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Changes in Auxin, Germination Inhibitors, Gibberellins, and Cytokinins during the Breaking of Seed Dormancy in Fagus sylvatica
Biochem. Physiol. Pflanzen 1'10, S. 51-58 (1976)
Changes in Auxin. Germination Inhibitors, Gibberellins, and Cytokinins during the Breaking of Seed Dormancy in Fagus sylvatica HAMED M. M. EL-ANTABLY Department of Botany, Faculty of Agriculture Ain Shams University, Cairo, Egypt Key Term Index: phytohormones, abscisic acid, auxin, gibberellins cytokinins, seed dormancy, plant development; Fagus sylvatica L.
Summary Studies on the effect of constant chilling temperature of 4 °C and normal fluctuating temperatures out-of-doors on germinating percentage and endogenous IAA, ABA, gibberellins and cytokinins during the breaking of dormancy of Fagus sylvatica seeds was investigated. Dormant seeds showed 40 and 64 per cent germination after 12 weeks stratification at constant chilling temperature and normal fluctuating temperatures, respectively. Marked increase in IAA levels occurred as a result of both treatments. The increase in IAA was found to be 64 per cent greater at normal fluctuating temperatures than at constant chilling temperature after 12 weeks stratification. The results also showed an equal loss in endogenous ABA after 12 weeks stratification at both treatments. Moreover, a marked increase in gibberellin-like activity at both treatments was found to be almost 75 per cent more at normal fluctuating temperatures than at constant chilling temperature after 12 weeks stratification. The results also showed a high yield in the butano soluble cytokinins with both treatments. The increase in cytokinins was found to be 50 per cent more at normal fluctuating temperatures than at constant chilling temperature.
Introduction
The seeds of Fagus sylvatica L. usually require a period of 3 months of after-ripening at constant chilling temperature of 2 -4°C before they were capable of germination. At natural fluctuating temperatures in winter, the seeds pass almost the same period of after-ripening. During this period, biochemical and physiological changes (PINFIELD 1965) usually occur to bring the seeds out of dormancy and germinate readily in the spring. VILLIERS and WAREING (1960) suggested that dormancy in Fraxinus excelsior seeds is controlled by the interaction of growth promotors and inhibitors. POUL et al. (1973) were able to demonstrate slight increase in auxin-like activity after 28 d of stratification in loblolly pine seeds, while the inhibitor level detected by Avena coleoptile bioassay was found to disappear almost completely. Chilling temperature after ripening resulted in a decrease in endogenous inhibitors in Prunus avium (LIPE and CRANE 1966), Juglans regia (MARTIN et al. 1969), Acer saccharum (WEBB et al. 1973), and Pinus taedar (POUL et al. 1973). In F. excelsior, VILLIERS and WAREING (1965) reported that the level of water soluble inhibitors do not seem to change markedly with stratification, whereas KENTZU (1966) was able to show a reduction in the levels of ether-soluble inhibitors 4*
H. M. 1\1. EL-ANL\BLY
52
with stratification in this species. In 1966 FRANKLAND and WAREING reported that stratification leads to small increase in the levels of cndogenous gibberellins in Carulus avellana seeds. When such chilled seeds transferred to 20°C there was a considerable rise in endogenous gibberellins estimated as 64-fold (GLC) or 79-fold (TLCjbioassay), this rise preceding the detection of measurable growth of the embryonic axis (Ross and BRAD BEER 1971). On the other hand, POUL et al. (1973) were able to detect gradual increase in endogenous gibberellin in Pinus taeda following 42-day stratification. KHAN (1971) has suggested that cytokinins play "a permissive role" in seed dormancy, Whereas VAN STADEN et al. (1972) suggested that cytokinins may play an important role in the dormancy of Acer saccharum seeds and concluded that the role of endogenous cytokinin in seed dormancy remains to be demonstrated. The present investigation was undertaken to follow changes in endogenous auxin, ABA, gibberellins and cytokinins levels in association with chilling and emergence from dormancy in seeds of Fagus sylvatica L. during constant chilling temperature of 4 °C and normal fluctuating temperatures, out-of-doors. Material and Methods
Plant material Nuts of F. sylvatica were collected from trees grown in the forest near Aarhus, Denmark, in November 1973. They were mixed with an equal volume of soil in polythene bags and closed with rubber bands. Half the bags were placed in the cold room at 4 DC to receive constant chilling temperature of 4 DC, whereas the other half were buried in a soil pit out-of-doors to receive normal fluctuating temperatures. The bags were checked weekly for moisture and fungal growth. For germination test and extraction, nuts were taken monthly from both cold room and out-ofdoor bags, in December, January and February i. e. after 1,2 and 3 months, respectively.
Extraction
of auxin and abscisic acid
(ABA)
The growth regulators IAA and ABA were extracted and identified by gas-liquid chromatography (GLC) using the same method described by LENTON et al. (1971) for IAA as for ABA. The seeds were extracted with 80 % cold methanol using an "Atomix" blender. The methanol was removed from the extract under vacuum at 35 DC, and the acidic, ethyl acetate soluble fractions were separated from the resultant aqueous solutions by solvent partition.
