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Changes in the level of endogenous gibberellins and auxins preceding the formation of adventitious roots on isolated epicotyls of pea plants
Flora, Aht. A, Bd. 160, S. 493-499 (1969)
Department of Biology, College of Agriculture, Jihlava, CSSR
Changes in the level of endogenous gibberellins and auxins preceding the formation of adventitious roots on isolated epicotyls of pea plants By MILADA BLA.HOVA With 2 figures (Received Juni 30, 1969)
Many years before discovery of auxin R. DOSTAL (1912) proclaimed his hypothesis on the basis of his correlation experiments that root regeneration is not stimulated by rough nutritional influences, but by specific regulative influences originating in growing axillary buds. At present first of all auxin is ranked among these regulative influences, for soon after the isolation of the principal substance of auxin nature indoleacetic acid (KOGL et al. 1934) - its positive influence on the formation of adventitious roots was proved after exogenous application of this regulant. There are, of course, relatively high non physiological concentrations of indoleacetic acid (IAA) which cannot corespond to the height of endogenous level of this regulative substance. Therefore, even doubt has arisen, whether adventitious root formation is really influenced by endogenous auxin (GAUTHERET 1944, VERLEYEN 1948). Optimum concentrations of exogenous auxin (IAA) are quite different of course, according to the stage of adventitious root formation. So according to LIBBERT (1956/57) a high concentration of IAA 10-5 to 10-4 g/cubic cm is necessary for the formation of adventitious root primordia "de novo" on epicotyls of pea plants; especially in the first internodium from the bottom 2-5 root primordia are usually already formed (SCHMIDT 1956) and much lower concentration of IAA, 10-7 g/cubic cm is sufficient . for stimulating their growth. At last, the optimum concentration for growth elongation of adventitious roots is an unusually low concentration, 10-11 to 10-13 g IAA/ cubic cm. Auxin, of course, is not the only substance influencing positively the formation of adventitious root primordia. The same is being proved for aneurin, nicotinic acid, nicotinamide, lactoflavin, and ascorbic acid (HITCHCOCK and ZIMMERMAN 1940, LIVINE and LEIN 1941, SCHEUERMANN 1951, FRIES 1955) as well as for vitamins K and H (HEMBERG 1952). From endogenous growth-regulative substances promoting adventitious root formation on the stem sections LIBBERT (1957) gives an evidence for
-
494 the existence of an inhibitor originating in the roots by which the initiation of adventitious roots is inhibited on an intact plant and the inhibiting influence of which disappears after the isolation of the stalk. Purine derivatives rather suppress the formation of adventitious roots as MAYER (1956) demonstrated for adenine and KAMINEK (1967) for kinetin. However, this suppression refers to the formation of root primordia only, because growth elongation of the roots already formed is supported by kinetin (SEBANEK 1969). Exogenous gibberellin regularly leads to the inhibition of adventitious root formation (BRIAN and RADLEY 1955, KATO 1958, DOSTAL 1960, BRIAN et al. 1960, TURECKAJA et al. 1963, BACHE LARD and STOWE 1963, LIEBERT and KRELLE 1966, MtTNZEL 1968, and others). The present investigation was designed to ascertain the change in the level of endogenous gibberellins in basal parts of pea epicotyls, namely from the moment of their isolation over three days following the isolation. The aim was to determine in time dependences not only the changes in the level of endogenous gibberellins, but also those of endogenous auxins being simultaneously in operation and accompanying the preparation for formation of adventitious roots on the isolated pea epicotyl. Materials, Methods and Results 1. Assays of endogenous gibberellins Pea seeds (variety Raman) have been swollen in water for 24 hr, thereafter put onto the moistened sawdust and kept for 10 days in the dark at laboratory temperature. Epicotyls approximately 10 cm long at that time were cut tightly above the cotyledons, immersed with their 2 cm long bases in a jar with distilled water and placed again in the dark at laboratory temperature. From that material samples were taken 24,48 and 72 hrs. after cutting for the detection of endogenous gibberellins. The samples were composed of basal parts of epicotyls in length of 2 cm only, the weight of each sample being 2 g (epicotyl bases from 20-23 plants). The homogenized samplp was extracted with methanol (24 hrs at 0° C), filtered and the filtrate was evaporated in vacuum in a water bath at 40 aC. The dry residue was dissolved in 5 ml of methanol, transferred onto an evaporating plate, evaporated in a stream of air to dryness, dissolved in a small amount of 70 "it) ethanol and in this form quantitatively applied to a thin layer (of silicagel G according to Stahl) for chromatographical analysis. Simultaneously with the sample examined a sample of gibberellin GAa was applied which was detected after development in the mixture chloroform - ethyl acetate - glacial acetic acid (60: 40: 5) - (SEMBDNER et a1. 1962) with sulphuric acid in ethanol (RF 0,3 -0,4). The extract from plant material was detected biologically by a lettuce test (FRANKLAND and WARIENG 1961, KREKULE and TELTSCHEROYA 1963). The stimulation of lettuce hypocotyls corresponded to RF for GAa. A detailed description of the method can be seen in SEB.~NEK (1965). The whole experiment was replicated three times. In all three replications it was shown uniformly that 24 hrs after separating the roots and cotyledons from epicotyls a decrease of endogenous gibberellin content appeared in the epicotyl base. However, after 48 hrs the decreasing was slower and after 72 hrs the content began to rise beyond the peak attainf'd immediately after the isolation of epicotyls (see Fig. 1).
