Planta 78,266~276 (1968)

Studies in Seed Dormancy IV. T h e Role of E n d o g e n o u s Inhibitors and Gibberellin in the D o r m a n c y and G e r m i n a t i o n of Corylus aveUana L. Seeds

J. W. BRAD:BEER Botany Department, King's College, London Received September 9, 1967

Summary. The dormancy of freshly harvested hazel seeds appears to be induced by inhibitors oceuring mainly in the testa and pericarp. Although d abseisic acid may not be one of the natural inhibitors involved, d,1 abscisic acid has been shown to strongly inhibit the germination of hazel seeds, probably through its antagonism towards the action of gibberellin. Dry storage of hazel nuts causes a deeper state of dormancy (secondary dormancy) to be superimposed on the primary dormancy. It is suggested that secondary dormancy consists of a block to gibberellin synthesis. The essential effect of chilling intact hazel seeds, which is the natural means of breaking their dormancy, may be to activate the mechanism for gibberellin synthesis, the subsequent synthesis of gibberellin being thought to occur at the germination temperature (20~ C) and not at the chilling temperature (5~ C). Introduction Hazel seed dormancy m a y be broken either b y an appropriate chilling t r e a t m e n t or b y application of gibberellin. FRANKLAND and W ~ E I N G (1966) found t h a t chilling induced a small increase in the concentration of gibberellin in hazel seeds and suggested t h a t gibberellin synthesized during chilling is the cause of the breakage of dormancy. As a result of further observations some modification of this hypothesis is proposed. g~_Rws (1966) found t h a t the germination of newly harvested hazel seeds was promoted b y removal of the testa or b y repeated leaching, thus implying t h a t dormancy m a y be imposed b y an inhibitor contained in the testa. Further investigations, reported here, support and extend this conclusion. As can be seen from the reviews of EVA~CA~I (1949) and W ~ E I N G (1965) inhibitory substances have been considered to play a p a r t in the imposition of dormancy in a large number of seeds, although little is known of their mechanism of action. The substance which appears to be the most inhibitory component of the "E-inhibitor" complex has been identified as d abscisic acid (abseisin, dormin: C O ~ O B T H c t a l . , 1965). The ability of synthetic abscisic acid (d,1 abseisie acid) to induce bud dormancy and inhibit bud growth (EL-A~TA:BLY et al., 1967) is in accord with the postulated role of d abscisie acid in bud dormancy. When CO~NrO~T~ et al. (1966) reported on the occurrence of d abseisie acid in

Inhibitors and Gibberellin in Hazel Seed Dormancy and Germination

267

a range of p l a n t s a n d tissues the highest c o n c e n t r a t i o n was f o u n d i n the pseudocarp of Rosa arvensis L. with smaller a m o u n t s i n the achenes, these seeds being deeply d o r m a n t . I t therefore appeared t h a t a s t u d y of the role of inhibitors i n hazel seed d o r m a n c y should include consideration of the possible p a r t i c i p a t i o n of d abscisic acid. C o n s e q u e n t l y a t t e m p t s have been m a d e to determine the d abscisic acid c o n t e n t of hazel seeds a n d to observe the effects of d,1 abscisie acid on hazel seed g e r m i n a t i o n . I t has Mready been reported t h a t d,1 abscisic acid i n h i b i t s the germin a t i o n of isolated embryos of F r a x i n u s ornus L. a n d F r a x i n u s americana L. (SoN])~[]~IME~ a n d GALSON, 1966) a n d CH~ISP]~LS a n d V~I~N]~R (1966, 1967) have studied the i n h i b i t o r y effect of d abscisic acid on the gibberellin-stimulated ~-amylase synthesis of b a r l e y - g r a i n aleurone cells.