PVP column The ethyl acetate soluble acids were further purified by being passed through a column of polyvinylpyrrolidone (PVP) and eluted with phosphate buffer at pH 8.0 at a flow rate of 91 drops (5.25 ml) per 12 min. The main ABA or IAA activities eluted at fraction 5-12 (between 26-60 ml) or at 16 to 24 (between 84-126 ml) respectively, were individually bulked for further extraction with diethyl ether and reduced to dryness for loading on TLC plates.
Thin-layer chromatography (T LC) The dried samples were dissolved in ethanol and loaded on 20 x 20 cm, 0.25 mm plates of Silica gel GE 254 (E. Merck, A. G.) which had been activated at 105 DC for one hour. Markers of authentic IAA or ABA were placed on either sides of the streak and the plates were developed with ethyl acetate: chloroform: acetic acid (15: 5: 1 v Iv) in tanks lined with filter paper. After developing, IAA and ABA were localized under U. V. radiation and the corresponding zones were marked. The individual
Growth Regulator Changes During Breaking Seed Dormancy
53
zones were luted with ethanol and the eluates reduced to dryness and placed in a vacuum desiccator until required.
Methylation The eluates from zones corresponding to IAA or ABA were methylated with diazomethane. The reagent was freshly prepared from p-tolylsulphonylmethylnitrosamide in a very clean, dry glass apparatus. After 16 h at room temperature, the reaction mixture was evaporated to dryness, and the methylated IAA or ABA were redissolved in a small, known volume of chloroform, when they were required for injection onto the GLC column.
Gas-liquid chromatography The GLC analyses were carried out with a Beckman GC 65 instrument with flame ionisation detector. The glass column, 6 feet X l/S inch, was packed with Epon 1001 on AW DMCS chromosorb W, 60-S0 mesh. The carrier gas was N 2 , 50 ml/min. Samples were analysed for both IAA and ABA at 200°C isotherm.
Extraction of gibberellins After passing the ethyl acetate soluble acids through a column of polyvinylpyrrolidone (PVP), eluted with phosphate buffer at pH S.O, the X purified material was extracted again with diethyl ether and reduced to dryness for loading on TLC plates.
T LC for gibber ellins Chromatograms intended for gibberellin bioassay, were developed with isopropanol, 35 % sp.gr. 0.91 ammonia, and water (10: 1: 1 v/v) and divided into 10 equal strips, each of which was scraped off, eluted with 2 ml distilled water, and tested for gibberellins activity using lettuce hypocotyl assay (FRANKLAND and WAREING 1960).
Extraction of cytokinins The seeds were extracted with SO % cold methanol, using the method described by HORGAN et al. (1973). The methanol was removed from the extract under vacuum at 35°C, filtered, and the filterates were adjusted to pH 2.5 with 2 N HCl. The methanolic extract was partitioned against ethyl acetate, and the ethyl acetate fraction discarded. The aqueous fraction was adjusted to pH 7.0, and partitioned against water-saturated n-butanol. After reduction to a small volume, the butanol fraction was chromatographed on Whatman's N. 3 MM paper in sec-butanol: 25% sp. gr. 0.91 ammonia (4: 1 v/v).
Soybean callus bioassay Paper chromatogram was cut into 10 equal RF strips and placed directly into 50 ml test tubes, to which 20 ml of medium (MILLER 1965) was added. After autoclaving and cooling, 100 mg pieces of soybean callus was added to each tube. The cultures were maintained at 30°C under continuous light, and the callus fresh weights determined after 21 days.
Results
Germination The results obtained in Table 1 show that no germination was occurred after 4 weeks stratification at either constant chilling temperature or normal fluctuating temperatures. After 8 weeks stratification, germination percentages were 9 and 17, whereas after 12 weeks, were 40 and 64 at constant and fluctuating temperatures respectively. At normal