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Fig. 1. Chromatographieal estimation of endogenous gibberellin content in basal parts of pea epicotyJs immediately after their isolation (0) and 24, 48 and 72 hrs after the isolation. Thp values on the ordinate express the content of endogenous gibberellins at R F 0,3 ~-0,4 in the per('entage of elongation of lettuce hypocotyls as compared with the control.
2. Assays of endogenous auxins Pea plants (variety Raman) were grown (as sub 1) up to the stage of being 8 days old, epicotyls cut again tightly above the cotyledons and analyses for auxin content were made from the matter of whole epicotyls in the same time intervals (as sub 1). Each sample was composed of 3 g of the matter. After the extraction with methanol (as sub 1) and filtration, the plant matter was extracted still three times shortly with methanol. Thereafter the combined methanol extracts were evaporated in a stream of air until the removing of alcohol, the water part was evaporated in vacuum evaporator to dryness. This second dry residue was extracted with unhydrous peroxidefree ethyl ether and after concentration the etherial extract was applied on the starting line of Whatman No.1 chromatography paper. Analysis was performed by ascending chromatography in the mixture isopropanol - 25 % ammonia - water (10: 1: 1). The zone of the chromatogram ('orresponding to the position of IAA according to RF (RF 0,2-0,3) was cut out, the paper was eut into small pieces and suspended in redistilled water. The solution obtained was tested biologically with coleoptile segments of wheat variety Kasticka osinata by a current method (NITSCH 1956). IAA position was ascertained for evidenee by Ehrlich reagent. The experiment was replicated three times. It was shown that 24 hrs. after separating the epicotyls from roots and cotyledons no significant changes occurred in epicotyls as regards the content of endogenous auxins. Nevertheless, after 48 hrs. there was a tendency to rising. After 72 hr. this rising was very distinct (see Fig. 2).
Discussion
Experimental results of the present work have shown that 24-48 hr. after severing epicotyls from roots and cotyledons in pea plants an expressive decrease of H4 Flora Aht. A, Bd. 160
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Fig. 2. Chromatographical estimation of endogenous auxin content in pea epicotyls immediately after their isolation (0) and 24, 48 and 27hrs after the isolation. The values on the ordinate express the content of endogenous auxins at RF 0,2 -0,3 in percentage of elongation of coleoptile segments in wheat as compared with the control.