Materials and Methods Hazel nuts (Kent cob nuts) were obtained directly from a grower, Mr. R. GOULD, of Mereworth, Kent, at the end of September 1966. The cupule was removed and the nuts were stored under dry and cool conditions (10~ C) until they were required. Germination tests were carried out after removal of the periearp, seeds showing any signs of damage or infection being rejected at this stage. The sound seeds were surface sterilized by 5 minutes immersion in sodium hypoch]orite solution (1% available chlorine), washed several times with sterile water, and each batch of 5 seeds was placed on a filter paper disc in 20 ml of water or other appropriate solution in a petri dish. The dishes of seeds were kept in the dark in an incubator at 20~ C. Germination, as shown by protrusion of the radicle, was recorded daily. Intact nuts were chilled in boxes of moist sand maintained at 5~ C in a tempel-ature controlled refrigerated cabinet. Alternatively the seeds were chilled after removal of the pericarp. In this case the seeds were prepared in an identical manner to that used in the germination tests, and the petri dishes of seeds were placed in the refrigerated cabinet. In this way treatment of seeds with an appropriate solution could be made during chilling, comparison of subsequent germination being made with seeds chilled in water. All germination tests have been carried out with samples of 100 seeds, 95 % confidence limits being determined from a table calculated by the use of the g2 test with a two by two contingency table (ROBERTS, 1963). These confidence limits are given in Figs. 1 to 4. Where mean fresh weights of cotyledons or embryonic axes have been determined, the 95% confidence limits have been calculated from the standard errors of the means by the use of "Students" t values. Results and Discussion I n t a c t n e w l y harvested hazel seeds of the 1966 crop gave only 7% g e r m i n a t i o n at 20 ~ C, b u t r e m o v a l of the testa i n d u c e d r a p i d germination, 64% g e r m i n a t i o n being recorded after 28 days {Fig. 1). W h e n the testa was stripped from the e m b r y o s b u t allowed to r e m a i n i n the dish with t h e m d u r i n g the g e r m i n a t i o n test the initial rate of g e r m i n a t i o n was significantly i n h i b i t e d b u t there was no i n h i b i t i o n of the final percentage g e r m i n a t i o n . A f u r t h e r significant fall i n the rate of germ i n a t i o n a n d a significant r e d u c t i o n i n the final percentage g e r m i n a t i o n

268

J.W. B~ADB~E~:

was found when both stripped testa and pericarp were placed together with the naked embryos. These results suggest that the dormancy of freshly harvested hazel nuts m a y be imposed by the presence of inhibitory substances which are found in the testa and pericarp. Of the 36 % of the naked embryos which did not germinate some are presumably not viable

%[

100

80

~ 9~

1

I

I~ Days Gt 2 0 ~

21

1

~o

2O

0

7

28

Fig. l. The effect of various treatments on the germination of freshly harvested hazel seeds at 20~ C. 9 testa intact; A testa removed; A testa stripped from embryo but left in dish with embryo; 9 testa stripped from embryo but left in dish with embryo and pericarp; o testa removed and embryo placed in 3 • 10-a M d,1 abscisic acid and some m a y contain sufficient inhibitory substances to prevent germination. Fig. 1 also shows that the inhibitory effects of testa and pericarp were somewhat exceeded by 3 • 10-5 M d,1 abscisic acid. Although the testa of m a n y seeds is thought to impose dormancy by means of its restriction of gaseous exchange (BA~ON, 1965), this would not appear to be important in hazel as extensive laceration of the testa, which is thin and papery, has been shown to have no effect on its germination. Further support for the presence of germination inhibitors in the testa has been provided by the demonstration that leaching, by daily change of the water in the petri dish, resulted in some stimulation of the germi-

