Hormonal Regulation of Germination and Early Seedling Development in Acer pseudoplatanus (L.)

Planta (Berl.) 104, 134-145 (1972) 9 by Springer-Vcrlag 1972 Hormonal Regulation of Germination and Early Seedling Development in Acer pseudoplatanus (L.) N. J. Pinfield and A. K. Stobart Plant Cell Metabolism Laboratory, Botany Department, The University Bristol, U. K. Received November 25 / December 17, 1971 Summary. Dormancy of intact sycamore (Acer pseudoplatanus) seeds was broken by chilling (5~ for several weeks in moist conditions. Treatment of unchilled seeds with kinetin induced some germination, but gibberellin was ineffective. This stimulation by kinetin was not suppressed by the added presence of abscisic acid during incubation. The chilling requirement of intact seeds was eliminated by removal of the testa, and the naked embryos developed with no morphological abnormalities. During early growth of isolated embryos in the light, two distinct developmental processes were recognised. One involved initial elongation of the radicle accompanied by geotropic curvature and was stimulated by kinetin but not by gibbercllin, while the other involved unrolling of the cotyledons, which was accelerated by gibberellin but much less by kinetin. Abscisic acid strongly suppressed both developmental processes when applied alone, inhibited cotyledon expansion in the presence of gibberellin, but failed to overcome the promotory effects of kinetin on radicle growth. Experiments with CCC indicated that under natural conditions the unrolling of the cotyledons is dependent upon endogenous gibberellin, t~adicle growth of isolated embryos was unimpaired by incubation in the dark, but cotyledon expansion of water incubated embryos was poor, and although it was accelerated by gibberellin, the responses in all treatments were slower than in the corresponding light grown samples. It is suggested that endogenous cytokinins are primary factors in the initiation of radicle growth, while gibberellins are important in cotyledon expansion. Abscisic acid appears to have an inhibitory role in both processes, and the interactions of these regulators in the control of germination and development are discussed. Introduction The seeds of many species which normally require a chilling period for the breaking of dormancy can be induced to germinate by the application of gibberellin (Frankland and Wareing, 1966; Bradbcer and Pinfield, 1967), and in a few cases by kinetin treatment (Frankland, 1961). Many cold requiring seeds show true embryo dormancy and the

N. J. Pinfield and A. K. Stobart: Hormonal Regulation of Germination 135 naked embryo fails to germinate under favourable growth conditions. This can usually be related to physiological and biochemical factors in the embryo itself (Pollock and Olney, 1959; Pinfield, 1968a, b). In other seeds with a chilling requirement, however, dormancy is imposed upon the embryo by the presence of one or more of the integuments which normally enclose it (Black and Wareing, 1959; Pinfield, Martin, and Stobart, 1972). Although inhibitory factors such as abseisie acid have frequently been implicated in such eases (Jackson and Blundell, 1966; Irving, 1968), less is known concerning the roles of growth promotory substances in seeds showing testa-imposed dormancy. The seed of Acer pseudoplatanus L., the sycamore, constitutes an example of this latter group, the isolated embryo germinating readily, while the seed remains dormant as long as the testa is still present (Wareing, 1969). The role of the covering integuments in the regulation of germination in sycamore has recently received intensive investigation (Wareing and Webb, 1972), but little is known concerning the control exerted on germination and subsequent seedling development by the hormonal balance within the seed. Materials and Methods Mature samaras of sycamore were collected locally during the Autumn and after drying for one week at room temperature were stored in a metal bin in a cool room. Periearps were removed and the intact seeds were soaked in water for 24 h. The seeds were then transferred either to 9 cm Petri dishes or to 2 cm specimen tubes. 10 seeds were sown in each Petri dish and single seeds were incubated in the specimen tubes. Both vessels were lined with a single thickness of Whatman no. 1 filter paper moistened with 5 ml or 0.5 ml distilled water or test solution respectively. Germination was measured as protrusion of the radicle. For germination of intact seeds samples of 50 seeds were used for each treatment, but samples of 20 isolated embryos were used because of the labour involved in removal of the testa. The germination of naked embryos was observed by stripping the testas from seeds soaked for 24 h in water, and incubating these embryos as described above. In these cases, the first geotropic curvature of the radicle was used as the criterion of germination (Wareing and Webb, 1972). The unrolling of the cotyledons was also used as a developmental criterion, and in this instance the embryos were scored on a system of nil for an embryo with fully rolled cotyledons to a maximum of 4 units for an embryo with fully expanded cotyledons, with three arbitrarily selected developmental stages between (Fig. 1). The figure derived from this scheme for a sample of 20 embryos is termed a cotyledon expansion index, the maximum value of which is 80 units. Both intact seeds and naked embryos were incubated at 20~ either in darkness or in continuous illumination of 6000 lux under fluorescent tubes. A gift of abseisic acid was generously provided by the Woodstock Agricultural Research Centre. Gibberellie acid and indole acetic acid were obtained from British Drug Houses Ltd., kinetin from Sigma Chemical Company, and (2-ehloroethyl) trimethylaminoninm chloride (CCC) from Eastman-Kodak Ltd.