endogenous gibberellin content occurs in basal parts of epicotyls. As late as 72 hrs. after severing the epicotyls a striking increase of the level of endogenous gibberellins can be observed in this part and this level can exceed that one attained immediately after the isolation. Thus the period of 0-48 hrs. after the isolation of epicotyls, associated with remeristematization of pericycle and formation of new root primordia is accompanied by a distinct decrease of the level of endogenous gibberellins. This is in agreement with experimental results indicating that the formation of adventituous roots is weakened by exogenous gibberellins (see the introduction). The decrease of endogenous gibberellins in basal parts of epicotyls after their separation from the roots is also in agreement with the works demonstrating the participation of roots in biosynthesis of endogenous gibberellins (JONES and PHILLIPS 1966, SEBANEK 1966, SITTON, RICHMOND and VAADIA 1967). Consequently, when the stem is separated from the root as an organ indisputably participating in gibberellin biosynthesis, a decrease of gibberellin level appears in the basal part of the stem in the course of 48 hrs. after the separation of the stem from the root. This decrease (similarly as the increase of endogenous auxin level already starting at that time) is most likely favourable for remeristematization of the pericycle being simultaneously in operation and for the formation of new root primordia. If there is a remarkable increase of endogenous gibberellins in the bases of epicotyls 72 hrs. after the isolation, then. the reason may be here that the endogenoUR
Changes in the level of endogenous gibberellins etc.
497
gibberellins begin to be newly biosynthetized in the growing primordia of adventitious roots at that time. The rise of the level of endogenous auxins in epicotyls 48 and 72 hrs. after their isolation is most likely connected with the fact that endogenous auxins activated in a stalk tip cannot be inactivated. Their inactivation takes place otherwise in the roots (PHILLIPS 1964a, b). We are aware of methodical shortcoming connected with the fact that endogenous gibberellins have been detected only in basal parts of epicotyls, whereas auxins in total epicotyls. It was done in this way because the basal parts of pea epicotyls by themselves possess such a small quantity of auxin that the biological test emplo~ in this study proved to be insufficiently sensitive for ascertaining the differences in the height of auxin level in idividual time intervals after the separation of epicotyls. Besides, the rise of endogenous auxin level as preparation for the formation of adventitious roots is already well known from other papers (see the introduction). The present study was rather undertaken in order that the same time intervals from epicotyl isolation in which endogenous gibberellin level had been detected might be completed with the changes of the level of endogenous auxins passing in the isolated stalk. Conclusions
Acknowledgment. This paper had its source at the cathedra lead by Prof. Dr. JIRI SEBANEK to whom I wish to express my thanks for advice and help. Etiolated pea seedlings were released from roots and cotyledons and thereafter the content of endogenous gibberellins was ascertained in basal parts of epicotyls 24, 48 and 72 hrs. after the isolation of epicotyls. After 24 and 48 hrs. the decrease of endogenous gibberellin level could be seen, after 72 hrs., however, its remarkable rise. The content of endogenous auxins in isolated epicotyls did not change significantly over 24 hrs. period from the isolation of epicotyls, but after 72 hrs. this content began to rise distinctly. Consequently, a 48 hrs. period from the isolation of epicotyls when the remeristematization of the pericycle and formation of new root primordia occur, is connected with the fall of endogenous gibberellin level and with initiating increase of endogenous auxin content. Thus it can be explained why the formation of adventitious roots is suppressed by endogenous gibberellins and conversely stimulated by exogenous auxins. Literature BACHELARD, E. P., and STOWE, B. B., 1963. Rooting of cuttings of Acer rubrum L. and Eucalyptus camaldulensis DEIIN. Austr. J. BioI. Sci., 16, 751-767. BRIAN, P. W., HEMMING, H. G., and LOWE, D., 1960. Inhibition of rooting of cuttings by gibberellic acid. Ann. Bot., 24, 407-419. BRIAN, P. W., and RADLEY, M., 1955. A physiQlogical comparison of gibberellic acid with some auxins. Physiologia Plant. (Copenh.), 8, 899-912. 34*
