Planta (Berl.) 104, 115-125 (1972) 9 by Springer-Verlag 1972
Seed Dormancy in Acer: Endogenous Germination Inhibitors and Dormancy in Acer pseudoplatanus L. D. P. W e b b a n d 1). F. W a r e i n g Botany Department, University College of Wales, Aberystwyth, U.K. Received December 28, 1971
Summary. Dormant seeds of Acer pseudoplatanus L. contain two zones of inhibition on paper chromatograms in " 1 0 : 1 : 1 " as detected by the lettuce and cress seed germination, and the wheat coleoptile bioassays. One zone at Rf 0.6-0.8 was partitioned into ethyl acetate at acid pH and was shown to contain ABA by its behaviour on GLC and isomerization under nltra-violet light. The other zone at Rf 0.9 was detected only in the germination bioassays and was partitioned into ethyl acetate over a range of pH indicating the presence of one or more neutral compounds. The inhibitors present in the embryo of dormant sycamore seeds inhibited the germination of non-dormant sycamore seeds at relatively low concentrations. A comparison with the effects of application of exogenous ABA indicated that endogenous ABA could not solely account for the inhibitory activity of seed extracts, which appeared to be due partly to the presence of ABA and partly to that of neutral compounds present in the embryo. Leaching treatments that removed dormancy led to a decrease in the level of inhibitors present mainly in the basic fraction. The exogenous application of kinetin to dormant sycamore seeds increased germination whereas gibberellic acid had no effect. Similar responses were obtained with lettuce seeds inhibited by the basic fraction of dormant sycamore seeds. I t is suggested that an inhibitor-eytokinin interaction may be involved in the dormancy of sycamore seeds. Introduction P r e v i o u s e x p e r i m e n t s on d o r m a n c y in seeds of Acer pseudoplatanus L. i n d i c a t e d t h a t whole fruits a n d seeds w i t h t h e t e s t a i n t a c t r e q u i r e a p e r i o d of chilling a t 5 C before d o r m a n c y is b r o k e n whereas b a r e e m b r y o s g e r m i n a t e i m m e d i a t e l y a t 20 C on m o i s t filter p a p e r w i t h o u t p r e t r e a t m e n t ( W e b b a n d W a r e i n g , 1972). Studies on t h e m a n n e r in which t h e t e s t a i m p o s e d d o r m a n c y on t h e e m b r y o i n d i c a t e d t h a t r e s t r i c t i o n on o x y g e n u p t a k e , w a t e r u p t a k e , m e c h a n i c a l r e s t r i c t i o n of e m b r y o e n l a r g e m e n t a n d t h e presence of g e r m i n a t i o n i n h i b i t o r s in t h e t e s t a are n o t limiting factors a t t h e e a r l y stages of d o r m a n c y . H o w e v e r , results f r o m leaching experim e n t s suggest t h a t d o r m a n c y is t h e result of t h e r e s t r i c t i o n b y t h e t e s t a to t h e o u t w a r d diffusion of a g e r m i n a t i o n inhibitor(s) p r e s e n t in t h e e m b r y o ( W e b b a n d W a r e i n g , 1972).
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D.P. Webb and P. F. Wareing:
This p a p e r describes e x p e r i m e n t s on t h e role of endogenous germination inhibitors in t h e d o r m a n c y of seeds of t h e s y c a m o r e maple.
Material and Methods Seed Storage and Germination. Sycamore fruits were collected from single opengrown trees in the Botany Gardens, Aberystwyth, in November, 1969. The fruits were air-dried and stored in sealed containers at 2-5 C. Seeds were germinated on two layers of Whatman No. 3 filter paper moistened with 7 ml of distilled water in petri dishes at 20 C. Protrusion of the radicle through the covering structures was used as the criterion for germination. Extraction o/Growth Regulators. Growth regulators were extracted by a modification of the methods of Hayashi and Rappaport (1962). The seed material was homogenized in a Virtis blendor with cold redistilled 80% methanol (10 ml per gram fresh weight). The slurry was continuously stirred for 12 h at 2 C and then filtered under vacuum. The residue was then re-extracted twice more with the same amount of methanol as before. The residue was dried at 80 C for 24 h and the dry weight recorded. The combined methanol extracts were reduced to aqueous and frozen at -12 C for approximately 18 h to precipitate chlorophyll. The aqueous fraction was thawed and centrifuged at 20,000 g for 30 min to remove suspended matter. The aqueous fraction was adjusted to pH 8.0 with 5% NaHCOa and partitioned 4 times with equal volumes of redistilled ethyl acetate. The bulked ethyl acetate fractions were dried over anhydrous Na2SO a and reduced to a small volume. This constituted the "Basic" fraction. The aqueous fraction was then adjusted to pH 2.5 with 1.0 N HCL and partitioned as before with equal volumes of redistilled ethyl acetate. The bulked fractions were dried over anhydrous Na~S04 and reduced to a small volume. This constituted the "Acidic" fraction with the "Aqueous" fraction remaining. Chromatography and Bioassay. The partitioned extracts were strip loaded onto Whatman ]qo. 1 chromatography paper and developed in a descending manner in the solvent system isopropanol:ammonia (sp g 0.88):water (10:1 : 1). The chromatograms were run 30 cm in the solvent, air-dried and cut into Rf zones for bioassay. In some cases the extracts were tested for inhibitory activity using germination of non-dormant sycamore seeds as the bioassay. However, because of the difficulty in storing sycamore seeds for long periods in this state the germination of lettuce seeds var. Grand Rapids was used routinely for the bioassay of germination inhibiters. Fifty lettuce seeds were added to petri dishes containing the segment of the chromatogram to be tested. This was moistened with 1.0 ml of distilled water. The seeds were germinated under continuous light at 25 C. Germination was recorded after 48 h. In all of the experiments reported the results are the means of at least two bioassays run at different times. The germination of cress seeds var. Plain were also used to detect inhibitory activity of extracts. :Fifty seeds were added to each dish with 1.0 ml of distilled water and placed in continuous light at 25 C. Germination was recorded after 48 h. In some experiments the extracts were assayed for inhibitory activity by means of the wheat var. Atle coleoptile "straight-growth" assay following the preeedure of Bentley and Honsley (1954). Results
Germination Inhibitors in Dormant Sycamore Seeds P r e v i o u s w o r k h a d shown t h a t d o r m a n c y in s y c a m o r e fruits was t h e result of t h e presence of g e r m i n a t i o n inhibitors in t h e e m b r y o which
Seed Dormancy in Acer
117
+2O 0
2