Fig. 1

Hormonal Regulation of Germination and Seedling Development 137 Results Germination o] Intact Fruits ancl Seeds Intact fruits and seeds of Aeer pseudoplatanus failed to germinate if incubated in water at 20 ~ C. Perforation of the testa in various sectors of the seed was also ineffective. These findings confirm those reported by Wareing and Webb (1972). Intact seeds, however, could be induced to germinate if the fruits were given a prior chilling treatment for a period of several weeks. The data shown in Fig. 2 were obtained from seeds incubated for 4 weeks at 20~ after various periods of chilling the intact fruits at 5~ Maximum values for germination in the three seasons investigated were 78% after 9 weeks chilling in 1969, 70% after 7 weeks chilling in 1970 and 84% after 7 weeks chilling in 1971. Most experiments described subsequently were carried out on seeds of two or more seasons and no substantial differences in the behaviour of the seeds from different crops were observed. For this reason, the data for different seasons are not presented separately. Concentrations of gibberellin between 3 10-7 M and 3 10-4M, indole acetic acid between 6 10-7 M and 6 10-4 M, and abscisic acid between 5 10-7 M and 5 10 5 M were all ineffective in inducing germination. Kinetin in the range 5 t0 6M--1 10-~M induced some germination and a maximum of 45% was recorded after 21 days incubation in a 5 10-5 M solution (Fig. 3). Fig. 3 also shows that no appreciable inhibition of this kinetin-stimulated germination resulted when seeds were incubated in mixtures containing 5 10-5 M kinetin and 5 10-5 M abscisic acid. In all instances where it occurred, germination followed the same pattern, namely an initial protrusion of the radicle, followed by radicle elongation and subsequently, after several days delay, by the emergence and expansion of the cotyledons from the ruptured testa. Incubation of seeds with previously perforated testas in gibberellin resulted in a stimulation of cotyledonary expansion, which was not preeeeded by any appreciable radicle growth, producing seedlings with fully expanded cotyledons but essentially embryonic radicles. In all cases incubation at 20~ was carried out both in the light and in the dark, with no appreciable differences in germination between comparable treatments. Fig. 1. Illustration of the scheme for the derivation of the cotyledonary expansion index. Individuals in the series A to E score 0 to 4 units respectively, as a measure of the degree of unrolling of the cotyledons. Cotyledonary expansion in all embryos was determined by reference to this scheme