498
)I. BLAHovA
DOSTAL, R. 1912. Liber die Korrelationsbeziehungen zwischen Wurzel lind SproJ.l (tschechiseh). Rozpravy Ces. akad. 11.,21,1-38. DOSTAL, R., 1960. tiber die Wechselwirkung der Gibberellinsiiure und anderer Stimulatoren in den Wachstumskorrelationen. BioI. Plant., 2, 48-60. FRANKLAND, B., and W.\REING, P. F., 1961. Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature, 185, 255-256. FlUES, N., 1955. The significance of thiamin and pyridoxin for the growth of the decotylised pea. seedlings. Physiol. Plantarum, 8, 859. GAUTHERET, R. J., 1944. Reeherches sur la polariM des tissus vegetaux. Rev. Cytol. Cytophysiol. Veget., 7, 45. HEMllERG, T., 1952. The effect of vitamin K and vitamin H'on the root formation in cuttings of Phaseolus vulgan:s L. Physiol. Plantarum, 6, 17 -20. HITCHCOCK, A. E., and ZIMMERMAN, P. W., 1940. Effects obtained with mixtures of rootinducing and other substances. ContI'. Boyce Thompson Inst., 11,143-146. JONES, R. L., and PlIILLIPS, I. D. J., 1966. Organs of gibberellin biosynthesis in lightgrown sunflower plants. Plant Physiol., 41, 1381-1386. lCnllNEK, M., 1967. Root formation in pea stem sections and its inhibition by kinetin, ethionine and chloramphenicol. BioI. Plant., 9, 86-91. KATO, J., 1958. Studies on the physiological effect of gibberellin. II. On the interaction of gibbprellin with auxins and growth inhibitors. Physiologia Plantarum, 11, 10-15. KOGL, F., HAAGEN-SMIT, A. J., und ERXLEBEN, H., 1934. tiber ein neues Auxin (Heteroauxin) aus Ham. Hoppe-Seylers Z. physiol. Chern., 228, 90-103. KI\EKULE, J., und TELTSCHEROVA, L., 1963. tiber den Gehalt an auxin- und gibberelliniihnlichen Stoffen bei jarowisierten und nichtjarowisierten Embryonen von Sommer- und Winterweizen. BioI. Plant., 5, 252-257. LEVINE, M., and LEIN, J., 1941. The effects of various growth substaces on the number and the length of roots of Allium cepa. Amer. J. Bot., 28, 163-168. LJIlBERT, E., 1956/57. Die hormonale und korrelative Steuerung der Adventivwurzelbildung. Wiss. Zeitschr. Humboldt-Univ. Berlin, Mat. Nat., 6, 315-347. LlBBEIlT, E., 1957. Untersuchungen liber die Physiologie der Adventivwurzelbildung. III. Untersuchung der Hemmstoffe, mittels derer eine Wurzel die Adventivwurzelbildung beeinfluJ.lt. Zeitsehr. f. Botanik, 45, 57 -76. LIBBERT, E., und KRELLE, E., 1966. Die Wirkung des "Gibberellinantagonisten" 2-Chloriithyltrimethylammoniumchlorid (CCC) auf die Stecklingsbewurzelung windender und nicht windender Pflanzen. Planta, 70, 95 -98. }L\YER, L., 1956. Wachstnm und Organbildung an in vitro knltivierten Segmenten von Pelargoninm zonale und Cyelamen persicum. Plant a 47, 401. :\lUNZEL, E., 1968. Hemmung der Regeneration and Begonia rex- Blattstlicken durch Gibberellin. Naturwiss., 55, 659. NITSCH, J. P., 1956. Methods for the investigation of natural auxins and growth inhibitors. In.: The Chemistry and mode of action of plant growth substances. London, Butterworths, 1956. PHILLIPS, 1. D. J., 1964a. Root-shoot hormone relations. I. The importance of an aerated root system in the regUlation of growth hormones levels in the shoot of Helianthus annuus. Ann. of Bot., 28, 17-35. PIlILLIPS, I. D. J., 1964 b. Root-shoot hormone relations. II. Changes in endogenous auxin concentration produ("ed by flooding of the root system in Helianthns annuus. Annals of Hot., 28, 37-45.
Changes in the level of endogenous gibberellins etc.