4
3
5
pH
6
-20 2 -40
8 -60E
o -808
+20] '6/o,8 0/
i.o'' 6.6 6.8 i,o' '6,6 0,8 i.o' ' 6.d 6.8 i.o' ' 6/0.d
7
8
9
10
0.6 0.8 t.0
0.6 0,8 1.0
i.o' pH
g -2o
0.5 0.8 1.0
0.6 0.8 1.0 Rf
Fig. 1. The effect of partitioning the aqueous fraction from dormant sycamore
seeds at different pHs on the distribution of germination inhibitors. The data represent the inhibition obtained from 1.0 g dry weight of seed chromatographed on paper in "10:1:1" and assayed with the lettuce seed germination bioassay
were restricted from being leached out b y the testa (Webb and Wareing, 1972). I t was further shown t h a t an aqueous solution containing the testas and periearps from dormant fruits had no inhibitory effect on the germination of non-dormant sycamore seeds. I t was concluded t h a t the covering structures do not significantly contribute to dormancy of the seed b y the presence of inhibitors they m a y contain. On the other hand, the embryo apparently contains substance(s) inhibitory to germination and these were initially investigated in dormant non-leached seeds. D o r m a n t seeds were extracted in 80 % redistflled methanol. The fractions were reduced to aqueous and partitioned with redistilled ethyl acetate. I t was found t h a t some degree of separation of inhibitors could be obtained b y altering the p H of the aqueous extract before partitioning with ethyl acetate. To examine this further the aqueous extract was divided into a number of ahquots (1.0 g dry weight) and each partitioned at a different p H ranging from p H 2.0 to p H 10.0. The ethyl acetate was reduced to a small volume, chromatographed on paper in "10:1 : 1 " and bioassayed with the lettuce seed germination bioassay. Germination inhibitors at different Rf values were extracted at all p H values (Fig. 1). Two zones of inhibition could be distinguished from
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D.P. Webb and P. F. Wareing:
the chromatograms. One zone at Rf 0.6-0.8 was extracted at acid p H and the other at Rf 0.9 was extracted over a range of pI-I from 4.0-10.0. To examine the nature of these inhibitors further, dormant seeds were extracted in methanol as before; this was reduced to aqueous and partitioned with ethyl acetate at p i t 8.0 and at p H 2.5. The basic, acidic and aqueous fractions so obtained were ehromatographed on paper in " 1 0 : 1 : 1 " and then bioassayed with the lettuce seed germination bioassay, the cress seed bioassay and the wheat coleoptile bioassay. As before, the acidic and basic fractions contained inhibitors. The acid fraction showed inhibition in the germination and straight growth bioassays at Rf 0.6-0.8. Similarly, if diethyl ether is used to partition the aqueous fraction at acid p H a zone of inhibition at the same l~f is found. This is characteristic of the fl inhibitor complex. I n the basic fraction which would also contain any neutral compounds, inhibition was found at Rf 0.9 in the lettuce and cress germination bioassay and at Rf 0.3-0.5 only in the wheat coleoptile bioassay. No inhibition was obtained in any bioassay when diethyl ether was used to partition the aqueous fraction at p H 8.0. The remaining aqueous fraction caused inhibition in all the bioassays used, but it was toxic as the tissue became soft and did not recover from the treatment on washing. Inhibition was located at Rf 0.1-0.3 which was highly pigmented and possibly contained tannins or polyphenols. Cornforth, et al. (1966) have shown that the fl inhibitor complex of sycamore leaves contains abscisic acid (ABA). The possibility t h a t the acidic ethyl acetate fraction obtained from sycamore seeds m a y also contain ABA as an active component was examined. The acidic fraction corresponding to 2.0 g fresh weight of dormant seeds was strip loaded onto a kieselgel GF2~~ thin-layer plate and developed 3 times in a mixture of toluene, ethyl acetate and acetic acid (50:5:5). The zone corresponding to the ABA marker was removed from the plate and eluted with ethyl acetate. Using the methods of Lenton et al. (1971), the sample was methylated with diazomethane and injected on the GLC fitted with the electron capture detectors. A peak with the same retention time as methyl abseisate (Me ABA) was obtained. Further, the sample was subjected to ultra-violet radiation for several hours and then reinjeered on the GLC. Under these conditions isomerization of Me ABA takes place to an equal mixture of cis-trans MeABA and its 2-trans isomer. The results indicated t h a t the peak corresponding to Me ABA was reduced and a similar increase in a second peak corresponding to the 2-trans isomer was observed, giving a ratio of 57.6% to 42.4%. This data strongly suggests that the acidic ethyl acetate fraction from sycamore seeds contains ABA. The above procedures were repeated with the basic ethyl acetate fraction. As expected no ABA was detected.
Seed Dormancy in Acer
119
A Abscisic acid 100
ISE
Seed extract
Control 1001/~.0mq/t . 80~
Control Testa extract 1.0seed/ml
//00
r o
4.0seeds/rnl
xlact
IcI r
O
O0.Omq/[
20t
20
2
4
6
8
&O. edsl t 10
Days :Fig. 2A and B. The effects of application of exogenous inhibitors to non-dormant sycamore seeds (A) the effects of abseisic acid. B the effects of the embryo extract and testa extract from dormant sycamore seeds
The E//ects o/Extracts/rein Dormant Seeds on the Germination o/NonDormant Sycamore Seeds Germination inhibitors as detected by the lettuce and cress seed germination bioassay can be extracted from dormant non-leached seeds. This does not mean, however, that they have a functional role in the seed from which they were extracted nor, in fact, that they will even inhibit the germination of these seeds. I t was necessary therefore, to examine the effects of inhibitors from dormant seeds on the germination of non-dormant sycamore seeds. Dormant sycamore seeds were divided into testas and embryos and these were extracted separately in 80% redistilled methanol as described previously. The bulked extracts were reduced to dryness on a rotary evaporator to remove all traces of solvent and then taken up in the appropriate volume of distilled water to give concentrations of 1 seed/ml and 4 seeds/ml. These extracts were then applied to non-dormant leached seeds on moist filter paper in petri dishes and germinated at 20 C. The maj or inhibition was associated with the embryo extract (Fig. 2 B). Significant inhibition was observed at a concentration of 1 seed/ml and at 4 seeds/ml almost total inhibition resulted. Significant inhibition was found with the testa extract at a concentration of 4 seeds/ml, but no significant inhibition was observed with the lower concentration of 1 seed/ml.