138 N.J. Pinfield and A. K. Stobart: 50. I00~ 90 80 70- t- O "~5, r- 0 60 84 4-O- 30-20- 10- o c 0 ho- 3o-.E o ~ 10- ~, "/f 0 5O I.~ 4.0- o 20 i 10 o-~ B r 10-6 10-5 10-4 tog molar kinetin concentration 0 0 2,4 6 8 10 12 Chilling period ( weeks )....,b.... 2'0... Time ( days ) Fig. 2 Fig. 3 Fig. 2. The effect of chilling at 5~ on the germination of intact sycamore seeds. The percentage germination is that found after 4 weeks subsequent treatment at 20~ o seeds of 1969 crop; = seeds of 1970 crop; ~ seeds of 1971 crop Fig. 3 A and B. The effect of kinetin on the germination of intact sycamore seeds at 2O~ A Final percentage germination after 4 weeks incubation plotted against the concentration of kinetin supplied. B The rate of germination in 5 10-5 M kinetin (o), and also in mixtures containing final concentrations of 5 10 ~-5 M kinetin and abseisic acid (.). Over this period, no germination occurred in the water controls Germination o/isolated Embryos When embryos were dissected from imbibed seeds by removal of the testa, and incubated in water in the light, germination and a renewal of growth occurred, which resulted in the production of morphologically normal seedlings with no sign of physiological dwarfing. This process can conveniently be divided into two parts, one involving extension of the radicle, and accompanied by a gcotropic curvature, used here as the criterion of germination. Subsequently the embryonic radicle differentiates into a true radicle and a hypocotyl. The second process, commencing simultaneously, or after a short delay of a few hours involved the unrolling of the previously tightly roiled cotyledons,

Hormonal Regulation of Germination and Seedling Development 139 100" 80. 6o- C E 4.O 0 20 84 4. A 80"1 X L.// f/<... glo, 2 /~ 6 8 10 12 1~ 0 Time (days) t,b D l~-' ptl//~,,, J 2 4 6 8 Time (days) i 10 Fig. 4A and B. The rate of germination (A) and cotyledon expansion (B) of isolated embryos in water (o), 3 10 -t M gibberellin (,), 5 10 -s M kinetin (.), and mixtures containing final concentrations of 3 1O -~ M gibberellin and 5 10-5 M kinetin (o). Embryos were incubated at 20~ in the light Table 1. Chlorophyll content of the cotyledons of sycamore seedlings grown from isolated embryos incubated for 10 days in water, kinetin, or gibberellin in the light. The values are averages of 5 determinations, each performed on a fully expanded pair of cotyledons, and a standard error for each mean is given Treatment Total chlorophyll Chlorophyll (mg/g fresh wt.) a/b ratio ~I20 0.89 0.13 3.22 ~ 0.28 Kineth~ 1.25 ~ 0.07 3.10 -E 0.23 Gibberellin 0.74 0.15 2.70 0.20 Freshly isolated embryos 0.11 -L 0.02 2.65 0.21 Although further extension of both radicle and hypocotyl occurred during the completion of cotyledonary expansion the data presented here are concerned only with the initial growth of the radicle associated with the geotropic curvature. In the experiments subsequently described, the final concentrations of growth regulators used were 3 10-4 M gibberellin, 5 10 -s M kinetin, 5 10 -~ M abseisic acid and 6 10 4 M CCC. Germination and initial extension of the radicle were markedly accelerated by incubation with kinetin, but no significant stimulation of these processes was observed as a result of gibberellin treatment (Fig. 4A). The unrolling of the cotyledons, however, was greatly stimulated by incubation with gibberellin, while treatment with kinetin was considerably less effective in this respect (Fig. 4B). Incubation of

