaminopurine on growth and 14 carbon translocation in excised mustard cotyledons. Physiol. Plant. 23, 179 (1970). NARAIN, A., and M. M. LALORAYA: Inhibition of chlorophyll synthesis by kinetin in Cucumis cotyledons. Plant and Cell Physiol. 11, 173 (1970). PERSON, C. D., D. ]. SAMBORSKI, and F. R. FORSYTH: Effects of benzimidazole on detached leaves. Nature 180, 1294 (1957). RIJVEN, A. H. G. C., and V. PARKASH: Cytokinin induced growth responses by fenugreek cotyledons. Plant Physiol. 46, 638 (1970). SANKHLA, N.: Expansion of isolated cotyledons of Ipomea pentaphylla in response to added auxins, cytokinins and morphectin. Biochem Physiol. Pflanz. 161, 183 (1970). SVESHNIKOVA, I. N., and V. A. KHOKHLOVA: Cytological study of the effect of 6-benzylaminopurine and kinetin on isolated flax cotyledons. Soviet Plant Physiol. 16, 570 (1969). WANG, D., and E. R. W AYGOOD: Effect of benzimidazole and nickel on the chlorophyll metabolism of detached leaves of khapli wheat. Can. ]. Bot. 37, 743 (1959).

A. NARAIN and MANMOHAN M. LALORAYA, Botany Department, Gujarat University, Ahmedabad/lndien.

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Pjlanzenphysiol. Bd. 71. S. 313-322. 1974.