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D.P. Webb and P. F. Wareing:
The effects of the extracts were compared to t h a t of abscisic acid on the germination of non-dormant sycamore seeds. Inhibition was observed at a concentration of 1.0 rag/1 ABA (Fig. 2A), but the equivalent of more than 100.0 rag/1 ABA was required to obtain the same inhibition as 1 seed/ml of the embryo extract. This suggests t h a t if ABA is the active component as shown in Malus by 1%udnicki (1969), and in Fraxinus americana by Sondheimer et al. (1968) an expectionally high concentration must exist in the embryo or more probably the inhibition is the result of some compound other than ABA. Neither ABA nor the embryo extract was toxic to the seeds. The inhibition could be removed in most cases by repeated washing of the treated seeds with water. E/leers o/Leaching Treatments on Inhibitor Levels If seeds with the testa torn are placed on moist filter paper so that the tear in the testa is in contact with free liquid and leaching of the embryo can occur these seeds will germinate whereas, seeds so treated but placed on moist filter paper so t h a t the tear in the testa is placed upwards and no direct leaching of the embryo can take place will not germinate (Webb and Wareing, 1972). I t was postulated that this effect was due to the restriction b y the testa to the outward diffusion of inhibitors from the embryo. If this hypothesis is correct, it should be possible to detect high levels of inhibitor in the aqueous eluatcs from seeds with the testa torn and a corresponding low level in the embryo. Also, from seeds with the testa intact low levels of inhibitors would be detected in the aqueous eluates and correspondingly high levels of inhibitors in the embryo. To test this hypothesis 50 seeds with the testa intact and 50 seeds with the testa torn were placed in distilled water at 20 C for 18 h. The aqueous eluates from the treatments were reduced to a small volume and bioassayed with the lettuce seed germination assay. The eluates from seeds with the testa torn inhibited germination by 87% whereas eluates from seeds with the testa intact inhibited germination by only 7.0%. The levels of inhibitors present in the embryos were also determined. The treated seeds were extracted with 80% methanol and partitioned into acidic and basic fractions with ethyl acetate . These fractions were dried onto filter paper and the equivalent of 0.33 g dry weight was bioassayed with lettuce seeds as before. Inhibition of germination was observed in both fractions. The acidic fraction caused high inhibition of germination in the bioassay, but no differences were found between treatments. On the other hand, the basic fraction from seeds with the testa intact inhibited germination by 98 % and in seeds with the testa torn inhibited germination by 33 %.
Seed Dormancy in Acer
121
Table 1. The effects of leaching seed with the testa intact and testa torn on inhibitors present in the aqueous eluates Treatment
Fraction
% Inhibition relative to controls
Testa intact
Basic Acidic
24.0 2.6
Testa torn
Basic Acidic
97.8 45.7
The aqueous eluates from seeds with the testa torn were chromatographed on paper in " 1 0 : 1 : 1" and bioassayed with lettuce seeds. The major inhibition was found at Rf 0.8-0.9 suggesting the presence of inhibitors that were previously detected in the basic fraction. This was examined in more detail. I n another experiment, seeds with the testa intact and with the testa torn were soaked in distilled water for 18 h at 20 C. The aqueous eluates were partitioned with ethyl acetate at p H 8.0 and 2.5 to obtain the basic and acidic fractions. The fractions were tested for inhibitory activity using the cress seed germination bioassay. As before the majority of the inhibition observed was associated with the testa torn treatment (Table 1). Although some inhibition was associated with the acidic fraction the major part of the inhibition of germination was found with the basic fraction. The above studies indicate that there seems to be a correlation between the level of inhibitors in the basic fraction and the state of dormancy of the seed.
E//ects o/ Application o/ Exogenous Gibberellic Acid and Kinetin on Germination o] Dormant Sycamore Seeds There is considerable evidence to suggest that gibberellins m a y be involved in the breaking of dormancy in many tree seeds (Wareing, 1969; Wareing and Saunders, 1971). More recently, the report by Van Staden et al. (1972) suggest that in seeds of Acer saccharum, cytokinins may play an important role in dormancy. The possibility that applications of exogenous gibberellic acid and kinetin m a y overcome the dormancy of sycamore seeds was examined. Gibberellic acid at concentrations ranging from 1.0 rag/1 to 1000.0 mg/l did not significantly affect germination of sycamore seeds relative to the controls (Fig. 3A). I t was first thought the reason that GA 3 had no effect on the dormant seed was because it was simply not getting past the testa and therefore was not reaching the embryo. Once the testa
122
D.P. Wobb and P. F. Wareing: A
Gibberellic acid
Kinetin
100" [SE
10.0mq/L
'~176 801t
80 .~_ 60 S
Control ~
E
~, 40. 20"
o
J 5
,r162 10.0~ / / 100.0, // andlO00.O 20 mq/[ 10
15
20 Days
/ 5
ylO
is
oL
20
Fig. 3A and B. The effects of application of exogenous (A) gibberellic acid and (B) kinctin upon germination of dormant sycamore seeds
is cut or removed and the seed leached, the seed will germinate. Therefore attempts were made to inject concentrated solutions of GA 8 into the embryo without leaching, but these as well as other methods of introducing the hormone had no stimulatory effect on germination as judged b y the protrusion of the radicle through the testa. An inhibitor of gibberellin synthesis (2-chloroethyl)trimethylammoninm chloride (CCC), had no effect on germination of sycamore seed with the testa torn and leached at concentrations of 1.0, 10.0 and 100.0 rag/1. However, at a higher concentration of 1000.0 rag/1 germination was inhibited, but this concentration also appeared to be toxic. Kinetin, in contrast to GAa, stimulated germination of dormant seeds with the testa intact (Fig. 3 B). Significant stimulation of germination was observed with a concentration of 1.0 rag/1. In all treatments germination appeared normal. A similar response to application of exogenous GA 3 and kinetin was observed with lettuce seeds treated with the basic fraction from dormant sycamore seeds. Lettuce seed germination was inhibited 100.0% b y the basic ethyl acetate fraction from 0.36 g dry weight of seed. Kinetin at a concentration of 1.0 mg/1 completely overcame the inhibition from the basic fraction (Fig. 4) whereas GA 3 at a concentration of 10.0 mg/l had no effect.
