ANATOMY, HISTOCHEMISTRY AND CYTOLOGY OF DORMANT AND STRATIFIED APPLE EMBRYOS

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1 Neio Phytol. (1981) 87, ANATOMY, HISTOCHEMISTRY AND CYTOLOGY OF DORMANT AND STRATIFIED APPLE EMBRYOS UL STRUCTURAL CHANGES DURING THE EARLY DEVELOPMENT OF SEEDLINGS IN RELATION TO EMBRYONIC DORMANCY BY A. DAWIDOWICZ-GRZEGORZEWSKA Institute of Botany, University of Warsaw, Krakowskie Przedmiescie 26/28, Warszawa, Poland (Accepted 16 June 1980) SUMMARY The following sequence of structural changes which are related to the utilization of storage materials have been shown to occur in the apple embryo: degradation of protein bodies, degradation of lipid bodies, appearance and degradation of starch. These processes take place in the embryonic a.xis during stratification and in the cotyledons during the germination of stratified embryos. Important differences were observed in the localization of these processes during the early post-embryonic (up to 12 days) growth of dormant and stratified embryos. It is concluded that during the germination of dormant embryos the embryonic axis infiuences certain hydrolytic processes in cotyledons by delaying and inhibiting their progress, whereas in non-dormant embryos these are stimulated by the embryonic axis. INTRODUCTION Embryos isolated from dormant apple seeds (Malus domestica Borb. cv. Antonovka) remain in a state of embryonic dormancy. They are able to germinate but their rate of germination is slower than the non-dormant, stratified seeds. The lower germination of dormant apple embryos is associated with several developmental anomalies (Wyziriska and Lewak, 1978), different metabolic activities (Lewak, Rychter and ^arska-maciejewska, 1975; Lewak and Rudnicki, 1977) and the development of photosynthetic activity (Maciejewska, 1979). Most of the previous studies on growth of seedlings from deep dormant seeds were carried out on species of the Rosaceae (Flemion, 1959; Come, 1970). However, comparative anatomical studies of seedlings obtained from dormant and stratified seeds are very scarce. The only other studies known to the author were made by Ledbetter (1959) on Rhodotypos tetrapetala and Primus persica and by Nikolaeva, Lodkina and Ljashuk (1974) on Acer tataricum seedlings. In this present study a comparison has been made of the anatomical and cytological changes that occur during development of young (up to 12 days) seedlings grown from dormant and non-dormant (stratified) embryos. It is also hoped that a comparison of the structural changes which take place during stratification (Dawidowicz-Grzegorzewska and Lewak, 1978; Dawidowicz- Grzegorzewska and Zarska-Maciejewska, 1979) with the data of this investigation, will enable a distinction to be made between the structural changes related to the germination process, from those related to the release of embryonic dormancy X/81/ S02.00/ The New Pliytologist

2 574 A. DAWIDOWICZ-GRZEGORZEWSKA MATERIALS AND METHODS Apple seeds (Malus domestica Borb. cv. Antonovka) collected in 1977 were used throughout this study. Embryos were isolated by removing the seed coat and endosperm from dormant seeds and from seeds which had been stratified for 70 days. The conditions of stratification have been described earlier (Dawidowicz- Grzegorzewska and Lewak, 1978). Isolated embryos were grown for 12 days on filter paper moistened with 5 ml of distilled water in Petri dishes (9 cm diam.), under the following conditions: at 25 C and a 12h photoperiod with a light intensity at 10"'' erg s~^ cm~^ from fiuorescent tubes, and with a 12 h photoperiod of 20 C. The optimal temperature for the germination of apple seeds is 15 C, however, at 25 C it is easier to distinguish between the more and less dormant embryos. Samples for cytological processing were taken daily from the culture of stratified embryos and at 3-day intervals from the culture of dormant embryos. Each sample contained 15 to 20 intact embryos or seedlings. The material was fixed and processed as described previously (Dawidowicz-Grzegorzewska and Lewak, 1978). For the localization of cell components the following procedures were performed: improved mercuric-bromophenol blue technique (Chapman, 1975) for proteins and PAS reaction for polysaccharides. RESULTS AND DISCUSSION As was shown earlier (Dawidowicz-Grzegorzewska and Lewak, 1978; Dawidowicz- Grzegorzewska and Zarska-Maciejewska, 1979), the mobilization and depletion of all storage materials during cold stratification is located predominantly in the embryonic axis and takes place in the following order: degradation of protein bodies, degradation of lipid bodies (unpublished data), increase in starch, then, degradation of starch. All of these processes start at the periphery of the hypocotyl and proceed inwards and longitudinally up and down in the embryonic axis. Germination and further growth of the stratified embryos is dependent on the mobilization and degradation of the cotyledonary storage materials, starting at the cotyledonary node and spreading as a wave along the axis. As in the axis, the activation starts in the protodermal cells and proceeds towards the central vascular bundle, progressing faster at the abaxial than in the adaxial side, similar to Pisum arvense and Phaseolus vulgaris (Smith and Elinn, 1967; Smith, 1974). The reserves from the cells around the main vascular bundle are degraded last. The breakdown of storage materials occurs at the same time in both cotyledons, ultimately reaching their apical parts. The histological pattern of the cellular activation processes occurring during stratification and subsequent culturing of the isolated embryos is shown diagrammatically in Figure 1. After the utilization of storage materials, the photosynthetic tissue underlying the abaxial and adaxial protoderm develops in both cotyledons, as described above. The direction of the degradation of cotyledonary reserves indicates that the process is stimulated by the embryonic axis. This observation agrees with many others on the stimulating infiuence exerted by the embryonic axis on different enzyme activities in various plants (Varner, 1964; Wiley and Ashton, 1967; Gientka-Rychter and Cherry, 1968; Chin, Poulson and Beevers, 1972; Hoffmannowa, 1978; Jarvis, Wilson and Fowler, 1978; Maciejewska, 1979). The hormonal nature of this stimulus has already been suggested by Varner (1964).

