Kéri, Ágnes

Nagy, Mária

Pataky, Szerén

English

Nagy Mária

Pataky Szerén

Kéri Ágnes

University of Szeged

Acta Biologica Szeged. 27 (1—4), pp. 127—137 (1981) DORMANCY IN FRUITS OF TILIA PLATYPHYLLOS SCOP. V. POSSIBLE ROLE OF CHILLING STRATIFICATION IN BREAKING DORMANCY MÁRIA NAGY, SZERÉN PATAKY a n d ÁGNES KÉRI Department of Plant Physiology and Department of Botany. Attila József University. Szeged (Received September 5, 1980) Abstract The sizes, germinative abilities and growth capabili t ies of embryos excised f r o m lime seeds stratified in a chilled and warm state, respectively, were compared . Though the embryos could grow both dur ing the warm and the chilled stratification, the e longat ion of the radicle proved to be more definite dur ing chilled stratification. Embryos excised f r o m seeds stratified in chilled state germinated quicker and grew more intensively than the embryos excised f rom seeds stratified in warm state. Chilled stratification plays a role also in the weakening of tissues sur rounding the embryo . Introduction Lime seeds require for the breaking of their dormancy a chilled stratification for 150—180 days. Similarly to most seeds requiring stratification the decrease of the amount of inhibitors and the increase of the amount of endogenous gibberellin-like substances during stratification can be observed also in the case of lime seeds (SZALAI and NAGY, 1974; NAGY, 1980). However, stratification cannot be replaced by exo-genous gibberellic acid, only the lenght of the stratification period can be shortened ( N A G Y and SZALAI, 1973). A further reduction of the stratification period is possible by the combined application of gibberellic acid and indole-acetic acid (1AA), (NAGY, 1976), which indicates that though these hormones may play an important role in the breaking of dormancy, during the stratification besides the rise of the level of endo-genous hormones also other alterations important from the aspect of the breaking of dormancy take place. On the effect of a hormonal stimulus arriving from the embryo a change may occur in the activity of metabolism, including the decomposition of the stored nutri-ents, ensuring in this way the nutrient supply of the embryo. Owing to the gluconeo-genesis the water potential of the embryo will be more negative and thus it may repre-sent a greater force against the surrounding tissues whose mechanical resistance re-mains unchanged. During the stratification also the mechanical resistance of the tis-sues surrounding the embryo may change or both processes of different direction may contribute to the breaking of dormancy. In the course of our study into the changes occurring during stratification the following problems were investigated: 128 M A R A I NAG Г. SZEREN PATAKY and AGNES KERI — observability of embryonal growth during stratification, — existence of a difference in growth intensity between seedlings developed from embryos excised at various periods of stratification, and — effect of the mechanical resistance of the surrounding tissues on the dormancy of the embryo. Materials and Methods M a t e r i a l of e x a m i n a t i o n : Our investigations were carried out with fruits of Tilia platy-phyllos S :op . obtained f rom the Forestry of County Csongrad. S t r a t i f i c a t i o n a n d e x c i s i o n of e m b r y o s : After the removal of the pericarp the seeds were scarified with sulphuric acid then stratified in culture pots containing washed sand wetted to 80% o( the full water capacity. Stratification was carried out in a refrigerator at 4 — 5 : C and in a thermostat at 25°C, respectively. At various periods of stratification the seeds were disinfcctcd with bromine water then the embryos were excised and kept in a Petri dish under semisterile condit ions on filter paper wetted with White nutrient (WHITE. 