Seed Dormancy in Acer
+20.
Basic fraction
123
Basic fraction plus 1.0mq/I kinetin
b')
-2
ga
"~E m o
0
-20.
~
-hO
o ~
-60. -8(
"(3
0.6 0.8 1.0 Rf
0.6 0.8 tO
Fig, 4. The effects of kinetin on the germination of lettuce seeds var. Grand
Rapids inhibited by the basic ethyl acetate fraction from 0.36 g dry weight of sycamore seeds chromatographed on paper in "10:1 : 1"
This is an important and interesting result since it suggests t h a t a cytokinin-inhibitor interaction m a y be involved in the breaking of dormancy in sycamore seeds. Discussion
According to Webb and Wareing (1972) dormancy in sycamore seeds is the result of the restriction b y the testa to the outward diffusion of germination inhibitors present in the embryo. The present results clearly indicate t h a t the dormant seed contains inhibitors as detected by the lettuce and cress seed bioassays. Some degree of separation of the inhibitors could be obtained by partitioning the aqueous extract at different p H s with ethyl acetate. The acidic fraction was shown to contain ABA b y its behaviour on GLC and b y isomcrization under ultra-violet light. The basic fraction did not contain ABA as would be expected, but nevertheless showed high inhibition in the germination bioassays. The inhibition from this fraction was found at Rf 0.9 on paper in " 1 0 : 1 : 1 " and was partitioned into ethyl acetate over a range of p t I from 4.0 to 10.0 suggesting the inhibitor(s) are neutral compounds. I t was further shown that the inhibitors present in the embryo of dormant sycamore seeds will inhibit the germination of non-dormant sycamore seeds at relatively low concentrations. The inhibitors obtained from the equivalent of 1.0 seed per ml caused greater inhibition of germination of sycamore seeds than did 100.0 mg/1 of ABA. This suggests t h a t if ABA is the active component a very high level of ABA is present in the embryo or, and more likely, the inhibition is the result of some compound other t h a n ABA. Thus, it is suggested that the inhibition is
124
D.P. Webb and P. F. Wareing:
partly due to the presence of ABA and partly to a neutral compound(s) present in the embryo. I n no case was the inhibition toxic as it could be overcome b y washing the seeds in distilled water. Wareing and Foda (1957) showed t h a t the coat effect in X a n t h i u m seed is the result of the presence of inhibitors located in the embryo that are restricted from being leached out by the testa. I n sycamore seeds it was found that leaching treatment t h a t breaks dormancy apparently leads to a decrease in inhibitors in the embryo. The aqueous eluates when partitioned with ethyl acetate and bioassayed were found to contain inhibitors mainly in the basic fraction. The application of exogenous kinetin to dormant sycamore seeds was found to increase germination whereas gibberellic acid apparently had no effect on germination. The lack of response to GA a does not appear to be due to it not penetrating to the embryo, although our results are not conclusive on this point. However, similar responses were obtained with lettuce seeds inhibited b y the basic fraction from sycamore seeds. Kinetin completely overcame the inhibition whereas GA a had no effect This suggests t h a t a possible cytokinin-inhibitor interaction m a y be important in the dormancy of sycamore seeds. Van Staden et al. (1972) found t h a t during the stratification of Acer 8accharum seeds the levels of cytokinins increased markedly after 20 d at 5 C. I t is possible t h a t the state of dormancy in sycamore seeds is dependent on the relative levels of cytokinins and inhibitors. Treatments which decrease the level of inhibitors, such as leaching, or increase the levels of cytokinins, as in the case of stratification, m a y lead to loss of dormancy. K h a n (1971) has suggested t h a t cytokinins play a permissive role in dormancy and germination. According to this hypothesis gibberellins are required for the breaking of dormancy. I t is well known t h a t increases in gibberellins take place during stratification of m a n y tree seeds (Wareing, 1969; Sinska and Lewak, 1970). Although application of exogenous GA s did not affect germination of dormant sycamore seeds, this by no means suggests that gibberellins are not important in dormancy in sycamore. Different gibberellins can v a r y markedly in their effects on germination (Ikuma and Thimann, 1963). References Bentley, J. A., Housley, S.: Bioassay of plant growth hormones. Physiol. Plant. 7, 405419 (1954). Cornforth, J. W., 1Vfilborrow,B. V., Ryback, G., Wareing, P. F. : Identity of sycamore "dormin" with abscisin II. Nature (Lond.) 205, 1269-1272 (1965). Hayashi, F., Rappaport, L.: Gibberellin-like activity of neutral and acidic substances in the potato tuber. Nature (Lond.) 195, 617-618 (1962). Ikuma, H., Thimann, K. V.: Activity of gibberellin " D " on the germination of photosensitive lettuce seeds. Nature (Load.) 197, 1313-1314 (1963).
Seed Dormancy in Acer
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Khan, A. A. : Cytokinins: Permissive role in seed germination. Science 171, 853-859 (1971). Lenton, J. P~., Perry, V. M., Saunders, P. F. : The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography. Planta (Berl.) 96, 271-280 (1971). Rudnicki, l~. : Studies on abscisic acid in apple seeds. Planta (Berl.) 86, 63-68 (1969). Sinska, I., Lewak, S.: Apple seed gibberellins. Physiol. Veg. 8, 661-667 (1970). Sondheimer, E., Tzou, D. S., Galson, E. D. : Abscisic acid levels and seed dormancy. Plant Physiol. 48, 1443-1447 (1968). Staden, J. van, Webb, D. P., Wareing, P. F. : The effect of stratification on endogenous cytokinin levels in seeds of Acer 8accharum. Planta (Berl.) (in press) (1972). Wareing, P. F. : Germination and dormancy. In: Physiology of plant growth and development, p. 605, ed. M. B. Wilkins. London: McGraw-Hill, 1969. Wareing, P. F., :Foda, H. A. : Growth inhibitors and dormancy in Xanthium seed. Physiol. Plant. 16, 266-280 (1957). Wareing, P. F., Saunders, P. F. : Hormones and dormancy. Ann. Rev. Plant Physiol. 22, 261-288 (1971). Webb, D. P., Wareing, P. F.: Seed dormancy in Acer: The role of the covering structures in dormancy of Acer pseudoplatanus L. J. exp. Bot. (in press) (1972). Dr. Paul Webb Botany Department University College of Wales Aberystwyth, Cards. U.K.