140 N.J. Pinfield and A. K. Stobart: Hormonal Regulation of Germination 100-80- ~ eo- E,,~ h0- A 80- x ~7o. 9 ~'f- '~ 60- o '~50- B o o 9 20- //, / [ [ /---~, ~10- o//~ u / 2 4 6 8 10 12 14 0 Time (days) 2 4 6 8 10 Time ( days } Fig. 5A and B. The rate of germination (A) and cotyledon expansion (B) of isolated embryos in water (o), 5 l0-5 M abseisic acid (,), mixtures with final concentrations of 5 10 -s M abscisie acid and 3 x 10-4 M gibberellin (=), and mixtures with final concentrations of 5 x 10-5 M abseisic acid and kinetin (o). Embryos were incubated at 20~ in the light embryos with mixtures of kinetin and gibberellin markedly stimulated both radicle growth (Fig. 4A) and cotyledonary expansion (Fig. 4B) compared with development in the water controls. A comparison of this result with those for incubation with gibberellin and Idnetin separately show that the response to the mixture was additive. Chlorophyll determinations carried out on fully expanded cotyledons of l0 day old light grown seedlings are given in Table 1. The rate of chlorophyll production during early seedling growth in the light was increased by treatment with kinetin, while the cotyledons of gibberellin treated material, although fully unrolled for a longer period, were not substantially different from the water controls. The significance of the slightly lower chlorophyll a:chlorophyll b ratio following gibbere]lin treatment is not clear. Both radicle growth and cotyledon expansion were completely blocked by abscisic acid (Fig. 5). Incubation of embryos in mixtures of abscisic acid and kinetin showed that abscisic acid was unable to overcome the promotory effect of kinetin on radicle growth (Fig. 5A), and both germination and radicle extension occurred at a rate similar to that recorded during incubation with kinetin alone. Fig. 5 B shows, however, that the gibberellin-stimulated unrolling of the cotyledons was suppressed if embryos were incubated in mixtures of abscisie acid and gibberellin. Fig. 6 shows the developmental stages occurring during early seedling growth as a result of treatment with water (Fig. 6A), kinetin (Fig. 6B) and gibberellin (Fig. 6C). The stimulation of radicle growth by kinetin

Fig. 6A C. The developmental stages of isolated sycamore embryos incubated in water (A), kinetin (B), or gibberellin (C) at 20~ in the light 10 Planta(Berl.),:Bd. 104

142 N.J. Piiffield and A. K. Stobart: 80- "X ~ 70,..- 60.o_ O- ~4o. A 4/ / B 8O 9 60 ~50 ~4o B 30. O ~2o- g 30. 0 8 10. 8 lo. 0 i i g ~ lb 0 Time (days) 2 4 6 8 10 Time (days) Fig. 7A and B. The effect of CCC on the gibberellin-stimulated expansion of the cotyledons of isolated embryos obtained from seeds imbibed for 24 hours in water (A) or 6 10-42V[ CCC (B). Embryos were subsequently incubated in water (o), 6 CCC (.), 3 gibberellin (-), or mixtures containing final concentrations of 6 10-4M CCC and 3 10-4M gibberellin (o) is readily apparent, while the effects of gibberellin can clearly be seen to be on cotyledon expansion, with the radicle remaining largely undeveloped. Kinetin, therefore, appears to be a primary factor in the initiation of radicle growth, gibberellin seems to be involved mainly with eotyledonary expansion, while abseisie acid appears to play an important role in both processes. It seems likely, therefore, that these phenomena are controlled in nature by corresponding naturally occurring endogenous regulators. Some evidence on the importance of endogenous gibberellin in the unrolling process has been obtained from experiments with CCC, an inhibitor of gibberellin biosynthesis. Incubation of isolated embryos from water imbibed seeds in CCC considerably retarded the unrolling of the cotyledons (Fig. 7). This inhibitory effect of CCC can readily be eliminated by simultaneous application of gibberellin. Furthermore, imbibition of seeds in CCC, followed by transfer of the isolated embryos to the appropriate test solution, resulted in a marked inhibition of cotyledon expansion in the water controls as well as in the embryos incubated in CCC. As before, this inhibition could be overcome by treatment with gibberellin, even when CCC was also present in the incubation medium. These results suggest that the unrolling phenomenon is dependent upon endogenous gibberellin and the necessity for gibberellin biosynthesis is removed when gibberellin is supplied exogenously. The experiments on isolated embryos described above were all conducted in the light, but similar experiments were also carried out in