Inhibitors and Gibbcrcllin in Hazel Seed Dormancy and Germination

269

nation of intact seeds. Although the percentage stimulation in Fig. 1 is not significant, JA~ws (1966) reported t h a t leaching increased the percentage germination of intact seeds of the 1965 crop from 5 to 22. D r y storage of intact newly harvested nuts causes a change in their state of dormancy. When naked embryos were obtained from nuts which had been dry-stored for four weeks the percentage germination was 30, the comparable values for embryos from newly harvested nuts and eight week stored nuts being 64 and 1O respectively. An a t t e m p t has been made to extract the inhibitors of 200 imbibed hazel nuts which were first separated into two fractions, namely (i) testa and pericarp and (ii) naked embryos. Both fractions were extracted three times with 80 % aqueous ethanol at room temperature and the ethanol was removed from the combined extracts b y distillation under reduced pressure. The p H of the aqueous residue was adjusted to 8.8 after which four extractions with redistilled ethyl acetate were made. The p H of the aqueous solution was then reduced to 2.4 and four further extractions with redistflled ethyl acetate were made. After removing the ethyl acetate b y distillation under reduced pressure the level of inhibitor in this fraction was determined b y means of the wheat embryo assay (Cold,CFORTH et al., 1965). The testa and pericarp of 200 imbibed seeds were found to contain inhibitor equivalent to 4 ~moles of d abscisie acid. The data shown in Fig. 1 indicates t h a t the inhibitor content of the testa and pericarp of 200 seeds is probably equivalent to something between 2 and 10 ~moles of d abscisie acid. Thus the acidic ethyl acetate fraction appears to contain sufficient inhibitory material to account for the inhibitory effects of testa and pericarp. Furthermore, as suggested from the data in Fig. 1, more inhibitory material was found in the testa and pericarp than in the embryo. Thin-layer chromatography of the acidic ethyl acetate fractions on Kieselgel G first in n-propanol: n-butanol: ammonia (specific gravity 0.88) : water (2 : 6 : 1:2) and subsequently in ethyl acetate has established t h a t they contained a number of inhibitory components, but t h a t no d abseisic acid could be detected by speetropolarimetry of the appropriate eluates from the thin layer chromatograms. Dr. G. RYBACK kindly performed the spectropolarimetry. If d abscisic acid was present in the testa and pericarp extract it accounted for less than 2 % of the inhibitory activity. The dormancy of intact hazel seeds can be broken by the application of gibberellin to the seeds (FI~A~KLAND, 1961; FRANKLA~TD and WAREI~G, 1962, 1966; B~ADBEER and PINFIELD, 1967). I n Fig. 2 it can be seen t h a t the promotive effect of 1 • 10-~ M gibberellin on hazel seed germination was significantly antagonized b y 3 • 10-5 M d,1 abseisic acid. I t seems to be particularly noteworthy t h a t CHRISP]~WLS and

270

J . W . BRADBEER:

VAgN~g (1966) f o u n d t h a t d abscisic a c i d will a n t a g o n i z e t h e gibberellins t i m u l a t e d ~ - a m y l a s e s y n t h e s i s of t h e aleurone cells of b a r l e y , a process which has a n i m p o r t a n t role in b a r l e y g e r m i n a t i o n . As d,1 abscisie a c i d also i n h i b i t s g i b b e r e l l i n - s t i m u l a t e d l e t t u c e g e r m i n a t i o n (BI%ADBEEI% a n d R o s s , in p r e p a r a t i o n ) i t is suggested t h a t t h e physiological role of abscisic a c i d m a y be to a n t a g o n i z e t h e a c t i o n of gibberellin. I t seems 100 ~

80

60 .tO "~

'~ 40

20

0 L------~ ~'

7

~

' I~ Days at 20 ~

, 21

, 28

Fig. 2. The effects of d,] abseisie acid and CCC on the gibbere]lin stimulated germination of intact hazel seed at 20 ~ C. 9 no gibberellin; 9 1 X 10-41K gibberellin; o 1 X ]0 -4 M gibberellin and 3 • 10-5 M d,1 abscisic acid; @ 1 X 10-4 M gibberellin and 3 X 10-~ d, 1 abseisic acid; [] 1 • ]0 -I M gibberellin and 6 • 10-* M CCC possible t h a t this role m a y be t h e basic f u n c t i o n of d abscisic acid in plants. Fig. 2 also shows t h a t 6 X 10 -4 M chlorocholine chloride (CCC) has no effect on t h e g i b b e r e l l i n - s t i m u l a t e d g e r m i n a t i o n of hazel seed, a n o b s e r v a t i o n which is in a c c o r d w i t h t h e r e p o r t e d role of CCG as being a n i n h i b i t o r of gibberellin-synthesis (~-IAI%ADAa n d LA~G, 1965). T h e course of g e r m i n a t i o n of i n t a c t hazel seeds a t 20 ~ C, after t h e y h a d b e e n chilled as i n t a c t n u t s a t 5 ~ for 28 d a y s in m o i s t sand, is shown in Fig. 3. T h e g e r m i n a t i o n of these chilled seeds was also o b s e r v e d in t h e presence of 3 x 10 -5 M d,1 abscisic a c i d a n d 6 X 10 -4 M CCC,