9 Planta(Berl.), Bd. 104:
Seed Dormancy in Acer: Endogenous Germination Inhibitors and Dormancy in Acer pseudoplatanus L. D. P. W e b b a n d 1). F. W a r e i n g Botany Department, University College of Wales, Aberystwyth, U.K. Received December 28, 1971
Summary. Dormant seeds of Acer pseudoplatanus L. contain two zones of inhibition on paper chromatograms in " 1 0 : 1 : 1 " as detected by the lettuce and cress seed germination, and the wheat coleoptile bioassays. One zone at Rf 0.6-0.8 was partitioned into ethyl acetate at acid pH and was shown to contain ABA by its behaviour on GLC and isomerization under nltra-violet light. The other zone at Rf 0.9 was detected only in the germination bioassays and was partitioned into ethyl acetate over a range of pH indicating the presence of one or more neutral compounds. The inhibitors present in the embryo of dormant sycamore seeds inhibited the germination of non-dormant sycamore seeds at relatively low concentrations. A comparison with the effects of application of exogenous ABA indicated that endogenous ABA could not solely account for the inhibitory activity of seed extracts, which appeared to be due partly to the presence of ABA and partly to that of neutral compounds present in the embryo. Leaching treatments that removed dormancy led to a decrease in the level of inhibitors present mainly in the basic fraction. The exogenous application of kinetin to dormant sycamore seeds increased germination whereas gibberellic acid had no effect. Similar responses were obtained with lettuce seeds inhibited by the basic fraction of dormant sycamore seeds. I t is suggested that an inhibitor-eytokinin interaction may be involved in the dormancy of sycamore seeds. Introduction P r e v i o u s e x p e r i m e n t s on d o r m a n c y in seeds of Acer pseudoplatanus L. i n d i c a t e d t h a t whole fruits a n d seeds w i t h t h e t e s t a i n t a c t r e q u i r e a p e r i o d of chilling a t 5 C before d o r m a n c y is b r o k e n whereas b a r e e m b r y o s g e r m i n a t e i m m e d i a t e l y a t 20 C on m o i s t filter p a p e r w i t h o u t p r e t r e a t m e n t ( W e b b a n d W a r e i n g , 1972). Studies on t h e m a n n e r in which t h e t e s t a i m p o s e d d o r m a n c y on t h e e m b r y o i n d i c a t e d t h a t r e s t r i c t i o n on o x y g e n u p t a k e , w a t e r u p t a k e , m e c h a n i c a l r e s t r i c t i o n of e m b r y o e n l a r g e m e n t a n d t h e presence of g e r m i n a t i o n i n h i b i t o r s in t h e t e s t a are n o t limiting factors a t t h e e a r l y stages of d o r m a n c y . H o w e v e r , results f r o m leaching experim e n t s suggest t h a t d o r m a n c y is t h e result of t h e r e s t r i c t i o n b y t h e t e s t a to t h e o u t w a r d diffusion of a g e r m i n a t i o n inhibitor(s) p r e s e n t in t h e e m b r y o ( W e b b a n d W a r e i n g , 1972).
116
D.P. Webb and P. F. Wareing:
This p a p e r describes e x p e r i m e n t s on t h e role of endogenous germination inhibitors in t h e d o r m a n c y of seeds of t h e s y c a m o r e maple.
Material and Methods Seed Storage and Germination. Sycamore fruits were collected from single opengrown trees in the Botany Gardens, Aberystwyth, in November, 1969. The fruits were air-dried and stored in sealed containers at 2-5 C. Seeds were germinated on two layers of Whatman No. 3 filter paper moistened with 7 ml of distilled water in petri dishes at 20 C. Protrusion of the radicle through the covering structures was used as the criterion for germination. Extraction o/Growth Regulators. Growth regulators were extracted by a modification of the methods of Hayashi and Rappaport (1962). The seed material was homogenized in a Virtis blendor with cold redistilled 80% methanol (10 ml per gram fresh weight). The slurry was continuously stirred for 12 h at 2 C and then filtered under vacuum. The residue was then re-extracted twice more with the same amount of methanol as before. The residue was dried at 80 C for 24 h and the dry weight recorded. The combined methanol extracts were reduced to aqueous and frozen at -12 C for approximately 18 h to precipitate chlorophyll. The aqueous fraction was thawed and centrifuged at 20,000 g for 30 min to remove suspended matter. The aqueous fraction was adjusted to pH 8.0 with 5% NaHCOa and partitioned 4 times with equal volumes of redistilled ethyl acetate. The bulked ethyl acetate fractions were dried over anhydrous Na2SO a and reduced to a small volume. This constituted the "Basic" fraction. The aqueous fraction was then adjusted to pH 2.5 with 1.0 N HCL and partitioned as before with equal volumes of redistilled ethyl acetate. The bulked fractions were dried over anhydrous Na~S04 and reduced to a small volume. This constituted the "Acidic" fraction with the "Aqueous" fraction remaining. Chromatography and Bioassay. The partitioned extracts were strip loaded onto Whatman ]qo. 1 chromatography paper and developed in a descending manner in the solvent system isopropanol:ammonia (sp g 0.88):water (10:1 : 1). The chromatograms were run 30 cm in the solvent, air-dried and cut into Rf zones for bioassay. In some cases the extracts were tested for inhibitory activity using germination of non-dormant sycamore seeds as the bioassay. However, because of the difficulty in storing sycamore seeds for long periods in this state the germination of lettuce seeds var. Grand Rapids was used routinely for the bioassay of germination inhibiters. Fifty lettuce seeds were added to petri dishes containing the segment of the chromatogram to be tested. This was moistened with 1.0 ml of distilled water. The seeds were germinated under continuous light at 25 C. Germination was recorded after 48 h. In all of the experiments reported the results are the means of at least two bioassays run at different times. The germination of cress seeds var. Plain were also used to detect inhibitory activity of extracts. :Fifty seeds were added to each dish with 1.0 ml of distilled water and placed in continuous light at 25 C. Germination was recorded after 48 h. In some experiments the extracts were assayed for inhibitory activity by means of the wheat var. Atle coleoptile "straight-growth" assay following the preeedure of Bentley and Honsley (1954). Results
Germination Inhibitors in Dormant Sycamore Seeds P r e v i o u s w o r k h a d shown t h a t d o r m a n c y in s y c a m o r e fruits was t h e result of t h e presence of g e r m i n a t i o n inhibitors in t h e e m b r y o which
Seed Dormancy in Acer
117
+2O 0
2
4
3
5
pH
6
-20 2 -40