1943) at a daily 16-hour illumination of 10 000 Lux, at a day-time temperature of 25°C and at a night temperature of 20°C. M e a s u r e m e n t of c l o r o p h y II c o n t e n t : the chlorophyll content of the leaves of the see-dlings was determined by photometry, using the method of WINTERMANS and MOTS (1965). T r e a t m e n t o f s e e d s w i t h e x o g e n o u s p e c t i n a s e a n d c e l l u l a s e Also the seed-coat was removed from a part of seeds disinfected with bromine water and they were treated for 7 days with a 1 % cellulase preparation (Fluka) prepared by a phosphate buffer of pH 5.0. Another part of the seeds was ireated with a 1 % pectinase preparation (Fluka) prepared by a phosphate buffer of pH 4.0 whereas a third part of the seeds similarly for 7 days with a preparat ion of 1 % cellulase + 1 % pectinase. During the treatment period the seeds were incubated at 25 °C, on exchanging each day twice the enzyme preparation. After the 7-day treatments with the enzyme preparation a part of the seeds was placed in a thermostat of 25 °C and another part in a refrigerator. In both cases also the influ-ence of a treatment with 3 .10" ' m OA.-, or with 3.10"" m GA S + 3.10"4 m 1AA on the germination of seeds af ter the exogenous enzyme treatment was investigated (NAGY, 1976). The result was eva-luated af ter the elapse of a month. H i s t o l o g i c a l i n v e s t i g a t i o n s . Af ter the removal of the seed-coat the seeds were fixed by the Juel-iype fixina agent (SARKANY and SZALAI, 1968) and cross and longitudinal cuttings were prepared by embedment in celloidin. Hematoxylin of Ehrlich type was used for the staining of the preparations. Results and discussion E f f e c t of c h i l l e d and warm s t r a t i f i c a t i o n on the g r o w t h of e m b r y o s p r e s e n t in i n t a c t s e e d s Since the embryos removed from dormant lime seeds are geminating quickly in a wet environment without any pretreatment and no signs of any morphological ab-normity can be observed on the developing seedlings whereas at the same time a chilled stratification is required for the breaking of dormancy of the intact seed, it appeared to be of interest to examine whether a difference exists between the size and the growth intensity of embryos removed from warm stratified and chilled stratified seeds. DORMANCY IN FRUITS OF TILIA PLATYPHYLLOS SCOP. V. POSSIBLE ROLE O F CHILLING 1 2 9 As shown by Fig. 1, the embryos are growing both during warm stratification and during chilled stratification. Though at the beginning the warm stratification appears to be more favourable for the growth of embryos, the elongation of the radicle i * > V * * * * # ¥ VZ weeks Fig. 1. Embryos cxcised f rom seeds chilled stratified (A) and warm stratified (B) for various periods. is more intensive during chilled stratification. The difference in the size of embryos at the various periods of stratification is due not only to water uptake because also the dry matter content of the embryos increases with the length of stratification periods, indicating that an actual growth takes place (Table 1). Table 1. Fresh mass and dry matter content of embryos excised f r o m seeds stratified fo r various periods, referred to 100 embryos 5 C 25 °C Stratification period, weeks Fresh mass. Dry mass, Fresh mass, Dry mass, 8 g g g 3 0.8442 0.3862 0.8585 0.3855 6 1.2132 0.4341 1.1971 0.3909 9 1.6972 0.5963 1.4039 0.4876 12 1.8584 0.6616 1.6308 0.6011 The more vigorous development of embryos stratified at 5 °C is proved also by the histological investigations (Fig. 2). Striking differences appear in the thickness of the cotyledons and cell-walls on the effect of treatments at both applied temperatures. The more favourable effect of stratification at 5 °C manifests itself also at the shoot apex in the size and in the differentiated nature of the shoot apex. However, this dif-ference is not as definite as in case of the cotyledones. 