Inhibitors and Gibberellin in gaze] Seed Dormancy and Germination

271

both of which can be seen to have significantly inhibited the rate and final level of germination attained. I t is deduced that the d,] abscisic acid inhibited germination through its antagonism of gibberellin action and that the CCC exerted its effect by inhibition of gibberellin synthesis. When FI~ANKLANDand WAxwinG (1966) proposed that the gibberellin synthesized during the chilling of hazel seeds may be responsible for the 100r%

75

= o

+5 E

50

(D

25

o~

~

5

I

10 Days at 20 ~

I

15

Fig. 3. The effects of d,l abscisic acid and CCC on the germination of previously chilled hazel seeds. Chilling carried ou~ in mois~ sand a~ 5~ C. Germination carried out at 20~ in: 9 water; 9 3• -SM d,1 abscisie acid; [] 6• -aM CCC subsequent breaking of dormancy they pointed out that only a very small quantity of gibberellin accumulated during the chilling treatment, this being very much less than the amount of exogenous gibberellin required to break dormancy. Since CCC supplied subsequently to chilling will inhibit germination it m a y be deduced that the gibberellin required for hazel seed germination was synthesized at 20 ~ C, or at least the later stages of gibberellin synthesis, including the CCC-sensitive step, occurred at 20 ~ C. I n a further experiment intact seeds were chilled at 5~ in petri dishes. As shown in Fig. 4 there were no significant differences between the subsequent germination at 20 ~ C of seeds previously chilled in water,

272

J . W . BRADBEER:

6 X 10 -4 M CCC or 3 x 10 -s M d,1 abscisic acid. T h e g e r m i n a t i o n tests a t 20 ~ C were carried o u t in w a t e r a n d t h e chilled seeds were rinsed in w a t e r p r i o r to t h e g e r m i n a t i o n tests. One e x p l a n a t i o n of t h e absence of i n h i b i t o r y effects of CCC a n d d,1 abscisie a c i d u n d e r these conditions is t h a t n e i t h e r gibberellin s y n t h e s i s n o r gibberellin a c t i o n are essential 100 %

9

75

.-o= 50

E 0

25

10

5

15

Days at 20~

Fig. 4. The effects of chilling hazel seeds in d,1 abscisic acid and CCC on their subsequent germination. Chilling carried out at 5~ in: 9 water; 9 3 X 10-5 M d,1 abseisie acid; [] 6 x 10-~ M CCC. Germination carried out at 20 ~ C in water p a r t s of t h e chilling process, b o t h t h e l a t e r stages of gibberellin synthesis a n d a c t i o n being essential processes a t t h e g e r m i n a t i o n t e m p e r a t u r e . I n a d d i t i o n to t h e r e c o r d i n g of t h e t i m e of radicle emergence, as shown in Figs. 1 to 4, t h e fresh weights of e m b r y o n i c axes a n d cotyledons of all g e r m i n a t e d seeds were d e t e r m i n e d on t h e f o u r t e e n t h d a y a f t e r t h e c o m m e n c e m e n t of t h e g e r m i n a t i o n tests. I n t h e T a b l e are given t h e m e a n fresh weights for each t r e a t m e n t t o g e t h e r w i t h t h e 95 % confidence limlts c a l c u l a t e d f r o m t h e s t a n d a r d error of t h e m e a n a n d " S t u d e n t s " t values. The T a b l e shows t h a t t h e p r o x i m i t y of s t r i p p e d t e s t a a n d p e r i c a r p h a d a h i g h l y significant i n h i b i t o r y effect on t h e g r o w t h of t h e axis of t h e