499
SCHEUERMANN, R., 1951. Der EinfluB wasserliislicher Vitamine auf die Wirksamkeit von Heteroauxin im WachstumsprozeB der hiiheren Pflanzen. Planta, 40,265-300. SCHMIDT, E., 195.6. Anatomische Untersuchung iiber das Vorkommen von Wurzelanlagen in verschiedenen Internodien von Pisum sativum. Flora, 144, 151-153. SEMDNER, G., GROSS, R., und SCHREiBER, K., 1962. Die Diinnschichtchromatographie von Gibberellinen. Experientia, 18, 584-585. SITTON, D., RICHMOND, A., and VAADIA, J., 1967. On the synthesis of gibberellins in roots. Phytochem., 6, 1101-1105. SEBANEK, J., 1965. Die Interaktion endogener Gibberelline in der Korrelation zwischen Wurzel und Epikotyl bei Pisum-Keimlingen. Flora A, 106, 303-311. SEBANEK, J., 1966. The effect of amputation of the epicotyl on the level of endogenous gibberellins in the roots of pea seedlings. BioI. Plant., 8, 470-475. SEBANEK, J., 1969. The effect of kinetin and CCC on the formation and growth of the adventitious roots (unpublish. results). TURECKA.JA, R. K., KEFELI, V. J., und KOF, E. M.,1963. Wechselwirkung von Heteroauxin und Gibberellin bei der Bewurzelung und SproBbildung von Weidenstecklingen. Doklady Akad. nauk SSSR, 148, 461-464. VERLEYEN, E. J. B., Le bouturage et les substances de croissance syntheti ques. Anvers 1948. Address of the author: MILADA BLAHOVA-SEMOTANOVA, DipI.-Biol. College of Agriculture, Jihlava (CSSR), Tolsteho 16.
Department of Biology, College of Agriculture, Jihlava, CSSR
Changes in the level of endogenous gibberellins and auxins preceding the formation of adventitious roots on isolated epicotyls of pea plants By MILADA BLA.HOVA With 2 figures (Received Juni 30, 1969)
Many years before discovery of auxin R. DOSTAL (1912) proclaimed his hypothesis on the basis of his correlation experiments that root regeneration is not stimulated by rough nutritional influences, but by specific regulative influences originating in growing axillary buds. At present first of all auxin is ranked among these regulative influences, for soon after the isolation of the principal substance of auxin nature indoleacetic acid (KOGL et al. 1934) - its positive influence on the formation of adventitious roots was proved after exogenous application of this regulant. There are, of course, relatively high non physiological concentrations of indoleacetic acid (IAA) which cannot corespond to the height of endogenous level of this regulative substance. Therefore, even doubt has arisen, whether adventitious root formation is really influenced by endogenous auxin (GAUTHERET 1944, VERLEYEN 1948). Optimum concentrations of exogenous auxin (IAA) are quite different of course, according to the stage of adventitious root formation. So according to LIBBERT (1956/57) a high concentration of IAA 10-5 to 10-4 g/cubic cm is necessary for the formation of adventitious root primordia "de novo" on epicotyls of pea plants; especially in the first internodium from the bottom 2-5 root primordia are usually already formed (SCHMIDT 1956) and much lower concentration of IAA, 10-7 g/cubic cm is sufficient . for stimulating their growth. At last, the optimum concentration for growth elongation of adventitious roots is an unusually low concentration, 10-11 to 10-13 g IAA/ cubic cm. Auxin, of course, is not the only substance influencing positively the formation of adventitious root primordia. The same is being proved for aneurin, nicotinic acid, nicotinamide, lactoflavin, and ascorbic acid (HITCHCOCK and ZIMMERMAN 1940, LIVINE and LEIN 1941, SCHEUERMANN 1951, FRIES 1955) as well as for vitamins K and H (HEMBERG 1952). From endogenous growth-regulative substances promoting adventitious root formation on the stem sections LIBBERT (1957) gives an evidence for
-
494 the existence of an inhibitor originating in the roots by which the initiation of adventitious roots is inhibited on an intact plant and the inhibiting influence of which disappears after the isolation of the stalk. Purine derivatives rather suppress the formation of adventitious roots as MAYER (1956) demonstrated for adenine and KAMINEK (1967) for kinetin. However, this suppression refers to the formation of root primordia only, because growth elongation of the roots already formed is supported by kinetin (SEBANEK 1969). Exogenous gibberellin regularly leads to the inhibition of adventitious root formation (BRIAN and RADLEY 1955, KATO 1958, DOSTAL 1960, BRIAN et al. 1960, TURECKAJA et al. 1963, BACHE LARD and STOWE 1963, LIEBERT and KRELLE 1966, MtTNZEL 1968, and others). The present investigation was designed to ascertain the change in the level of