8 -60E
o -808
+20] '6/o,8 0/
i.o'' 6.6 6.8 i,o' '6,6 0,8 i.o' ' 6.d 6.8 i.o' ' 6/0.d
7
8
9
10
0.6 0.8 t.0
0.6 0,8 1.0
i.o' pH
g -2o
0.5 0.8 1.0
0.6 0.8 1.0 Rf
Fig. 1. The effect of partitioning the aqueous fraction from dormant sycamore
seeds at different pHs on the distribution of germination inhibitors. The data represent the inhibition obtained from 1.0 g dry weight of seed chromatographed on paper in "10:1:1" and assayed with the lettuce seed germination bioassay
were restricted from being leached out b y the testa (Webb and Wareing, 1972). I t was further shown t h a t an aqueous solution containing the testas and periearps from dormant fruits had no inhibitory effect on the germination of non-dormant sycamore seeds. I t was concluded t h a t the covering structures do not significantly contribute to dormancy of the seed b y the presence of inhibitors they m a y contain. On the other hand, the embryo apparently contains substance(s) inhibitory to germination and these were initially investigated in dormant non-leached seeds. D o r m a n t seeds were extracted in 80 % redistflled methanol. The fractions were reduced to aqueous and partitioned with redistilled ethyl acetate. I t was found t h a t some degree of separation of inhibitors could be obtained b y altering the p H of the aqueous extract before partitioning with ethyl acetate. To examine this further the aqueous extract was divided into a number of ahquots (1.0 g dry weight) and each partitioned at a different p H ranging from p H 2.0 to p H 10.0. The ethyl acetate was reduced to a small volume, chromatographed on paper in "10:1 : 1 " and bioassayed with the lettuce seed germination bioassay. Germination inhibitors at different Rf values were extracted at all p H values (Fig. 1). Two zones of inhibition could be distinguished from
118
D.P. Webb and P. F. Wareing:
the chromatograms. One zone at Rf 0.6-0.8 was extracted at acid p H and the other at Rf 0.9 was extracted over a range of pI-I from 4.0-10.0. To examine the nature of these inhibitors further, dormant seeds were extracted in methanol as before; this was reduced to aqueous and partitioned with ethyl acetate at p i t 8.0 and at p H 2.5. The basic, acidic and aqueous fractions so obtained were ehromatographed on paper in " 1 0 : 1 : 1 " and then bioassayed with the lettuce seed germination bioassay, the cress seed bioassay and the wheat coleoptile bioassay. As before, the acidic and basic fractions contained inhibitors. The acid fraction showed inhibition in the germination and straight growth bioassays at Rf 0.6-0.8. Similarly, if diethyl ether is used to partition the aqueous fraction at acid p H a zone of inhibition at the same l~f is found. This is characteristic of the fl inhibitor complex. I n the basic fraction which would also contain any neutral compounds, inhibition was found at Rf 0.9 in the lettuce and cress germination bioassay and at Rf 0.3-0.5 only in the wheat coleoptile bioassay. No inhibition was obtained in any bioassay when diethyl ether was used to partition the aqueous fraction at p H 8.0. The remaining aqueous fraction caused inhibition in all the bioassays used, but it was toxic as the tissue became soft and did not recover from the treatment on washing. Inhibition was located at Rf 0.1-0.3 which was highly pigmented and possibly contained tannins or polyphenols. Cornforth, et al. (1966) have shown that the fl inhibitor complex of sycamore leaves contains abscisic acid (ABA). The possibility t h a t the acidic ethyl acetate fraction obtained from sycamore seeds m a y also contain ABA as an active component was examined. The acidic fraction corresponding to 2.0 g fresh weight of dormant seeds was strip loaded onto a kieselgel GF2~~ thin-layer plate and developed 3 times in a mixture of toluene, ethyl acetate and acetic acid (50:5:5). The zone corresponding to the ABA marker was removed from the plate and eluted with ethyl acetate. Using the methods of Lenton et al. (1971), the sample was methylated with diazomethane and injected on the GLC fitted with the electron capture detectors. A peak with the same retention time as methyl abseisate (Me ABA) was obtained. Further, the sample was subjected to ultra-violet radiation for several hours and then reinjeered on the GLC. Under these conditions isomerization of Me ABA takes place to an equal mixture of cis-trans MeABA and its 2-trans isomer. The results indicated t h a t the peak corresponding to Me ABA was reduced and a similar increase in a second peak corresponding to the 2-trans isomer was observed, giving a ratio of 57.6% to 42.4%. This data strongly suggests that the acidic ethyl acetate fraction from sycamore seeds contains ABA. The above procedures were repeated with the basic ethyl acetate fraction. As expected no ABA was detected.
Seed Dormancy in Acer
119
A Abscisic acid 100
ISE
Seed extract
Control 1001/~.0mq/t . 80~
Control Testa extract 1.0seed/ml
//00
r o
4.0seeds/rnl
xlact
IcI r
O
O0.Omq/[
20t
20
2
4
6
8
&O. edsl t 10
Days :Fig. 2A and B. The effects of application of exogenous inhibitors to non-dormant sycamore seeds (A) the effects of abseisic acid. B the effects of the embryo extract and testa extract from dormant sycamore seeds
The E//ects o/Extracts/rein Dormant Seeds on the Germination o/NonDormant Sycamore Seeds Germination inhibitors as detected by the lettuce and cress seed germination bioassay can be extracted from dormant non-leached seeds. This does not mean, however, that they have a functional role in the seed from which they were extracted nor, in fact, that they will even inhibit the germination of these seeds. I t was necessary therefore, to examine the effects of inhibitors from dormant seeds on the germination of non-dormant sycamore seeds. Dormant sycamore seeds were divided into testas and embryos and these were extracted separately in 80% redistilled methanol as described previously. The bulked extracts were reduced to dryness on a rotary evaporator to remove all traces of solvent and then taken up in the appropriate volume of distilled water to give concentrations of 1 seed/ml and 4 seeds/ml. These extracts were then applied to non-dormant leached seeds on moist filter paper in petri dishes and germinated at 20 C. The maj or inhibition was associated with the embryo extract (Fig. 2 B). Significant inhibition was observed at a concentration of 1 seed/ml and at 4 seeds/ml almost total inhibition resulted. Significant inhibition was found with the testa extract at a concentration of 4 seeds/ml, but no significant inhibition was observed with the lower concentration of 1 seed/ml.