3 Anatomy of apple embryos III 575 Fig, 1, Diagrammatic representation of the histological pattern of the progress of cellular activation in the apple embryo during stratification (continuous arrows) and after the culture of isolated embryos for 2 days (interrupted arrow), ID, non-activated area; D, activated area. It seems possible that the utilization of storage materials which occurs (during stratification) in the embryonic axis, can be related to the release of embryonic dormancy, whereas utilization of the same reserves in cotyledons, which constitute the main bulk of seed storage materials, can be attributed to germination and to processes taking place during the early growth period of the seedlings. The post-embryonic growth of embryos isolated from dormant seeds differs significantly from the growth pattern described above. Degradation of storage materials is markedly delayed and proceeds according to the different histological pattern (Fig. 2). However, the cells lying in close proximity to the apical root meristem are activated in a manner which shows that the activation is exerted by the apical root meristem cells. So, the protein bodies undergo breakdown and become replaced by vacuoles, thus appearing as a light part of the bromophenol-blue stained section, when compared with the dark peripheral region of the hypocotyl [Fig. 3(a)]. Even in those few embryos which sometimes grow more intensively, cellular activation is always less advanced in the peripheral tissues of the hypocotyl than in the central core. Degradation of storage materials depleted in the cotyledons proceeds from the apical to the basal part and is always faster in the lower cotyledon, which remains in direct contact with the growth medium, and slower in the upper cotyledon [Fig, 4(a), (b)]. After degradation of the storage materials. Fig, 2, Diagrammatic representation of the histological pattern of the progress of cellular activation which occurs during the culture of isolated dormant embryos for 12 days, ED, non-activated area; D, activated area.

4 576 A. DAWIDOWICZ-GRZEGORZEWSKA Fig. 3. The pattern of breakdown of storage protein bodies in a 12-day-old seedling grown from an embryo isolated from a dormant (non-stratitied) seed. Note the presence of light areas (arrowed), devoid of protein bodies at the apical part of the cotyledons and in the cells of the central cylinder near the root apical meristem. Stained with mercuric-bromophenol-blue x 15.

5 Anatomy of apple embryos III 577. '^S- V..... r ; m&. (c) Wk V ^^ Fij;. 4. (a) Loii(,'itudinal section through the shoot apex and adjacent parts of the axis and cotyledons, from a 12-day old seedling grown from a dormant apple emhryo. Note the presence of a dwarfed epicotyl with three pairs of leaf primordia (only one leaf of Hrst pair is present in the section). Note the dark stained, non-degraded protein bodies in the axial tissues as well as in the cotyledon on the left (arrowed). Stained with mercuric-hromophenol-hlue. x45. (b) Median longitudinal section through the apical shoot meristem and fragments of two adjacent cotyledons. Note the absence of any leaf initiation activity in the shoot apical meristem. The protein bodies are only degraded in the righthand cotyledon which is in contact with the growth medium, (c) and (d) Longitudinal median section through the shoot apex with associated leaf primordia and parts of cotyledons from the seedlings grown from stratified apple embryos for 4 days (c) and 7 days (d). Note the expanded, long internodes as compared with (a) and the absence of storage materials in the tissues. Stained with mercuric-bromophenol-blue. x45.