54
H. M. :\1.
EL-A)lTABLY
Table 1. Effect of Stratification all genm:nation percentage of Fagus sylvatica, seeds. were stratified at constant chilling temperature of 4 °C and at normal fluctuating temperatures, out-of-doors, for 12 weeks. The germination of samples of 50 test nuts for each case. ~uts
Stratification in week
Sampling time
4 8 12
December January February
Germination % At constant chilling Temp. (4°C)
At normal fluctuating Temp. (out-of-doors)
9 40
17 64
fluctuating temperatures, the seeds show 60% increase in germination more than at constant chilling temperature after 12 weeks stratification. Endogenous IAA Table 2 represent the results of the GLC analyses of the acidic ethyl acetate fractions corresponding to 5 g dry weight of extracted seeds, developed on TLC plates, in a mixture of ethyl acetate, chloroform, acetic acid (15: 5: 1 vjv). The zone corresponding to IAA marker was scraped and eluted with ethanol. 0.5,tt1 of the methylated sample equivalent to 2 mg dry weight was chromatographed on the GLC column. A peak cochromatographed with methylated authentic IAA and with same retention time was Table 2. Endogenous contents of fAA, ABA, Gibberellins and Cytokinins. Extracted seeds of Fagus sylvatica, stratified at constant chilling temp. of 4°C, and normal fluctuating temp., out-of-doors, for 12 weeks. Samples for extraction were taken in December, January and February, after 4, 8 and 12 weeks respectively. ng/g dry weight At normal fluctuating Temp. (out-of-doors)
At constant chilling Temp. (4°C)
IAA ABA Gibberellins Cytokinins
December
January
February
December
January
35 126.5 22
90 24.3 86 243
140 12.5 215 675
30 120 39 25
105 33.3 70 210
77
February 230 12.5 376 1010
detected in each extract. It can be seen that there is remarkable ij1crease in IAA levels as a result of both stratification treatments. The amount of IAA detected after 12 weeks stratification was found to be more in seeds subjected to normal fluctuating temperatures than in seeds kept at constant chilling temperature in cold room. Such increase at normal fluctuating temperatures was found to be 64.3 % as much as constant chilling temperature. Endogenous ABA Table 2 also represent the data of GLC analyses of the acidic ethyl acetate fraction corresponding to 5 g dry weight of extracted tissue developed on TLC plates in same
Growth Regulator Changes During Breaking Seed Dormancy
55
solvent as IAA. The zone corresponding to an ABA marker was scraped and eluted with ethanol. 0.5,u1 of methylated sample equivalent to 2 mg dry weight was chromatographed on GLC column. A peak co-chromatographed with authentic sample of cis-trans-methyl abscisate and with same retention time was detected in each extract. A marked reduction was observed in the level of endogenous ABA in seeds stratified at both constant and fluctuating temperatures. The results show that same level of endogenous ABA was detected in February in both samples after 12 weeks stratification. Endogenous gibberellins The results obtained for the bioassay of gibberellin activities as detected by the lettuce hypocotyl assay are shown in Fig. 1 and Table 2. An increase in gibberellin activity was
At normal fluctuating temperatures 13
December
January
February
11
9
E 7 E
>.
-0 5 v o
Cl.
~
At
constant
chilling
temperature
~ 11
"
~
9
7
5
o
1.0
o
1.0
o
RF_
Fig. 1. Lettuce hypocotyl bioassay of eluates from thin layer chromatogram of purified extracts of Fagus sylvatica seeds. Seeds stratified at constant chilling temperature of 4 °C and normal fluctuating temperatures, out-ofdoors, for 12 weeks. Samples for extraction were taken in December, January and February after 4, 8 and 12 weeks stratification respectively. Each extract equivalent to 1 g dry weight of extracted seeds. The least significant differences between any 2 readings is 2.16 mm at the 1 % level (base of block parts).
56
H. M. M.
EL-ANTABLY
occurred at both constant chilling temperature of 4°C, and normal fluctuating temperatures, out-of-doors. This increase was found to be about 75 % more at normal fluctuating temperatures than at constant chilling temperature, after 12 weeks stratification. On the other hand, the activity was located at RF values 0.9-1,0, 0.5-0.6 and 0.0-0.6, and 0.7-1.0 at normal fluctuating temperatures, after 4, 8, and 12 weeks stratification respectively, whereas at constant chilling temperature, no significant activity was detected after 4 weeks (in December), but after 8 and 12 weeks, it appeared at RF values 0.0-0.2 and 0.5-0.6 and RF 0.0-0.5 and 0.8-0.9, respectively. Endogenous cytokinin fl. The results obtained by the soybean callus bioassay for F. sylvatica seeds stratified at constant chilling temperature and normal fluctuating temperatures, are shown in Fig. 2 and Table 2. Cytokinin activity in each treatment was estimated as ng kinetin per g dry weight of extracted seeds. A marked increase in cytokinins yield was occurred At normal fluctuating temperatures 600
Febnary
January
Decr:mbcr
500
400
300
At constant chilling temperature
III U
300
200
100
0.5
1.0
0.5
1.0
o.s
1.0
Rf-
Fig. 2. Soybean callus bioassay of an extmet of Fagus sylvatiea. seeds. Seeds stratified at constant chilling temperature and normal fluctuating temperatures for 12 weeks. 2 g dry weight equivalent chromatographed in Butan-2-01: 25 % sp. gr. 0.91 ammonia (4: 1 v/v).