Inhibitors and Gibberellin in Hazel Seed Dormancy and Germination

273

Table. Effects of various treatments on the /resh weights o/ the embryonic axes and cotyledons ol hazel seeds. Except ]or the ungerminated sample each value represents the mean/or those seeds which had germinated, as determined on the lath day o] treatment at 20 ~ C. 95 % con/idenee limits are also given Treatment

mean fresh weight in mg Embryonic Cotyledons axis

Imbibed ungerminated seed Freshly harvested seed Testa removed Testa stripped but left beside embryo Testa stripped but left with periearp beside embryo Testa removed and 3 • 10-5 M d,1 abscisic acid added

5 4- 1

1,970 4- 70

162 4- 26 142 4- 28 73 =J=37 109 4- 43

2,570 -b 100 2,520 • 130 2,380 4- 220 2,360 4- 180

Fruits prechilled at 5~ C for 28 days Intact seeds in water Intact seeds in 3 • 10-5 M d,1 abscisic acid Intact seeds in 6 X 10-4 1 CCC

235 4- 28 153 q- 22 181 ::[=25

2,830 4- 140 2,670 4- 130 2,830 • 160

Intact seeds in 1 • 10-4 N gibberellin No further additions With 3 • 10-5 M d,1 abscisic acid With 3 X 10-~ M d,1 abscisic acid With 6 • 10-4 M CCC

173 :J=42 1054-28 169 4- 37 155 • 33

3,720 4- 190 3,540~210 3,540 • 170 3,580 ~ 210

n e w l y h a r v e s t e d n a k e d e m b r y o , t h e i n h i b i t i o n being g r e a t e r t h a n t h a t o b t a i n e d w i t h 3 • 10 -5 M, d,1 abscisie acid. I t should be n o t e d t h a t this c o n c e n t r a t i o n of d,1 abscisie a c i d was clearly m o r e effective t h a n t h e t e s t a a n d p e r i c a r p i n h i b i t o r s on radicle emergence (Fig. 1). Pret r e a t m e n t of hazel n u t s a t 5 ~ for 28 d a y s r e s u l t e d in significantly g r e a t e r axis g r o w t h d u r i n g t h e 14 d a y s of t h e g e r m i n a t i o n t e s t t h a n d i d r e m o v a l of t h e t e s t a of unehilled seeds or t r e a t m e n t of unehilled seeds w i t h gibberellin. The i n h i b i t i o n of t h e e m b r y o n i c axis g r o w t h of p r e v i o u s l y chilled seeds b y d,1 abseisie a c i d a n d CCC was in b o t h cases significant a t t h e 99 % level. The e m b r y o n i c axis g r o w t h resulting from gibberellin t r e a t m e n t c o r r e s p o n d e d to t h e results of radicle emergence (Fig. 2), o n l y 3 x 10 .5 M, d,1 abscisic acid a n t a g o n i s i n g t h e gibberellin action, in this case t h e i n h i b i t i o n being significant a t t h e 99 % level. I n general t h e results of e m b r y o n i c axis g r o w t h show close a g r e e m e n t w i t h results o b t a i n e d f r o m t h e t i m e of radicle emergence. The T a b l e also shows t h a t g e r m i n a t i o n of hazel seeds is a c c o m p a n i e d b y a considerable rise of c o t y l e d o n fresh weight, b y far t h e g r e a t e s t increases occurring as a r e s u l t of t h e v a r i o u s gibberellin t r e a t m e n t s . M e a s u r e m e n t s of cell size h a v e e s t a b l i s h e d t h a t t h i s c o t y l e d o n e x p a n s i o n can be w h o l l y a c c o u n t e d for b y increases in cell volume. The o n l y