1 3 0 MÁRIA NAGY, SZERÉN PATAKY and ÁGNES KÉRI Fig. 2. State of development of embryos stratified for two months at various temperatures A: cross section of the cotyledon of an embryo stratified at 5 °C . e=ep ide rmis , m = meso phyllum, v=vascular tissue; x270 B: cross section of the cotyledon of an embryo stratified at 25 °C. x270 C — D : median longitudinal section of the shoot apex of an embryo stratified at 5 "C. c = c o r p u s , cot = cotyledon. proc=procambium, t = tunica. C = x 2 7 0 , D = x l 4 0 . E : median longitudinal section of the shoot apex of an embryo stratified at 25 °C. x ! 4 0 DORMANCY IN FRUITS OF TILIA PLATYPHYLLOS SCOP. V. POSSIBLE ROLE OF CHILLING 131 G r o w t h i n t e n s i t y o f e m b r y o s e x c i s e d at v a r i o u s p e r i o d s of s t r a t i f i c a t i o n Embryos excised at various periods of stratification differ from each other in the speed of germination (Fig. 3) (in the case of excised embryos the first geotropic cur-vature of the radicle is considered as germination). The longer was the period of chilled stratification, the higher is the percentage of germination in the first 24 hours. A parti-C o D C Q> c n 90-70-50" 30-I 0 i • chilled stratification (5°C) • warm stratification (25°C) 0 3 6 9 12 s t r a t i f i c a t i o n s h ' m e i n w e e k s Fig. 3. Germination of embryos stratified for various periods, observed 24 hours af ter excision cularly great change is observable after six weeks of chilled stratification. Embryos warm stratified for various periods are germinating much slower and hardly differ from each other from the aspect of their vigour of germination. The growth activity of embryos excised at various periods of stratification is presented in Fig. 4. where the size of seedlings is shown at the seventh day after exci-sion, expressed as percentages of the non-stratified controls. Similarly to the results of germination tests, the growth intensity of embryos warm stratified for various periods is very similar but even in case of a stratification of the shortest duration higher than that of the non-stratified control. 132 MÁRIA NAGY, SZERÉN PATAKY and ÁGNES KÉRI I chilledslrotiiïcaKon (5°C) • warm stra h" Pica hon (25°C) Fig. 4. Size of seedlings developed from embryos excised at various periods of stratification, observed on the seventh day after excision and expressed as percentages of the non-stratified control Seedlings originating from chilled stratified embryos were at all investigated pe-riods larger than seedlings developed from warm stratified embryos, indicating clearly that chilled stratification plays an important role in the increase of the capability of growth of the embryo. These statements are supported also by our results concerning the investigation of the degree of verdure (Fig. 5, A and B). As shown by these figures, in the leaves of the seedlings developed from chilled stratified embryos the formation of chlorophyll is more intensive than in the seedlings developed from warm stratified embryos. DORMANCY IN FRUITS OF TILIA PLATYPHYLLOS SCOP. V. POSSIBLE ROLE OF CHILLING 133 E f f e c t of the m e c h a n i c a l r e s i s t a n c e of the s u r r o u n d i n g t i s s u e s on the d o r m a n c y of the e m b r y o Low temperature may exert a favourable effect on the germination of seeds in two directions: 1. it may increase the capability of growth of the embryo which may represent at the end of the period of chilled stratification a greater stretching force against the surrounding tissues whose mechanical resistance remained unchanged, and thus the radicle will be capable of breaking through the surrounding tissues, 2. during the chilled stratification also the mechanical resistance of the surround-ing tissues may decrease and this promotes the breakthrough of the radicle. As indicated by our results presented thus far, the capability of growth of the embryo increases during the chilled stratification. However, the capability