Growth Regulator Changes During Breaking Seed Dormancy
at both treatments after 8 weeks stratification (in January). After 12 weeks stratification the increase in cytokinins yield was found to be 49.6 % more at normal fluctuating temperatures out-of-doors than at constant chilling temperature at 4°C in the cold room. Discussion
Recently WEBB, STADEN and WAREING (1973), suggested that the breaking of seed dormancy at low temperature involved the initiation of an ordered sequence of events that include the changes in growth hormones necessary to determine the course of development for the removal of dormancy. It was reported by several investigators that marked decrease in inhibitor content was occurred as a result of low temperature after ripening. This was observed in Prunus avium (LIPE and CRANE 1966), Juglans regia (MARTIN et al. 1969), Acer saccharum (WEBB et al.1973), and Pbnus taeda(PoUL et al.1973). However, in F. excelsior, VILLIERiil and WAREING (1965) were unable to detect any changes in inhibitor content with stratification, whereas KENTZU (1966) reported a reduction in the levels of ether-soluble inhibitors with stratification in this species. On the other hand, WEBB and WAREING (1972) reported that leaching sycamore seeds resulted in germination of dormant seeds, possibly by decreasing the level of inhibitor in the embryo. Regarding endogenous auxin and breaking of dormancy, BLOMMAERT (1955) noted that during the dormant period of Prunus persica buds, auxin content was low and the inhibitor was high and the situation was reversed as dormancy was broken. On the other hand, POUL et al. (1973) were able to demonstrate in Pinus taeda seeds slight increase in auxin-like activity after 28 d of stratification. The present result showed that 12 weeks stratification resulted in increasing germination percentage but was higher following normal fluctuating temperatures than at constant chilling temperature. Moreover, stratification whether at constant chilling temperature or normal fluctuating temperatures, showed an equal decrease in endogenous ABA after 12 weeks, whereas endogenous IAA increased remarkably in both treatments but was found to be 64.3 % greater at normal fluctuating temperatures than at constant chilling temperature. Such result confirm other observations and support previous findings that ABA is involved in seed dormancy. The results also showed that both endogenous gibberellins and cytokinins increased after 12 weeks stratification with a greater response at normal fluctuating temperatures. This increase on endogenous gibberellins and cytokinins at normal fluctuating temperatures may be related to rising temperatures out-of-doors at the end of February following an earlier period of chilling in December and January, exactly as for IAA. These results of gibberellins are in agreement with those obtained by POUL et al. (1973), and WEBB et al. (1973). The increase in endogenous cytokinins observed in this work confirm previous findings by WEBB and WAREING (1972) for seed of Acer pseudoplatanus, VAN STADEN et al. (1972) and WEBB et al. (1973) for seeds of Acer saccharum, and BBOWN and VAN STADEN (1973) for seeds of Protea compacta and Leucadendron daphnoides. However, these results
58
H. }I. M. EL-AxLUILY, Growth Regulator Changes During Breaking Seed Dormancy
suggested that the increase in growth promoters at normal fluctuating temperatures than at constant chilling temperature could be due to the activity of the cotyledons or the radicles in synthesizing such substances after dormancy in their seeds was broken. It is well-known that both auxin and cytokinins are required for the growth of callus and other plant tissues (SKOOG and ARMSTRONG 1970). On the other hand, the observations of BrswAs et al. (1972) that kinetin failed to improve the germination of unstratified seeds, while the 21-day stratified seeds responded favourably to this treatment, indicate that another substance is present in the 21-day stratified seeds which interact with kinetin and improve germination. Thus, if account is taken of the present findings, IAA in combination with cytokinins should be also involved in seeds dormancy particularly in later stage of development. References BISWAS, P. K., BONAMY, P. A., and POUL, K. B., Physiol. Plant. 27, 71-76 (1972). BLOMMAERT, K. L. J., Union S. Africa, Dept. Agric. Sci. Bull. 368, 1-23 (1955). BROWN, N. A. C., and VAN STADEN, J., Physiol. Plant. 28, 388-392 (1973). FRANKLAND, B., and WAREING, P. F., Nature 185, 255-256 (1960). - - J. Exp. Bot. 17, 596-611 (1966). HORGAN, R., HEWETT, E. W., PURSE, J. G., HORGAN, J. M., and WAREING, P. F., Plant Sci. Letters 1
321- 324 (1973). KHAN, A. A., Science 171, 853-859 (1971). KENTZU, T., Acta Soc. Bot. Pol. 35, 575-~85 (1966). LENTON, J. R., PERRY, V. M., and SAUNDERS, P. F., Planta 96,271-280 (1973). LIPE, W. N., and CRANE, J. C., Science 103, 541-542 (1966). MARTIN, G. C., MASON, M. 1. R., and FORDE, H. E., J. Amer. Soc. Hort. Sci. 94, 13-17 (1969). MILLER, C. 0., In: F. Wightman and G. Seterfield (eds), Biochem. and Physiol. of Plant Growth Regulators, Runge Press, Ottawa pp. 33 (1968). PINFIELD, N., PH. D. Thesis, Univ. of Wales, (1965). POUL, K. B., PETER, C. S., and BISWAS, P. K., Physiol. Plantar. 28, 530-534 (1971). Ross, J. D., and BRADBEER, J. W., Planta 100, 288-302 (1971). SKOOG, F., and ARMSTRONG, D. J., Plant Physiol. 21, 359-384 (1970). VAN STADEN, J., WEBB, D. P., and WAREING, P. F., Planta 104, 110-114 (1972). TUKEY, J. W., Transact. New York Acad. Sci. 16, 88-97 (1953). VILLIERS, T. A., and WAREING, P. F., Nature, 185, 112 (1960). - - J. Exp. Bot. 16, 519-531 (1965). WEBB, D. P., and WAREING, P. F., Planta 104, 115-125 (1972). - VAN STADEN, J., and WAREING, P. F., J. Exp. Bot. Vol. 24, 78, 105-116 (1973). Received December 17, 1975; Revision March 2, 1976. Author's address: Dr. HAMED M. M. EL-ANTABLY, Department of Botany, Faculty of Agriculture, Ain Shams University, Shoubra EI-Kheima, Cairo, Egypt.