endogenous gibberellins in basal parts of pea epicotyls, namely from the moment of their isolation over three days following the isolation. The aim was to determine in time dependences not only the changes in the level of endogenous gibberellins, but also those of endogenous auxins being simultaneously in operation and accompanying the preparation for formation of adventitious roots on the isolated pea epicotyl. Materials, Methods and Results 1. Assays of endogenous gibberellins Pea seeds (variety Raman) have been swollen in water for 24 hr, thereafter put onto the moistened sawdust and kept for 10 days in the dark at laboratory temperature. Epicotyls approximately 10 cm long at that time were cut tightly above the cotyledons, immersed with their 2 cm long bases in a jar with distilled water and placed again in the dark at laboratory temperature. From that material samples were taken 24,48 and 72 hrs. after cutting for the detection of endogenous gibberellins. The samples were composed of basal parts of epicotyls in length of 2 cm only, the weight of each sample being 2 g (epicotyl bases from 20-23 plants). The homogenized samplp was extracted with methanol (24 hrs at 0° C), filtered and the filtrate was evaporated in vacuum in a water bath at 40 aC. The dry residue was dissolved in 5 ml of methanol, transferred onto an evaporating plate, evaporated in a stream of air to dryness, dissolved in a small amount of 70 "it) ethanol and in this form quantitatively applied to a thin layer (of silicagel G according to Stahl) for chromatographical analysis. Simultaneously with the sample examined a sample of gibberellin GAa was applied which was detected after development in the mixture chloroform - ethyl acetate - glacial acetic acid (60: 40: 5) - (SEMBDNER et a1. 1962) with sulphuric acid in ethanol (RF 0,3 -0,4). The extract from plant material was detected biologically by a lettuce test (FRANKLAND and WARIENG 1961, KREKULE and TELTSCHEROYA 1963). The stimulation of lettuce hypocotyls corresponded to RF for GAa. A detailed description of the method can be seen in SEB.~NEK (1965). The whole experiment was replicated three times. In all three replications it was shown uniformly that 24 hrs after separating the roots and cotyledons from epicotyls a decrease of endogenous gibberellin content appeared in the epicotyl base. However, after 48 hrs the decreasing was slower and after 72 hrs the content began to rise beyond the peak attainf'd immediately after the isolation of epicotyls (see Fig. 1).
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Fig. 1. Chromatographieal estimation of endogenous gibberellin content in basal parts of pea epicotyJs immediately after their isolation (0) and 24, 48 and 72 hrs after the isolation. Thp values on the ordinate express the content of endogenous gibberellins at R F 0,3 ~-0,4 in the per('entage of elongation of lettuce hypocotyls as compared with the control.
2. Assays of endogenous auxins Pea plants (variety Raman) were grown (as sub 1) up to the stage of being 8 days old, epicotyls cut again tightly above the cotyledons and analyses for auxin content were made from the matter of whole epicotyls in the same time intervals (as sub 1). Each sample was composed of 3 g of the matter. After the extraction with methanol (as sub 1) and filtration, the plant matter was extracted still three times shortly with methanol. Thereafter the combined methanol extracts were evaporated in a stream of air until the removing of alcohol, the water part was evaporated in vacuum evaporator to dryness. This second dry residue was extracted with unhydrous peroxidefree ethyl ether and after concentration the etherial extract was applied on the starting line of Whatman No.1 chromatography paper. Analysis was performed by ascending chromatography in the mixture isopropanol - 25 % ammonia - water (10: 1: 1). The zone of the chromatogram ('orresponding to the position of IAA according to RF (RF 0,2-0,3) was cut out, the paper was eut into small pieces and suspended in redistilled water. The solution obtained was tested biologically with coleoptile segments of wheat variety Kasticka osinata by a current method (NITSCH 1956). IAA position was ascertained for evidenee by Ehrlich reagent. The experiment was replicated three times. It was shown that 24 hrs. after separating the epicotyls from roots and cotyledons no significant changes occurred in epicotyls as regards the content of endogenous auxins. Nevertheless, after 48 hrs. there was a tendency to rising. After 72 hr. this rising was very distinct (see Fig. 2).