Hormonal I~egulation of Germination and Seedling Development 143 Table 2. The effects of kinetin, gibberellin, and abscisie acid on germination and cotyledon expansion of isolated embryos incubated in the dark Treatment Incubation time (days) % germination cotyledon expansion index 4 7 10 4 7 10 H~O 10 35 40 4 12 12 Kinetin 45 60 80 23 30 43 Gibberellin 15 25 45 33 49 72 Abscisic acid 0 0 0 2 6 6 the dark. In all cases radicle growth was similar to that in the corresponding light grown material, but cotyledon expansion in dark grown embryos incubated in water was much slower than in the comparable light grown controls. Treatment with gibberellin and, to a much lesser extent, kinetin, however, induced unrolling of the cotyledons in the dark, but in all cases the responses were slower and less marked than in the corresponding light grown samples. These results are summarised in Table 2. Discussion Although cytoldnins appear to be less widely effective than gibberellins in the promotion of seed germination, a number of examples are known (Fox, 1969). Haber and Luippold (1960) suggested that this promotory effect resulted from a stimulation of cell enlargement in the radicle. More recently, however, Ikuma and Thimarm (1963) have presented evidence for the occurrence of a primary response to cytokinins in the cotyledons. In the present report, it is clear that the initial macroscopic response occurs in the radicle, although it is possible that this may be preceded by physiological or biochemical changes in the cotyledons. Although kinetin causes a marked increase in chlorophyll levels during early seedling growth, kinetin also stimulates germination in the dark, where no chlorophyll synthesis is occurring, and it would seem, therefore, that germination is not directly connected with a kinetin-accelerated greening of the cotyledonary tissues. Although gibberellin failed to induce germination in intact seeds it greatly stimulated the unrolling of the cotyledons of isolated embryos. Its failure to cause germination appears to be related to its ineffectiveness in stimulating any appreciable growth of the radicle. Nevertheless, it appears to play an important role in the early stages of seedling development and the experiments with CCC indicate that under natural conditions this role is taken by endogenous gibberellins produced in the 10"

144 5I. J. Pinfield and A. K. Stobart: embryo itself. It is quite clear from the results of experiments with isolated embryos that unrolling of the cotyledons in the presence of added gibberellin can occur unaccompanied by renewed radicle growth. It would seem Likely, therefore, that incubation of intact seeds with gibberellin would result in expansion of the cotyledons, causing rupture of the testa, and germination. That this is not the case indicates that the testa may be acting as a barrier to gibberellin uptake. It has recently been established that the seed integuments of some Acer species act as effective barriers to water uptake (Dnmbroff and Webb, 1969; Webb and Dumbroff, 1970), and thus uptake of more complex molecules such as gibberellin may also be impaired. In the present report, results of experiments on seeds with perforated testas indicate that the intact testa may well be blocking uptake of gibberellin, while the sub-maximal germination response of intact seeds to kinetin may also be related to restricted kinetin uptake. Active meristematie regions of a plant are the major sites of gibberellin biosynthesis (Jones and Phillips, 1966), and it can be argued that under natural conditions endogenous cytokinins induce a renewal of meristematic activity in the radicle apex, which leads to increased gibbercllin biosynthesis, resulting in cotyledon expansion. However, the poor development of the cotyledons of dark grown water incubated embryos with actively growing radicles indicates that this explanation may be too simple. Abscisic acid also appears to play a role of primary importance in both germination and development. Examples of antagonism between abscisic acid and both cytokinins and gibberellins are numerous (Addicott and Lyon, 1969; Wareing and Saunders, 1971), but the mechanisms of these interactions are not clear. The involvement of phytochrome in the unrolling of cereal leaves is now well established, and more recently (Beevers, Loveys, Pearson, and Wareing, 1970; Pearson and Wareing, 1970), it has been demonstrated that cytokinins, gibberellins and abscisic acid are all implicated in this process. From the results in the present report, it appears that the same factors are involved in the unrolling of the embryonic leaves of sycamore, and that the unrolling process in these taxonomically distinct species may well be controlled by a similar mechanism. References Addicott, F. T., Lyon, J. L.: Physiology of abscisic acid and related substances. Ann. Rev. Plant Physiol. 20, 139-164 (1969). Beevers, L., Loveys, B., Pearson, J.A., Wareing, P. F. : Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (]3erl.) 90, 286-294 (1970).