Changes in Auxin. Germination Inhibitors, Gibberellins, and Cytokinins during the Breaking of Seed Dormancy in Fagus sylvatica HAMED M. M. EL-ANTABLY Department of Botany, Faculty of Agriculture Ain Shams University, Cairo, Egypt Key Term Index: phytohormones, abscisic acid, auxin, gibberellins cytokinins, seed dormancy, plant development; Fagus sylvatica L.
Summary Studies on the effect of constant chilling temperature of 4 °C and normal fluctuating temperatures out-of-doors on germinating percentage and endogenous IAA, ABA, gibberellins and cytokinins during the breaking of dormancy of Fagus sylvatica seeds was investigated. Dormant seeds showed 40 and 64 per cent germination after 12 weeks stratification at constant chilling temperature and normal fluctuating temperatures, respectively. Marked increase in IAA levels occurred as a result of both treatments. The increase in IAA was found to be 64 per cent greater at normal fluctuating temperatures than at constant chilling temperature after 12 weeks stratification. The results also showed an equal loss in endogenous ABA after 12 weeks stratification at both treatments. Moreover, a marked increase in gibberellin-like activity at both treatments was found to be almost 75 per cent more at normal fluctuating temperatures than at constant chilling temperature after 12 weeks stratification. The results also showed a high yield in the butano soluble cytokinins with both treatments. The increase in cytokinins was found to be 50 per cent more at normal fluctuating temperatures than at constant chilling temperature.
Introduction
The seeds of Fagus sylvatica L. usually require a period of 3 months of after-ripening at constant chilling temperature of 2 -4°C before they were capable of germination. At natural fluctuating temperatures in winter, the seeds pass almost the same period of after-ripening. During this period, biochemical and physiological changes (PINFIELD 1965) usually occur to bring the seeds out of dormancy and germinate readily in the spring. VILLIERS and WAREING (1960) suggested that dormancy in Fraxinus excelsior seeds is controlled by the interaction of growth promotors and inhibitors. POUL et al. (1973) were able to demonstrate slight increase in auxin-like activity after 28 d of stratification in loblolly pine seeds, while the inhibitor level detected by Avena coleoptile bioassay was found to disappear almost completely. Chilling temperature after ripening resulted in a decrease in endogenous inhibitors in Prunus avium (LIPE and CRANE 1966), Juglans regia (MARTIN et al. 1969), Acer saccharum (WEBB et al. 1973), and Pinus taedar (POUL et al. 1973). In F. excelsior, VILLIERS and WAREING (1965) reported that the level of water soluble inhibitors do not seem to change markedly with stratification, whereas KENTZU (1966) was able to show a reduction in the levels of ether-soluble inhibitors 4*
H. M. 1\1. EL-ANL\BLY
52
with stratification in this species. In 1966 FRANKLAND and WAREING reported that stratification leads to small increase in the levels of cndogenous gibberellins in Carulus avellana seeds. When such chilled seeds transferred to 20°C there was a considerable rise in endogenous gibberellins estimated as 64-fold (GLC) or 79-fold (TLCjbioassay), this rise preceding the detection of measurable growth of the embryonic axis (Ross and BRAD BEER 1971). On the other hand, POUL et al. (1973) were able to detect gradual increase in endogenous gibberellin in Pinus taeda following 42-day stratification. KHAN (1971) has suggested that cytokinins play "a permissive role" in seed dormancy, Whereas VAN STADEN et al. (1972) suggested that cytokinins may play an important role in the dormancy of Acer saccharum seeds and concluded that the role of endogenous cytokinin in seed dormancy remains to be demonstrated. The present investigation was undertaken to follow changes in endogenous auxin, ABA, gibberellins and cytokinins levels in association with chilling and emergence from dormancy in seeds of Fagus sylvatica L. during constant chilling temperature of 4 °C and normal fluctuating temperatures, out-of-doors. Material and Methods
Plant material Nuts of F. sylvatica were collected from trees grown in the forest near Aarhus, Denmark, in November 1973. They were mixed with an equal volume of soil in polythene bags and closed with rubber bands. Half the bags were placed in the cold room at 4 DC to receive constant chilling temperature of 4 DC, whereas the other half were buried in a soil pit out-of-doors to receive normal fluctuating temperatures. The bags were checked weekly for moisture and fungal growth. For germination test and extraction, nuts were taken monthly from both cold room and out-ofdoor bags, in December, January and February i. e. after 1,2 and 3 months, respectively.
Extraction
of auxin and abscisic acid
(ABA)
The growth regulators IAA and ABA were extracted and identified by gas-liquid chromatography (GLC) using the same method described by LENTON et al. (1971) for IAA as for ABA. The seeds were extracted with 80 % cold methanol using an "Atomix" blender. The methanol was removed from the extract under vacuum at 35 DC, and the acidic, ethyl acetate soluble fractions were separated from the resultant aqueous solutions by solvent partition.