274

J.W. BRAI)BEER:

significant inhibitions of cotyledon expansion were induced by applying 3 • 10-5 M d,1 abscisie acid to intact chilled seeds and to freshly harvested naked seeds. General Discussion I t has been demonstrated that the embryo of the newly harvested hazel nut is not dormant, but that inhibitors present in the testa and pericarp are thought to be carried to the embryo during imbibition, thus imposing a state of dormancy on the intact seed or the intact nut. It is proposed to describe dormancy imposed in this way as primary dormancy. The testa and pericarp of hazel seeds have been found to contain a number of substances which are inhibitory to wheat embryo growth, but these substances have yet to be identified and it is not known which metabolic reactions in hazel seeds are inhibited by them. d Abscisie acid does not appear to account for any appreciable part of the inhibitory activity of hazel testa and pericarp. Nevertheless the effects of d,1 abscisic acid on hazel seed germination have been investigated. Abscisic acid appears to be of value as an antagonist of gibberellin action (CJ~RISF~ELS and V A ~ E g , 1967), and it seems to function in this way in hazel seeds. Gibberellin has been found to play an important part in hazel seed germination. Studies with exogenous gibberellin have established that gibberellin induces embryonic axis growth (BRADBEEt~ and PINFIELD, 1967), eotyledonary cell expansion (Table), an increase in the activity of the cotyledonary oil to sucrose conversion system including an increase in the isocitrate lyase activity (Dr. N. J. PI~FIELD, pers. commn.), increases labelling of glutamate at the expense of tricarboxylic acid cycle acids and increases the labelling of nucleotides (BRADBEERand t:)IlgFIELD, 1967). I t has not yet been possible to locate the actual site or sites of gibberellin action, nor is it known whether gibberellin acts in the embryonic axis or in the cotyledons or in both. However the marked effects of exogenous gibberellin on cotyledon cell expansion does suggest that gibberellin may react directly on the cotyledons. I t has also been pointed out that the sites of action of the testa and pericarp inhibitors are unknown, but it is possible that they may antagonize the action of gibberellin. However, the possibility that these compounds inhibit gibberellin synthesis cannot be precluded. Identification of the inhibitors and their sites of action is the aim of further investigations. I t has been shown that dry storage of hazel nuts causes a rapid loss in the ability of the naked embryos to germiuate. On the other hand dry storage for up to twelve weeks has no detectable effect on the response of hazel seeds to exogenous gibberellin, neither the rate of germination in 3 • 10-4 IV[ gibberellin (PI~FIELD, 1965) nor the response to a range

Inhibitors and Gibberellin in Hazel Seed Dormancy and Germination

275

of gibberellin concentrations (B~ADBWE~ and PINFIELD, 1967) showing any change. However, dry storage has been found to increase the chillLug requirement of hazel seeds, i.e. with increased dry storage an increased period of chilling is required for the subsequent achievement of a given level of germination (CoLMAN, 1961; JA~VIS, 1966; B~ADB~n~ and COL~A~, 1967). The experiments with CCC reported in the present paper suggest t h a t the role of chilling m a y be to prepare the seed for gibbercllin synthesis, which subsequently occurs most rapidly at the germination temperature. The effects of dry storage on the chilling requirement of hazel seeds and on the dormancy of the naked embryos, together with the absence of an effect of dry storage on the response of seeds to exogenous gibberellin, suggest t h a t dry storage m a y either induce a block in the p a t h w a y of the synthesis of endogenous gibberellin or promote the activity of a mechanism for the destruction of gibberellin. Chilling presumably either overcomes a block to gibberellin synthesis or by prorooting the subsequent rate of gibberellin synthesis enables gibberellin synthesis to exceed gibberellin consumption. The t e r m secondary dormancy, which has been used to describe the dormancy induced b y exposure to unfavourable environmental conditions (BARTON, 1965) would appear to be an appropriate description for the deeper state of dormancy induced b y dry storage. I n the newly harvested hazel seed, or in a prematurely fallen hazel seed, a state of primary dormancy occurs through the inhihitors conrained in the testa and pericarp. Some dry storage would normally occur prior to natural dehiscence and the development of secondary dormancy would in this circumstance reinforce primary dormancy. Natural breaking of dormancy, during overwintering in a moist substratum, would remove any block to gibberellin synthesis and the subsequent synthesis of gibberellin which occurs on attainment of the germination temperature, would provide sufficient gibberellin to set in motion the germination processes. I wish to thank Mr. K. A. MAYBU~Yfor technical assistance, the Royal Society and the University of London Central Research Fund for financial support, Dr. T. W. COR~FORT~ for a gift of d,1 abscisie acid, Dr. J. E. KEYS for discussions and Dr. E. H. ROBERTS for statistical information.