6 578 A. DAWIDOWICZ-GRZEGORZEWSKA development of the photosynthetic tissue and differentiation of the cotyledonary vascular bundles can also be observed basipetally. Finally, after 12 days of culture, a sharp boundary between 'activated' cotyledones and the 'non-activated' embryonic axis can be observed [Fig, 4(a)], A similar boundary was observed by Nikolaeva et al. (1974) during the culture of dormant Acer tataricum L, embryos. The development of the young shoot, if it occurs, is strongly perturbed. At the cellular level these perturbations can be attributed to a reduction in cell elongation, which results in a shortening of the internodes and a reduction in the length of young leaves in relation to their width, as in the dwarf seedlings (Pelton, 1964). There is also an increase in the period of leaf initiation in comparison to normally developed stratified seedlings [Fig. 4(c), (d)]. These observations suggest that some 'inhibitory factor' responsible for the maintenance of dormancy is located in the embryonic axis and is not removed during culture at 25 C, A similar conclusion was made by Maciejewska (1979) in relation to the mode of development of photosynthetic activity in dormant apple seedlings. Additionally, it can be assumed from cytological observations, that some stimulation is also produced by the cells of the apical root meristem. Thus, observed developmental anomalies in seedlings cultured from dormant embryos can be ascribed, not only to the presence of inhibitors but also to a disturbance of the normal equilibrium between inhibiting and stimulating factors. In summary, the anatomical development of young seedlings seems to be controlled by the embryonic axis but its effect is dependent on the physiological state of the embryo; post-embryonic growth of the dormant embryo is inhibited, while that of the non-dormant embryo is stimulated by the embryonic axis. ACKNOWLEDGEMENTS I gratefully acknowledge the constant encouragement and advice given to me by Professor Dr Stanislaw Lewak, This project was supported by funds made available from the Marie Sklodowska-Curie fund established by contributions of the United States and Polish Governments, Grant No, FG-Po-353 (JB-20), REFERENCES CoMK, D, (1970), Les obstacles a la germination. Masson et Cie, Paris, CHAPMAN, D, M, (1975), Dichromatism of bromophenol blue with an improvement in the mercuric bromophenol blue techique for protein. Stain Technology, 50, 25-30, CHIN, T, Y,, POUI.SON, R, & BEEVERS, L, (1972), The influence of axis removal on protein metabolism of Pisum sativum L, Plant Physiology, 49, , DAwmowicz-GRZEGORZEWSKA, A, & LEWAK, S, (1978), Anatomy, histo-chemistry and cytology of dormant and stratified apple embryos, I, General observations and changes in the starch content during and after ripening of seeds. New Phytologist, 81, , DAWir)owicz-GRZEGORZEW,SKA, A, & J^ARSKA-MACIEJEWSKA, B, (1979), Anatomy histochemistry and cytology of dormant and stratified apple embryos, II, Storage protein degradation and correlated nucleoli development. New Phytologist, 83, , Fi.UMlON, F, (1959), Effects of temperature, light and gibberellic acid on stem elongation and leaf development in physiologically dwarfed seedlings of peach and Rhodotypos. Contributions from Boyce-Thompson Institute, 20, 57-70, GiENTKA-RvciniiR, A, & CHERRY, J, M, (1968), Df HHI'O synthesis of isocitratase in peanut (Arachis hypogaea h.) cotyledons. Plant Physiology, 43, , HoFFMANNOWA, A, (1978), Control of starch mobilization in cotyledons of germinating yellow lupin {Lupinus luteus L,), Biochemie und Physiologie der Pflanzen, 173, , JARVIS, B, C, WH.SON, D, A, & FOWI.ER, M, W, (1978), Growth of isolated embryonic axes from dormant seeds of hazel {Corylus avallana L,), New Phytologist, 80, ,

7 Anatomy of apple embryos III 579 LEDBETTER, M. C.( 1959). Anatomical and morphological comparisons of normal and physiologically dwarfed seedlings of Rhodotypos tetrapetala and Prunus persica. Contributions from Boyce-Tliompson Institute, 20, LEW.\K, S., RYCHTER, A. & ZARSKA-MACIEJEWSKA, B. (1975). Metabolic aspects of embryonal dormancy in apple seeds. Physiologie Vegetale, 13, LEWAK, S. & RUDNICKI, R. M. (1977). After ripening in cold requiring seeds. In: The Physiology and Biochemistry of Seed Dormancy and Germination. (Ed. by A. A. Khan), pp North-Holland Biomedical Press, Amsterdam. MACIEJEWSKA, U. (1979). The effect of embryonal axis on tbe development of photosynthetic activity in apple seedlings. Neiv Phytologist, 82, NiKOLAEVA, M. G., LODKINA, M. M. & LjASHUK, A. J. (1974). On the nature of physiological dwarfing of the dormant seeds embryo. Botanitcheskij Journal, 59, 14. PELTON, J. S. (1964). Genetic and morphogenetic studies of Angiosperm single-gene dwarfs. The Botanical Review, SMITH, D. L. (1974). A histological and histochemical study of the cotyledons oi Phaseolus vulgaris L. during germination. Protoplasma, 79, SMITH, D. L. & FI.INN, A. M. (1967). Histology and histochemistry of the cotyledons of Pisum arvense L. during germination. Planta (Berl.), 74, VARNER, J. E. (1964). Gibberellic acid controlled synthesis of a-amylase in barley endosperm. Plant Physiology, 39, WYZINSKA, D. & LEWAK, S. (1978). Morphological aspects of apple seedling early development in relation to embryonal dormancy. Biologia Plantarum (Praha), 20, WILEY, L. & ASHTON, F. M. (1967). Influence of the embryonic axis on protein hydrolysis in cotyledons of Cucurbita maxima. Physiologia Plantarum, 20,

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