PVP column The ethyl acetate soluble acids were further purified by being passed through a column of polyvinylpyrrolidone (PVP) and eluted with phosphate buffer at pH 8.0 at a flow rate of 91 drops (5.25 ml) per 12 min. The main ABA or IAA activities eluted at fraction 5-12 (between 26-60 ml) or at 16 to 24 (between 84-126 ml) respectively, were individually bulked for further extraction with diethyl ether and reduced to dryness for loading on TLC plates.
Thin-layer chromatography (T LC) The dried samples were dissolved in ethanol and loaded on 20 x 20 cm, 0.25 mm plates of Silica gel GE 254 (E. Merck, A. G.) which had been activated at 105 DC for one hour. Markers of authentic IAA or ABA were placed on either sides of the streak and the plates were developed with ethyl acetate: chloroform: acetic acid (15: 5: 1 v Iv) in tanks lined with filter paper. After developing, IAA and ABA were localized under U. V. radiation and the corresponding zones were marked. The individual
Growth Regulator Changes During Breaking Seed Dormancy
53
zones were luted with ethanol and the eluates reduced to dryness and placed in a vacuum desiccator until required.
Methylation The eluates from zones corresponding to IAA or ABA were methylated with diazomethane. The reagent was freshly prepared from p-tolylsulphonylmethylnitrosamide in a very clean, dry glass apparatus. After 16 h at room temperature, the reaction mixture was evaporated to dryness, and the methylated IAA or ABA were redissolved in a small, known volume of chloroform, when they were required for injection onto the GLC column.
Gas-liquid chromatography The GLC analyses were carried out with a Beckman GC 65 instrument with flame ionisation detector. The glass column, 6 feet X l/S inch, was packed with Epon 1001 on AW DMCS chromosorb W, 60-S0 mesh. The carrier gas was N 2 , 50 ml/min. Samples were analysed for both IAA and ABA at 200°C isotherm.
Extraction of gibberellins After passing the ethyl acetate soluble acids through a column of polyvinylpyrrolidone (PVP), eluted with phosphate buffer at pH S.O, the X purified material was extracted again with diethyl ether and reduced to dryness for loading on TLC plates.
T LC for gibber ellins Chromatograms intended for gibberellin bioassay, were developed with isopropanol, 35 % sp.gr. 0.91 ammonia, and water (10: 1: 1 v/v) and divided into 10 equal strips, each of which was scraped off, eluted with 2 ml distilled water, and tested for gibberellins activity using lettuce hypocotyl assay (FRANKLAND and WAREING 1960).
Extraction of cytokinins The seeds were extracted with SO % cold methanol, using the method described by HORGAN et al. (1973). The methanol was removed from the extract under vacuum at 35°C, filtered, and the filterates were adjusted to pH 2.5 with 2 N HCl. The methanolic extract was partitioned against ethyl acetate, and the ethyl acetate fraction discarded. The aqueous fraction was adjusted to pH 7.0, and partitioned against water-saturated n-butanol. After reduction to a small volume, the butanol fraction was chromatographed on Whatman's N. 3 MM paper in sec-butanol: 25% sp. gr. 0.91 ammonia (4: 1 v/v).
Soybean callus bioassay Paper chromatogram was cut into 10 equal RF strips and placed directly into 50 ml test tubes, to which 20 ml of medium (MILLER 1965) was added. After autoclaving and cooling, 100 mg pieces of soybean callus was added to each tube. The cultures were maintained at 30°C under continuous light, and the callus fresh weights determined after 21 days.
Results
Germination The results obtained in Table 1 show that no germination was occurred after 4 weeks stratification at either constant chilling temperature or normal fluctuating temperatures. After 8 weeks stratification, germination percentages were 9 and 17, whereas after 12 weeks, were 40 and 64 at constant and fluctuating temperatures respectively. At normal
54
H. M. :\1.
EL-A)lTABLY
Table 1. Effect of Stratification all genm:nation percentage of Fagus sylvatica, seeds. were stratified at constant chilling temperature of 4 °C and at normal fluctuating temperatures, out-of-doors, for 12 weeks. The germination of samples of 50 test nuts for each case. ~uts
Stratification in week
Sampling time
4 8 12
December January February
Germination % At constant chilling Temp. (4°C)
At normal fluctuating Temp. (out-of-doors)
9 40
17 64
fluctuating temperatures, the seeds show 60% increase in germination more than at constant chilling temperature after 12 weeks stratification. Endogenous IAA Table 2 represent the results of the GLC analyses of the acidic ethyl acetate fractions corresponding to 5 g dry weight of extracted seeds, developed on TLC plates, in a mixture of ethyl acetate, chloroform, acetic acid (15: 5: 1 vjv). The zone corresponding to IAA marker was scraped and eluted with ethanol. 0.5,tt1 of the methylated sample equivalent to 2 mg dry weight was chromatographed on the GLC column. A peak cochromatographed with methylated authentic IAA and with same retention time was Table 2. Endogenous contents of fAA, ABA, Gibberellins and Cytokinins. Extracted seeds of Fagus sylvatica, stratified at constant chilling temp. of 4°C, and normal fluctuating temp., out-of-doors, for 12 weeks. Samples for extraction were taken in December, January and February, after 4, 8 and 12 weeks respectively. ng/g dry weight At normal fluctuating Temp. (out-of-doors)
At constant chilling Temp. (4°C)
IAA ABA Gibberellins Cytokinins
December
January
February
December
January
35 126.5 22
90 24.3 86 243
140 12.5 215 675
30 120 39 25
105 33.3 70 210
77
February 230 12.5 376 1010
detected in each extract. It can be seen that there is remarkable ij1crease in IAA levels as a result of both stratification treatments. The amount of IAA detected after 12 weeks stratification was found to be more in seeds subjected to normal fluctuating temperatures than in seeds kept at constant chilling temperature in cold room. Such increase at normal fluctuating temperatures was found to be 64.3 % as much as constant chilling temperature. Endogenous ABA Table 2 also represent the data of GLC analyses of the acidic ethyl acetate fraction corresponding to 5 g dry weight of extracted tissue developed on TLC plates in same
Growth Regulator Changes During Breaking Seed Dormancy
55
solvent as IAA. The zone corresponding to an ABA marker was scraped and eluted with ethanol. 0.5,u1 of methylated sample equivalent to 2 mg dry weight was chromatographed on GLC column. A peak co-chromatographed with authentic sample of cis-trans-methyl abscisate and with same retention time was detected in each extract. A marked reduction was observed in the level of endogenous ABA in seeds stratified at both constant and fluctuating temperatures. The results show that same level of endogenous ABA was detected in February in both samples after 12 weeks stratification. Endogenous gibberellins The results obtained for the bioassay of gibberellin activities as detected by the lettuce hypocotyl assay are shown in Fig. 1 and Table 2. An increase in gibberellin activity was
At normal fluctuating temperatures 13
December
January
February
11
9
E 7 E
>.
-0 5 v o
Cl.
~
At
constant
chilling
temperature
~ 11
"
~
9
7
5
o
1.0
o
1.0
o
RF_
Fig. 1. Lettuce hypocotyl bioassay of eluates from thin layer chromatogram of purified extracts of Fagus sylvatica seeds. Seeds stratified at constant chilling temperature of 4 °C and normal fluctuating temperatures, out-ofdoors, for 12 weeks. Samples for extraction were taken in December, January and February after 4, 8 and 12 weeks stratification respectively. Each extract equivalent to 1 g dry weight of extracted seeds. The least significant differences between any 2 readings is 2.16 mm at the 1 % level (base of block parts).
56
H. M. M.
EL-ANTABLY
occurred at both constant chilling temperature of 4°C, and normal fluctuating temperatures, out-of-doors. This increase was found to be about 75 % more at normal fluctuating temperatures than at constant chilling temperature, after 12 weeks stratification. On the other hand, the activity was located at RF values 0.9-1,0, 0.5-0.6 and 0.0-0.6, and 0.7-1.0 at normal fluctuating temperatures, after 4, 8, and 12 weeks stratification respectively, whereas at constant chilling temperature, no significant activity was detected after 4 weeks (in December), but after 8 and 12 weeks, it appeared at RF values 0.0-0.2 and 0.5-0.6 and RF 0.0-0.5 and 0.8-0.9, respectively. Endogenous cytokinin fl. The results obtained by the soybean callus bioassay for F. sylvatica seeds stratified at constant chilling temperature and normal fluctuating temperatures, are shown in Fig. 2 and Table 2. Cytokinin activity in each treatment was estimated as ng kinetin per g dry weight of extracted seeds. A marked increase in cytokinins yield was occurred At normal fluctuating temperatures 600
Febnary
January
Decr:mbcr
500
400
300
At constant chilling temperature
III U
300
200
100
0.5
1.0
0.5
1.0
o.s
1.0
Rf-
Fig. 2. Soybean callus bioassay of an extmet of Fagus sylvatiea. seeds. Seeds stratified at constant chilling temperature and normal fluctuating temperatures for 12 weeks. 2 g dry weight equivalent chromatographed in Butan-2-01: 25 % sp. gr. 0.91 ammonia (4: 1 v/v).