of growth 134 MÁRIA NAGY, SZERÉN PATAKY and ÁGNES KÉRI 'ncreases also during warm stratification in comparison to the capability of the non-stratified control, furthermore even on the effect of a treatment with exogenous GA 3 , in certain cases to such an extent that the embryo gets crushed in the seed as an accor-dion ( S Z A L A I and N A G Y , 1 9 6 8 ) , and the radicle cannot break through the surrounding tissues. In connection with the possible role of the mechanical resistance of the tissues surrounding the embryo in dormancy the effect of the breaking up of the ends of the seeds at the radicle or the apex, respectively, was investigated on the germination of seeds. This operation was carried out after disinfecting the seeds by bromine water. This was done manually at diffuse light, with the use of a scalpel, a lance-shaped needle and a dissecting needle, then the seeds were placed in a Petri dish on sterile filter paper wetted with water containing 100/ig/ml streptomycin and incubated in a 25 °C ther-mostat. The result visible on the fourth day after this operation is shown in Fig. 6. Seeds operated at their end near the radicle germinated in all cases, quite inde-pendently of being treated previously by chilling. The higher growth intensity ob-servable in chilled stratified seeds is quite in accordance with our earlier results ob-DORMANCY IN FRUITS OF TILIA PLATYPHYLLOS SCOP. V. POSSIBLE ROLE OF CHILLING 135 L * 1 1 • J V i r > ra Fig. 6. Effec t of operat ive t rea tment on the germinat ion of seeds A : seeds stratif ied a t 5 °C f o r 2 m o n t h s B : seeds stratif ied a t 25 °C f o r 2 m o n t h s . tained with excised embryos. In contrast to that, germination was not observed at all in seeds operated at their ends near the apex. Attempts were made to clear up the role of the mechanical resistance of the tis-sues surrounding the embryo by experiments where the seeds were treated with exo-genous cellulase and pectinase. Our aim was here to weaken the mechanical resistance of tissues surrounding the embryo and to observe the germination of seeds at 5 °C and at 25 °C. The best results were obtained at the treatment with 1 % cellulase. Experimental data are shown in Fig. 7, where the dark columns refer to seeds with intact seed-coat and the light columns to results obtained with seeds whose seed-coat was previously removed. It can be seen in Fig. 7 that 100% germination could be observed in case of seeds whose seed-coats were removed and which were kept after their treatment with the cellulase preparation for one month at 5 °C and were subsequently treated with GA3 + + IAA. The percentage of germination was in case of seed-coat-less seeds in all the cases higher than in tests with intact seeds. The results indicated as well that at low temperature the treatment with hormones is more efficient than at higher temperature. This may be in correlation with the conditions of the entrance of the hormones. Thus, the primary role of chilled stratification may be likely not the ensuring of the growth of the embryo but rather the increase of the capability of germination of the embryo. Simultaneously with this process a weakening of the mechanical resis-tance of the tissues surrounding the embryo occurs on the effect of enzymes of key importance whose synthesis or activation starts at the impulse of chilled stratification. This presumption is confirmed also by the results obtained with other species requir-ing chilled stratification ( O L N E Y and POLLOCK, 1 9 6 0 ; VILLIERS and W A R E I N G , 1 9 6 5 ; VANSTONE and L A CROIX, 1 9 7 5 ) , in experiments wherein the growth of embryos could be experienced during warm stratification but germination started only after chilled stratification. It can be imagined that similarly to the observations made in case of celery (JACOBSON et al., 1976) also in the lime seeds a quicker degradation of the cells located in the environment of the radicle occurs at low temperature. 