120
D.P. Webb and P. F. Wareing:
The effects of the extracts were compared to t h a t of abscisic acid on the germination of non-dormant sycamore seeds. Inhibition was observed at a concentration of 1.0 rag/1 ABA (Fig. 2A), but the equivalent of more than 100.0 rag/1 ABA was required to obtain the same inhibition as 1 seed/ml of the embryo extract. This suggests t h a t if ABA is the active component as shown in Malus by 1%udnicki (1969), and in Fraxinus americana by Sondheimer et al. (1968) an expectionally high concentration must exist in the embryo or more probably the inhibition is the result of some compound other than ABA. Neither ABA nor the embryo extract was toxic to the seeds. The inhibition could be removed in most cases by repeated washing of the treated seeds with water. E/leers o/Leaching Treatments on Inhibitor Levels If seeds with the testa torn are placed on moist filter paper so that the tear in the testa is in contact with free liquid and leaching of the embryo can occur these seeds will germinate whereas, seeds so treated but placed on moist filter paper so t h a t the tear in the testa is placed upwards and no direct leaching of the embryo can take place will not germinate (Webb and Wareing, 1972). I t was postulated that this effect was due to the restriction b y the testa to the outward diffusion of inhibitors from the embryo. If this hypothesis is correct, it should be possible to detect high levels of inhibitor in the aqueous eluatcs from seeds with the testa torn and a corresponding low level in the embryo. Also, from seeds with the testa intact low levels of inhibitors would be detected in the aqueous eluates and correspondingly high levels of inhibitors in the embryo. To test this hypothesis 50 seeds with the testa intact and 50 seeds with the testa torn were placed in distilled water at 20 C for 18 h. The aqueous eluates from the treatments were reduced to a small volume and bioassayed with the lettuce seed germination assay. The eluates from seeds with the testa torn inhibited germination by 87% whereas eluates from seeds with the testa intact inhibited germination by only 7.0%. The levels of inhibitors present in the embryos were also determined. The treated seeds were extracted with 80% methanol and partitioned into acidic and basic fractions with ethyl acetate . These fractions were dried onto filter paper and the equivalent of 0.33 g dry weight was bioassayed with lettuce seeds as before. Inhibition of germination was observed in both fractions. The acidic fraction caused high inhibition of germination in the bioassay, but no differences were found between treatments. On the other hand, the basic fraction from seeds with the testa intact inhibited germination by 98 % and in seeds with the testa torn inhibited germination by 33 %.
Seed Dormancy in Acer
121
Table 1. The effects of leaching seed with the testa intact and testa torn on inhibitors present in the aqueous eluates Treatment
Fraction
% Inhibition relative to controls
Testa intact
Basic Acidic
24.0 2.6
Testa torn
Basic Acidic
97.8 45.7
The aqueous eluates from seeds with the testa torn were chromatographed on paper in " 1 0 : 1 : 1" and bioassayed with lettuce seeds. The major inhibition was found at Rf 0.8-0.9 suggesting the presence of inhibitors that were previously detected in the basic fraction. This was examined in more detail. I n another experiment, seeds with the testa intact and with the testa torn were soaked in distilled water for 18 h at 20 C. The aqueous eluates were partitioned with ethyl acetate at p H 8.0 and 2.5 to obtain the basic and acidic fractions. The fractions were tested for inhibitory activity using the cress seed germination bioassay. As before the majority of the inhibition observed was associated with the testa torn treatment (Table 1). Although some inhibition was associated with the acidic fraction the major part of the inhibition of germination was found with the basic fraction. The above studies indicate that there seems to be a correlation between the level of inhibitors in the basic fraction and the state of dormancy of the seed.
E//ects o/ Application o/ Exogenous Gibberellic Acid and Kinetin on Germination o] Dormant Sycamore Seeds There is considerable evidence to suggest that gibberellins m a y be involved in the breaking of dormancy in many tree seeds (Wareing, 1969; Wareing and Saunders, 1971). More recently, the report by Van Staden et al. (1972) suggest that in seeds of Acer saccharum, cytokinins may play an important role in dormancy. The possibility that applications of exogenous gibberellic acid and kinetin m a y overcome the dormancy of sycamore seeds was examined. Gibberellic acid at concentrations ranging from 1.0 rag/1 to 1000.0 mg/l did not significantly affect germination of sycamore seeds relative to the controls (Fig. 3A). I t was first thought the reason that GA 3 had no effect on the dormant seed was because it was simply not getting past the testa and therefore was not reaching the embryo. Once the testa
122
D.P. Wobb and P. F. Wareing: A
Gibberellic acid
Kinetin
100" [SE
10.0mq/L
'~176 801t
80 .~_ 60 S
Control ~
E
~, 40. 20"
o
J 5
,r162 10.0~ / / 100.0, // andlO00.O 20 mq/[ 10
15
20 Days
/ 5
ylO
is
oL
20
Fig. 3A and B. The effects of application of exogenous (A) gibberellic acid and (B) kinctin upon germination of dormant sycamore seeds
is cut or removed and the seed leached, the seed will germinate. Therefore attempts were made to inject concentrated solutions of GA 8 into the embryo without leaching, but these as well as other methods of introducing the hormone had no stimulatory effect on germination as judged b y the protrusion of the radicle through the testa. An inhibitor of gibberellin synthesis (2-chloroethyl)trimethylammoninm chloride (CCC), had no effect on germination of sycamore seed with the testa torn and leached at concentrations of 1.0, 10.0 and 100.0 rag/1. However, at a higher concentration of 1000.0 rag/1 germination was inhibited, but this concentration also appeared to be toxic. Kinetin, in contrast to GAa, stimulated germination of dormant seeds with the testa intact (Fig. 3 B). Significant stimulation of germination was observed with a concentration of 1.0 rag/1. In all treatments germination appeared normal. A similar response to application of exogenous GA 3 and kinetin was observed with lettuce seeds treated with the basic fraction from dormant sycamore seeds. Lettuce seed germination was inhibited 100.0% b y the basic ethyl acetate fraction from 0.36 g dry weight of seed. Kinetin at a concentration of 1.0 mg/1 completely overcame the inhibition from the basic fraction (Fig. 4) whereas GA 3 at a concentration of 10.0 mg/l had no effect.
Seed Dormancy in Acer
+20.