Growth Regulator Changes During Breaking Seed Dormancy
at both treatments after 8 weeks stratification (in January). After 12 weeks stratification the increase in cytokinins yield was found to be 49.6 % more at normal fluctuating temperatures out-of-doors than at constant chilling temperature at 4°C in the cold room. Discussion
Recently WEBB, STADEN and WAREING (1973), suggested that the breaking of seed dormancy at low temperature involved the initiation of an ordered sequence of events that include the changes in growth hormones necessary to determine the course of development for the removal of dormancy. It was reported by several investigators that marked decrease in inhibitor content was occurred as a result of low temperature after ripening. This was observed in Prunus avium (LIPE and CRANE 1966), Juglans regia (MARTIN et al. 1969), Acer saccharum (WEBB et al.1973), and Pbnus taeda(PoUL et al.1973). However, in F. excelsior, VILLIERiil and WAREING (1965) were unable to detect any changes in inhibitor content with stratification, whereas KENTZU (1966) reported a reduction in the levels of ether-soluble inhibitors with stratification in this species. On the other hand, WEBB and WAREING (1972) reported that leaching sycamore seeds resulted in germination of dormant seeds, possibly by decreasing the level of inhibitor in the embryo. Regarding endogenous auxin and breaking of dormancy, BLOMMAERT (1955) noted that during the dormant period of Prunus persica buds, auxin content was low and the inhibitor was high and the situation was reversed as dormancy was broken. On the other hand, POUL et al. (1973) were able to demonstrate in Pinus taeda seeds slight increase in auxin-like activity after 28 d of stratification. The present result showed that 12 weeks stratification resulted in increasing germination percentage but was higher following normal fluctuating temperatures than at constant chilling temperature. Moreover, stratification whether at constant chilling temperature or normal fluctuating temperatures, showed an equal decrease in endogenous ABA after 12 weeks, whereas endogenous IAA increased remarkably in both treatments but was found to be 64.3 % greater at normal fluctuating temperatures than at constant chilling temperature. Such result confirm other observations and support previous findings that ABA is involved in seed dormancy. The results also showed that both endogenous gibberellins and cytokinins increased after 12 weeks stratification with a greater response at normal fluctuating temperatures. This increase on endogenous gibberellins and cytokinins at normal fluctuating temperatures may be related to rising temperatures out-of-doors at the end of February following an earlier period of chilling in December and January, exactly as for IAA. These results of gibberellins are in agreement with those obtained by POUL et al. (1973), and WEBB et al. (1973). The increase in endogenous cytokinins observed in this work confirm previous findings by WEBB and WAREING (1972) for seed of Acer pseudoplatanus, VAN STADEN et al. (1972) and WEBB et al. (1973) for seeds of Acer saccharum, and BBOWN and VAN STADEN (1973) for seeds of Protea compacta and Leucadendron daphnoides. However, these results
58
H. }I. M. EL-AxLUILY, Growth Regulator Changes During Breaking Seed Dormancy
suggested that the increase in growth promoters at normal fluctuating temperatures than at constant chilling temperature could be due to the activity of the cotyledons or the radicles in synthesizing such substances after dormancy in their seeds was broken. It is well-known that both auxin and cytokinins are required for the growth of callus and other plant tissues (SKOOG and ARMSTRONG 1970). On the other hand, the observations of BrswAs et al. (1972) that kinetin failed to improve the germination of unstratified seeds, while the 21-day stratified seeds responded favourably to this treatment, indicate that another substance is present in the 21-day stratified seeds which interact with kinetin and improve germination. Thus, if account is taken of the present findings, IAA in combination with cytokinins should be also involved in seeds dormancy particularly in later stage of development. References BISWAS, P. K., BONAMY, P. A., and POUL, K. B., Physiol. Plant. 27, 71-76 (1972). BLOMMAERT, K. L. J., Union S. Africa, Dept. Agric. Sci. Bull. 368, 1-23 (1955). BROWN, N. A. C., and VAN STADEN, J., Physiol. Plant. 28, 388-392 (1973). FRANKLAND, B., and WAREING, P. F., Nature 185, 255-256 (1960). - - J. Exp. Bot. 17, 596-611 (1966). HORGAN, R., HEWETT, E. W., PURSE, J. G., HORGAN, J. M., and WAREING, P. F., Plant Sci. Letters 1
321- 324 (1973). KHAN, A. A., Science 171, 853-859 (1971). KENTZU, T., Acta Soc. Bot. Pol. 35, 575-~85 (1966). LENTON, J. R., PERRY, V. M., and SAUNDERS, P. F., Planta 96,271-280 (1973). LIPE, W. N., and CRANE, J. C., Science 103, 541-542 (1966). MARTIN, G. C., MASON, M. 1. R., and FORDE, H. E., J. Amer. Soc. Hort. Sci. 94, 13-17 (1969). MILLER, C. 0., In: F. Wightman and G. Seterfield (eds), Biochem. and Physiol. of Plant Growth Regulators, Runge Press, Ottawa pp. 33 (1968). PINFIELD, N., PH. D. Thesis, Univ. of Wales, (1965). POUL, K. B., PETER, C. S., and BISWAS, P. K., Physiol. Plantar. 28, 530-534 (1971). Ross, J. D., and BRADBEER, J. W., Planta 100, 288-302 (1971). SKOOG, F., and ARMSTRONG, D. J., Plant Physiol. 21, 359-384 (1970). VAN STADEN, J., WEBB, D. P., and WAREING, P. F., Planta 104, 110-114 (1972). TUKEY, J. W., Transact. New York Acad. Sci. 16, 88-97 (1953). VILLIERS, T. A., and WAREING, P. F., Nature, 185, 112 (1960). - - J. Exp. Bot. 16, 519-531 (1965). WEBB, D. P., and WAREING, P. F., Planta 104, 115-125 (1972). - VAN STADEN, J., and WAREING, P. F., J. Exp. Bot. Vol. 24, 78, 105-116 (1973). Received December 17, 1975; Revision March 2, 1976. Author's address: Dr. HAMED M. M. EL-ANTABLY, Department of Botany, Faculty of Agriculture, Ain Shams University, Shoubra EI-Kheima, Cairo, Egypt.