Discussion
Experimental results of the present work have shown that 24-48 hr. after severing epicotyls from roots and cotyledons in pea plants an expressive decrease of H4 Flora Aht. A, Bd. 160
496
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Fig. 2. Chromatographical estimation of endogenous auxin content in pea epicotyls immediately after their isolation (0) and 24, 48 and 27hrs after the isolation. The values on the ordinate express the content of endogenous auxins at RF 0,2 -0,3 in percentage of elongation of coleoptile segments in wheat as compared with the control.
endogenous gibberellin content occurs in basal parts of epicotyls. As late as 72 hrs. after severing the epicotyls a striking increase of the level of endogenous gibberellins can be observed in this part and this level can exceed that one attained immediately after the isolation. Thus the period of 0-48 hrs. after the isolation of epicotyls, associated with remeristematization of pericycle and formation of new root primordia is accompanied by a distinct decrease of the level of endogenous gibberellins. This is in agreement with experimental results indicating that the formation of adventituous roots is weakened by exogenous gibberellins (see the introduction). The decrease of endogenous gibberellins in basal parts of epicotyls after their separation from the roots is also in agreement with the works demonstrating the participation of roots in biosynthesis of endogenous gibberellins (JONES and PHILLIPS 1966, SEBANEK 1966, SITTON, RICHMOND and VAADIA 1967). Consequently, when the stem is separated from the root as an organ indisputably participating in gibberellin biosynthesis, a decrease of gibberellin level appears in the basal part of the stem in the course of 48 hrs. after the separation of the stem from the root. This decrease (similarly as the increase of endogenous auxin level already starting at that time) is most likely favourable for remeristematization of the pericycle being simultaneously in operation and for the formation of new root primordia. If there is a remarkable increase of endogenous gibberellins in the bases of epicotyls 72 hrs. after the isolation, then. the reason may be here that the endogenoUR
Changes in the level of endogenous gibberellins etc.
497
gibberellins begin to be newly biosynthetized in the growing primordia of adventitious roots at that time. The rise of the level of endogenous auxins in epicotyls 48 and 72 hrs. after their isolation is most likely connected with the fact that endogenous auxins activated in a stalk tip cannot be inactivated. Their inactivation takes place otherwise in the roots (PHILLIPS 1964a, b). We are aware of methodical shortcoming connected with the fact that endogenous gibberellins have been detected only in basal parts of epicotyls, whereas auxins in total epicotyls. It was done in this way because the basal parts of pea epicotyls by themselves possess such a small quantity of auxin that the biological test emplo~ in this study proved to be insufficiently sensitive for ascertaining the differences in the height of auxin level in idividual time intervals after the separation of epicotyls. Besides, the rise of endogenous auxin level as preparation for the formation of adventitious roots is already well known from other papers (see the introduction). The present study was rather undertaken in order that the same time intervals from epicotyl isolation in which endogenous gibberellin level had been detected might be completed with the changes of the level of endogenous auxins passing in the isolated stalk. Conclusions
Acknowledgment. This paper had its source at the cathedra lead by Prof. Dr. JIRI SEBANEK to whom I wish to express my thanks for advice and help. Etiolated pea seedlings were released from roots and cotyledons and thereafter the content of endogenous gibberellins was ascertained in basal parts of epicotyls 24, 48 and 72 hrs. after the isolation of epicotyls. After 24 and 48 hrs. the decrease of endogenous gibberellin level could be seen, after 72 hrs., however, its remarkable rise. The content of endogenous auxins in isolated epicotyls did not change significantly over 24 hrs. period from the isolation of epicotyls, but after 72 hrs. this content began to rise distinctly. Consequently, a 48 hrs. period from the isolation of epicotyls when the remeristematization of the pericycle and formation of new root primordia occur, is connected with the fall of endogenous gibberellin level and with initiating increase of endogenous auxin content. Thus it can be explained why the formation of adventitious roots is suppressed by endogenous gibberellins and conversely stimulated by exogenous auxins. Literature BACHELARD, E. P., and STOWE, B. B., 1963. Rooting of cuttings of Acer rubrum L. and Eucalyptus camaldulensis DEIIN. Austr. J. BioI. Sci., 16, 751-767. BRIAN, P. W., HEMMING, H. G., and LOWE, D., 1960. Inhibition of rooting of cuttings by gibberellic acid. Ann. Bot., 24, 407-419. BRIAN, P. W., and RADLEY, M., 1955. A physiQlogical comparison of gibberellic acid with some auxins. Physiologia Plant. (Copenh.), 8, 899-912. 34*
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