136 M Á R I A N A G Y , SZERÉN PATAKY and ÁGNES KÉRI C _ o a c Ë í-QJ CP 6 A 3 + IAÁ C o n l r Con IT. GA3 GA3+IAA 5°C 25° C Fig. 7. Effect of exogenous cellulase t reatment on the germinat ion of seeds. Dark co lumns ind ica te the results obtained with scarified seeds whereas light co lumns show the results ob ta ined wi th seeds whose seed-coats were removed previously. References AMEN, R . D . (1968): A model of seed do rmancy . — Bot. Rev. 34, 1—31. BASKIN, J . M. and BASKIN, C. C. (1970): Replacement of chilling requirement in seeds of Ruellia humilis by gibberellic acid. — Planta 94, 250—258. FRANKLAND, B. (1961): Effect of gibberellic acid, kinetin and o the r substances on seed d o r m a n c y — N a t u r e , 192, 678. FRANKLAND, B . a n d WAREING, P . F . ( 1 9 6 2 ) : C h a n g e s in e n d o g e n o u s g i b b e r e l l i n s in r e l a t i o n t o c h i l l i n g of d o r m a n t seeds. — N a t u r e 194, 313—314. FRANKLAND, B. and WAREING, P. F. (1966): Hormona l regulat ion of seed d o r m a n c y in hazel and beech. — J. Exp. Bot. 17, 596—61. JACOBSEN, J . V . , PRESSMAN, E . a n d PYLIOTTC, N . A . ( 1 9 7 6 ) : G i b b e r e l l i n - i n d u c e d s e p a r a t i o n o f c e l l s in isolated endosperm of celery seed. — Planta 129, 113—122. JUNTILLA, O. (1970): Effects of stratif ication, gibberellic acid and germinat ion t empera tu re o n the germinat ion of Betula nana. — Phys. Plant . 23, 425—433. NAGY, MÁRIA (1976): Dormancy in f rui ts of Tillaplatyphyllos Scop. III . T h e possible role of auxin and cytokinin in the breaking of do rmancy . — Acta Biol. Szeged. 22, 47 55. NAGY, MÁRIA ( 1 9 8 0 ) : D o r m a n c y in f r u i t s o f Tiliaplatyphyllos SCOP. I V . C h a n g e s in t h e e n d o g e n o u s gibberellin content dur ing the s trat i f icat ion of the seeds. — Acta Agron . 29, 1 11. NAGY, MÁRIA and SZALAI, I. (1973): D o r m a n c y in f rui ts of Tilia platyphvllos Scop . I. — Acta Biol S z e g e d , 19, 7 1 — 7 7 . OLNEY, H. O . and POLLOCK, B. M. (1960): Studies of rest period. II . Nitrogen and p h o s p h o r u s chan -ges in embryonic organs of af ter-r ipening cherry seed. — Plant Physiol. 35, 970—975. D O R M A N C Y I N F R U I T S O F TILIA PLATYPHYLLOS SCOP. V. POSSIBLE R O L E O F C H I L L I N G 1 3 7 SÁRKÁNY, S . a n d SZALAI, I . ( 1 9 5 7 ) : N ö v é n y s z e r v e z e t t a n i g y a k o r l a t o k ( P h y t o m o r p h o l o g i c a l e x e r c i s e s ) (in Hungarian). — Tankönyvkiadó, Budapest, Hungary, pp. 524—577. SZALAI, I . a n d NAGY. MÁRIA ( 1 9 6 8 ) : T h e c a u s e s o f d o r m a n c y i n Fraxinus excelsior L. f r u i t s a n d t h e role of gibberellic acid in breaking dormancy. — Acta Bot. Acad. Sci. Hung. — 14, 4?5—433. SZALAI, I . a n d NAGY, MÁRIA ( 1 9 7 4 ) : D o r m a n c y in f r u i t s o f Tilia plaiyphyllos SCOP. I I . T h e i n h i b i t o r substance content of dormant fruits. — Acta Bot. Acad. Sci. Hung. — 20, 389—394. VANSTONE, D . E . a n d LA CROIX, L . J . ( 1 9 7 5 ) : E m b r y o i m m a t u r i t y a n d d o r m a n c y o f b l a c k a s h . — J . Am. Soc. Hort . Sci. 100, 630—632. VILUERS, T. A. and WAREINO, P. F . (1965): The possible role of low temperature in breaking t h e dormancy of seeds of Fraxinus excelsior L. — J . Exp. Bot. 16, 519—531. WHITE, P. R. (1943): A handbook of plant tissue culture. — Catell Press Lancaster. Address of the au thors : D R . MÁRIA NAOY ÁGNES KÉRI Department of Plant Physiology D R . SZERÉN PATAKY Department of Botany, A. J. University, H-6701 Szeged, Hungary 

Domancy in fruits o f Tilia platyphyllos Scop. V. Possible role of chilling stratification in breaking dormancy

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Domancy in fruits o f Tilia platyphyllos Scop. V. Possible role of chilling stratification in breaking dormancy

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