References BARTON,L.V. : Dormancy in seeds imposed by the seed coat, In: Handbueh der Pflanzenphysiologie, Bd. XV/2, S. 727 (A. LA~G, Hrsg.). Berlin-HeidelbergNew York: Springer 1965. BRADBEnR, J. W., and B. COLMA~: Studies in seed dormancy. I. The metabolism of [2-14C] acetate by chilled seeds of Corylus aveUana L. New Phytologist 66, 5--15 (1967). --, and N. J. ~I~IELD-" Studies in seed dormancy. IIL The effects of gibberellin on dormant seeds of Corylus avellana L. New Phytologist 66, 515--523 (1967).

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CHRISPEELS, M. J., and J. E. VAI~XEI~:Inhibition of gibbcrellic acid induced formation of ~-amylase by abscisin II. :Nature (Lond.) 212, 1066--1067 (1966). - - - - Hormonal control of enzyme synthesis: on the mode of action of gibberellic acid and abscisin in aleurone layers of barley. Plant Physiol. 42, 1008--1016 (1967). COLlAr, ]3. : Metabolic aspects of dormancy. Ph.D. Thesis, University o~ Wales (1961). COR~FO~T~, J. W., ]3. V. M I ~ o ~ o w , and G. RYBACK: Identification and estimation of (~-)-abscisin I I ('Dormin') in plant extracts by spectropolarimetry. Nature (Lond.) 210, 627--628 (1966). , and P. F. WA~EI~G: Identity of sycamore dormin with abscisin II. Nature (Lond.) 205, 1269--1270 (1965). EL-ANTABLY,H. M.M., P . F . WA~EI~G, and J. HILL~A~: Some physiological responses to d,1 abscisin (dormin). Planta (]3erl.) 73, 74--90 (1967). EvA~A~I, M.: Germination inhibitors. ]3ot. Rev. 1~, 153--194 (1949). FRA~KLA~n,]3.: Effect of gibberellic acid, kinetin and other substances on seed dormancy. Nature (Lond.) 192, 678--679 (1961). --, and P. F. WAREI~G: Changes in endogenous gibberellins in relation to chilling of dormant seeds, lX~ature (Lond.) 194, 313--314 (1962). - - - - Hormonal regulation of seed dormancy in hazel (Corylus avellana L.) and beech (Fagus sylvatlca L.). J. exp. ]3ot. 17, 596--611 (1966). HARADA,H., and A. LA~G: Effect of some (2-chloroethyl) trimethylammoninm chloride analogs and other growth retardants on gibberellin biosynthesis in Fusarium monili]orme. Plant Physiol. 40, 176--183 (1965). JA~vis, ]3. C. : Nucleotide metabolism in dormant and non-dormant seeds. Ph.D. Thesis, University of Wales (1966). P~FI~LD, N. J.: Gibberellic acid Seed dormancy. Ph. D. Thesis, University of Wales (1965). ROBERTS, E. H. : The effects of inorganic ions on dormancy in rice seed. Physiol. Plant., Kobenhaven 16, 732--744 (1963). SOND~EIME~, E., and E. C. GALso~: Effects of abseisin I I and other plant growth substances on germination of seeds with stratification requirements. Plant Physiol. 41, 1397--1398 (1966). WA~EI~G, P. 1%: Endogenous inhibitors in seed germination and dormancy. In: Handbuch der Pflanzenphysiologie, ]3d. XV/2, S. 909 (A. L A ~ , Hrsg.). ]3erlinHeidelberg-New York: Springer 1965. Dr. J. W. ]3RADBEER ]3otany Department, King's College 68 Half Moon Lane, London, S.E. 24, England