Hormonal Regulation of Germination and Seedling Development 145 Black, M., Wareing, P. F.: The role of germination inhibitors and oxygen in the dormancy of the light sensitive seed of Betula spp. J. exp. Bot. 10, 134-145 (1959). Bradbcer, J.W., Pinfield, N. J.: Studies in seed dormancy. III. The effects of gibberellin on dormant seeds of Corylus avellana L. ~qew Phytologist 66, 515-523 (1967). Dumbroff, E. B., Webb, D. P.: Factors influencing the stratification process in seeds of Acer ginnala. Canad. J. Bot. 48, 2009-2015 (1970). Fox, J. E. : The cytokinins. In: Physiology of plant growth and development, p. 84, ed. M. B. Wilkins. London: McGraw-Hill 1969. Frankland, B. : Effect of gibberellic acid, kinetin and other substances on seed dormancy. =Nature (Lond.) 192, 678-679 (1961). Frankland, B., Warcing, P. F. : Hormonal regulation of seed dormancy in hazel (Corylus avellana L.) and beech (Fagus sylvatica L.). J. exp. Bot. 17, 596-611 (1966). :Haber, A. H., Luippold, H. J.: Separation of mechanisms initiating cell division and cell expansion in lettuce seed germination. Plant Physiol. 35, 168-173 (1960). Ikuma, H., Thimann, K. V.: Action of kinetin on photosensitive germination of lettuce seed as compared with that of gibberellic acid. Plant and Cell Physiol. 4, 113-128 (1963). Irving, R. M. : Study on dormancy, germination, and growth of seeds and buds of Acer negundo. Plant Physiol. 43, Suppl. 49 (1968). Jackson, G. A. D., Blundell, J. B. : Effects of dormin on fruit-set in Rosa. Nature (Lond.) 212, 1470-1471 (1966). Jones, R.L., Phillips, I. D.J.: Organs of gibbereltin synthesis in light-grown sunflower plants. Plant Physiol. 41, 1381-1386 (1966). Pearson, J.A., Warcing, P. F. : PolysomM changes in developing wheat leaves. Planta (Berl.) 93, 309-313 (1970). Pirgield, N. J.: The promotion of isocitrate lyase activity in hazel cotyledons by exogenous gibberellin. Flanta (Berl.) 82, 337-341 (1968a). Pinfield, N. J.: The effects of gibberellin on the metabolism of ethanol-soluble constituents in the cotyledons of hazel seeds (Corylus avellana L.). J. exp. Bot. 19, 452-459 (1968b). Pinfield, N.J., Martin, M.H., Stobart, A. K. : The control of germination in Stachys alpina (L.). New Phytologist 71, 99-104 (1972). Pollock, B. M., Olney, H. O. : Studies of the rest period. I. Growth, translocation and respiratory changes in the embryonic organs of the after-ripening cherry seed. Plant Physiol. 34, 131-142 (1959). 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., Saunders, P.F.: Hormones and dormancy. Ann. l~ev. Plant Physiol. 22, 261-288 (1971). Wareing, P. F., Webb, D. P. : Seed dormancy in Acer: The role of the covering structures in the dormancy of Acer pseudoplatanus L. J. exp. Bot. 23 (in press) (1972). Webb, D.P., ])umbroff, E. B. : Factors influencing the stratification process in seeds of Acer saccharum. Canad. J. Bot. 47, 1555-1563 (1969). Dr. N. J. Pinfield Plant Cell Metabolism Laboratory Botany Department The University Bristol BS8 lug, U.K.