Basic fraction
123
Basic fraction plus 1.0mq/I kinetin
b')
-2
ga
"~E m o
0
-20.
~
-hO
o ~
-60. -8(
"(3
0.6 0.8 1.0 Rf
0.6 0.8 tO
Fig, 4. The effects of kinetin on the germination of lettuce seeds var. Grand
Rapids inhibited by the basic ethyl acetate fraction from 0.36 g dry weight of sycamore seeds chromatographed on paper in "10:1 : 1"
This is an important and interesting result since it suggests t h a t a cytokinin-inhibitor interaction m a y be involved in the breaking of dormancy in sycamore seeds. Discussion
According to Webb and Wareing (1972) dormancy in sycamore seeds is the result of the restriction b y the testa to the outward diffusion of germination inhibitors present in the embryo. The present results clearly indicate t h a t the dormant seed contains inhibitors as detected by the lettuce and cress seed bioassays. Some degree of separation of the inhibitors could be obtained by partitioning the aqueous extract at different p H s with ethyl acetate. The acidic fraction was shown to contain ABA b y its behaviour on GLC and b y isomcrization under ultra-violet light. The basic fraction did not contain ABA as would be expected, but nevertheless showed high inhibition in the germination bioassays. The inhibition from this fraction was found at Rf 0.9 on paper in " 1 0 : 1 : 1 " and was partitioned into ethyl acetate over a range of p t I from 4.0 to 10.0 suggesting the inhibitor(s) are neutral compounds. I t was further shown that the inhibitors present in the embryo of dormant sycamore seeds will inhibit the germination of non-dormant sycamore seeds at relatively low concentrations. The inhibitors obtained from the equivalent of 1.0 seed per ml caused greater inhibition of germination of sycamore seeds than did 100.0 mg/1 of ABA. This suggests t h a t if ABA is the active component a very high level of ABA is present in the embryo or, and more likely, the inhibition is the result of some compound other t h a n ABA. Thus, it is suggested that the inhibition is
124
D.P. Webb and P. F. Wareing:
partly due to the presence of ABA and partly to a neutral compound(s) present in the embryo. I n no case was the inhibition toxic as it could be overcome b y washing the seeds in distilled water. Wareing and Foda (1957) showed t h a t the coat effect in X a n t h i u m seed is the result of the presence of inhibitors located in the embryo that are restricted from being leached out by the testa. I n sycamore seeds it was found that leaching treatment t h a t breaks dormancy apparently leads to a decrease in inhibitors in the embryo. The aqueous eluates when partitioned with ethyl acetate and bioassayed were found to contain inhibitors mainly in the basic fraction. The application of exogenous kinetin to dormant sycamore seeds was found to increase germination whereas gibberellic acid apparently had no effect on germination. The lack of response to GA a does not appear to be due to it not penetrating to the embryo, although our results are not conclusive on this point. However, similar responses were obtained with lettuce seeds inhibited b y the basic fraction from sycamore seeds. Kinetin completely overcame the inhibition whereas GA a had no effect This suggests t h a t a possible cytokinin-inhibitor interaction m a y be important in the dormancy of sycamore seeds. Van Staden et al. (1972) found t h a t during the stratification of Acer 8accharum seeds the levels of cytokinins increased markedly after 20 d at 5 C. I t is possible t h a t the state of dormancy in sycamore seeds is dependent on the relative levels of cytokinins and inhibitors. Treatments which decrease the level of inhibitors, such as leaching, or increase the levels of cytokinins, as in the case of stratification, m a y lead to loss of dormancy. K h a n (1971) has suggested t h a t cytokinins play a permissive role in dormancy and germination. According to this hypothesis gibberellins are required for the breaking of dormancy. I t is well known t h a t increases in gibberellins take place during stratification of m a n y tree seeds (Wareing, 1969; Sinska and Lewak, 1970). Although application of exogenous GA s did not affect germination of dormant sycamore seeds, this by no means suggests that gibberellins are not important in dormancy in sycamore. Different gibberellins can v a r y markedly in their effects on germination (Ikuma and Thimann, 1963). References Bentley, J. A., Housley, S.: Bioassay of plant growth hormones. Physiol. Plant. 7, 405419 (1954). Cornforth, J. W., 1Vfilborrow,B. V., Ryback, G., Wareing, P. F. : Identity of sycamore "dormin" with abscisin II. Nature (Lond.) 205, 1269-1272 (1965). Hayashi, F., Rappaport, L.: Gibberellin-like activity of neutral and acidic substances in the potato tuber. Nature (Lond.) 195, 617-618 (1962). Ikuma, H., Thimann, K. V.: Activity of gibberellin " D " on the germination of photosensitive lettuce seeds. Nature (Load.) 197, 1313-1314 (1963).
Seed Dormancy in Acer
125
Khan, A. A. : Cytokinins: Permissive role in seed germination. Science 171, 853-859 (1971). Lenton, J. P~., Perry, V. M., Saunders, P. F. : The identification and quantitative analysis of abscisic acid in plant extracts by gas-liquid chromatography. Planta (Berl.) 96, 271-280 (1971). Rudnicki, l~. : Studies on abscisic acid in apple seeds. Planta (Berl.) 86, 63-68 (1969). Sinska, I., Lewak, S.: Apple seed gibberellins. Physiol. Veg. 8, 661-667 (1970). Sondheimer, E., Tzou, D. S., Galson, E. D. : Abscisic acid levels and seed dormancy. Plant Physiol. 48, 1443-1447 (1968). Staden, J. van, Webb, D. P., Wareing, P. F. : The effect of stratification on endogenous cytokinin levels in seeds of Acer 8accharum. Planta (Berl.) (in press) (1972). Wareing, P. F. : Germination and dormancy. In: Physiology of plant growth and development, p. 605, ed. M. B. Wilkins. London: McGraw-Hill, 1969. Wareing, P. F., :Foda, H. A. : Growth inhibitors and dormancy in Xanthium seed. Physiol. Plant. 16, 266-280 (1957). Wareing, P. F., Saunders, P. F. : Hormones and dormancy. Ann. Rev. Plant Physiol. 22, 261-288 (1971). Webb, D. P., Wareing, P. F.: Seed dormancy in Acer: The role of the covering structures in dormancy of Acer pseudoplatanus L. J. exp. Bot. (in press) (1972). Dr. Paul Webb Botany Department University College of Wales Aberystwyth, Cards. U.K.
9 Planta(Berl.), Bd. 104:
