The Effect of Stratification on Endogenous Cytokinin Levels in Seeds of Acer saccharum

Transcription

1 Planta (Berl.) 14, (1972) 9 by Springer-Verlag 1972 The Effect of Stratification on Endogenous Cytokinin Levels in Seeds of Acer saccharum J. van Staden, D. P. Webb and P. F. Warcing Botany Department, University College of Wales, Aberystwyth, U.K. Received December 1, 1971 Summary. Stratification of sugar maple seed resulted in high levels of eytokinins after 2 days. A further increase in the chilling period led to a decrease in cytokinins. Most of the cytokinin activity detected was due to compound(s) that co-chromategraphed with zeatin. t is concluded that this increase in cytokinins may result in the breaking of dormancy, perhaps by overcoming the effect of endogenous inhibitors. ntroduction Many seeds have a chilling requirement for germination. This may involve an interplay between germination promoters and inhibitors (Villiers and Wareing, 196; Amen, 1968). n several species the levels of endogenous inhibitors decreased during stratification (Lipe and Crane, 1966; Martin, Mason, and Forde, 1969; Rudnicki, 1969) whereas in others significant increases in the levels of endogenous gibberellins have been reported (Frankland and Wareing, 1966; Kentzer, 1966; Ross and Bradbeer, 1968; Sifiska and Lewak, 197). No changes in endogenous cytokinins of stratified seed have apparently yet been reported. Webb and Dumbroff (1969) showed that the dormant embryos of the sugar maple require a period of chilling prior to germination. Kinetin and gibberellic acid were effective in overcoming the dormancy of unstratified seed, suggesting that endogenous cytokinins and gibberellins may play a role in overcoming seed dormancy in this species. The object of the present study was to investigate whether stratification has any effect on the endogenous cytokinins of sugar maple seeds. Materials and Methods Fruits of Acer saccharum Marsh. were soaked in distilled water for 24 hr and incubated at 5 ~ and 2~ for, 2, 4 and 5 days respectively. At the completion of the treatments the seeds were removed from the pericarps and extracted for eytokinins. Twenty five grams of non-germinated seed was homogenized with 5 ml 8 % methanol. The homogenate was extracted for 12 hr at 4~ and then filtered. The

2 Stratification and Endogenous Cytokinin Levels 111 residue was washed with a further 5 ml of methanol and then discarded. The combined filtrates were concentrated to the aqueous phase under vacuum at 35~ and left at -- 2~ overnight. Samples were thawed and then centrifuged at 1, g (max.) for 3 mill. The precipitate was washed twice with water, recentrifuged and discarded. The combined supernatan~s were brought to 2 ml, adjusted to ph 9. and extracted as follows: Petroleum ether Aqueqas Extract Partitioned with 2 equal volumes of petroleum ether (b.p. 6-8~ Ethyl acetate Aqueous/faction Adjusted to ph 2.5 and shaken with 3 equal volumes of ethyl acetate l Butanol ]raction Aqueous ]raction Adjusted to ph 7. and shaken with 3 equal volumes of water saturated n- butanol Aqueous ]ractiou Treated with alkaline phosphatase (i mg enzyme/1 ml extract) for 24 hr at 3~ at pit 8.2 in.1 M Tris buffer and.1 M MgCl 2. Partitioned with 3 equal volumes of water saturated n-butanol F Aqueous/raction Redistilled solvents were used for partitioning. All fractions were taken to dryness under vacuum at 35~ redissolved in 9% ethanol, streaked on Whatman No. 1 chromatography paper, and separated with iso-propanol: ammonia :water (1:1 : 1 v/v). The dried chromatograms were divided into 1 equal strips and assayed for cytokinin activity with the soybean callus bioassay (Miller, 1965). Fifty ml of medium was added to each 1 ml flask and three callus explants were transferred to each flask. These were maintained at 26~ under continuous illumination. Results and Discussion Sugar maple seed incubated at 2~ for various periods do not germinate when transferred to suitable germination conditions. Furthermore no cytokinin activity could be detected in them. n contrast, seed subjected to periods of chilling showed an increased ability to germinate. Fifteen, 38 and 65 % of the seeds germinated after 2, 4 and 5 days of 8*

3 112 J. van Staden, D. P. Webb and P. F. Wareing:.2 A B C Day,_,--..-,._.._.~ '-L.-.--,_q ~,-m..., Day O.8.6 o.4 22 q ~ m ~. 6.~.2 ~ j ~.2 t Day ~ 1.2- ;L "~.8 f ~ t...l ~.6- Day 5 ~ ~.4-.2-~ c f~-_ t~ O d i, i, i i i, P, i ,1,i, i, i.i 1,1,, i, i, i Fig. 1. Soybean callus bioassay of seeds stratified for, 2, 4 and 5 days. The petroleum ether (A), n-butanol (B), and aqueous (C) extracts were chromatographed on Whatman No. 1 paper in isopropanol: ammonia: water (1:1 : 1 v/v). Cultures were grown for 28 days chilling respectively. Cytokinin activity was found in the petroleum ether, butanol and aqueous extracts of stratified seed (Fig. 1). The ethyl acetate extracts showed no signs of any activity and are not included in the figure. The highest concentration of eytokinins was found in the butanol extract after 2 days of chilling. A further increase in the chilling period resulted in a decrease in eytokinins. n fact no cytokinin activity could be detected in the germinated seed. This was surprising as the roots

4 Stratification and Endogenous Cytokinin Levels 113 r k , ~ 2.2 Zeotin i i E~ E td o. o ~ 1.8 1,6 9 ~ o.8.6 O.Z~ Fk]sk number Fig. 2. The distribution of cytokinin activity of a Sephadex LH-2 fractionation of a butanol extract of non-germinated seed stratified for 4 days. Cultures were grown for 28 days are considered to be one of the major sites of cytokinin synthesis (Kende, 1965; Sitton, tai, and Kende, 1967). The fact that the petroleum ether extract of stratified seed showed cytokinin activity is noteworthy since none of the authentic cytokinins available to us partitioned into this solvent at pt 9.. We are of the opinion that the activity is probably not due to a purine derivative. An attempt was made to determine whether the eytokinin activity present at gf.6-.8 of the butanol extracts was due to more than one natural compound. An extract from non-germinated seed stratified for 4 days was separated on a Sephadex LH-2 column (3 cm 9 cm) using the technique of Armstrong, Burrows, Evans and Skoog (1969). The column was eluted with 1.6 litres of 35 % ethanol at a flow rate of 25 ml/ hr. After discarding the void volume 4 ml fractions were evaporated to dryness at 45~ and assayed for cytokinin activity. The results in Fig. 2 indicate that most of the cytokinin activity was due to compound(s) which eo-chromatographed with zeatin. The inability of unehitled (dormant) sugar maple seeds to germinate may be the result of a deficiency of endogenous cytokinins, which during low temperature treatment are either synthesized or released from a bound form. Moreover, this increase in cytokinins during chilling may be responsible for overcoming the effect of endogenous inhibitors present in the seed. t has been shown that application of exogenous eytokinins

5 114 J. van Staden et al. : Stratification and Endogenous Cytokinin Levels can overcome the effect of endogenous inhibitors and release the seeds from the dormant state (Khan, 1966; Khan and HeR, 1969). However, before any definite conclusions regarding the possible role of endogenous cytokinins in breaking the dormancy of sugar maple seeds can be made, the levels of endogenous inhibitors and gibberellins will also have to be investigated. These are presently under study. This research was supported in part by a grant from the Council for Scientific and ndustrial Research, Pretoria, Republic of South Africa. References Amen, R. D. : A model of seed dormancy. Bot. Rev. 34, 1-3 (1968). Armstrong, D. J., Burrows, W. J., Evans, P. K., Skoog, F. : solation of cytokinins from trna. Biochem. biophys. Res. Commun. 87, (1969). Frankland, B., Wareing, P.F.: Hormonal regulation of seed dormancy in hazel (Corylus avellana L.) and beech (Fagus sylvatica L.). J. exp. Bot. 17, Kende, H.: Kinetin-like factors in the root exudate of sunflowers. Proc. nat. Acad. Sci. (Wash.) 53, (1965). Kentzer, T. : The dynamics of gibberellin-like and growth-inhibiting substances during seed development of Fraxinus excelsior L. Acta Soc. Bot. Pol. 85, Khan, A. A. : Breaking of dormancy in Xanthium seeds by kinetin mediated by light and DNA dependent RNA synthesis. Physiol. Plantarum (Cph.) 19, Khan, A. A., Heir, C. E. : Selective effect of hormones on nucleic acid metabolism during germination of pear embryos. Biochem. J. 118, (1969). Lipe, W. N., Crane, J. C. : Dormancy regulation in peach seeds. Science 158, Martin, G. C., Mason, M.. R., Forde, H. E. : Changes in endogenous growth substances in the embryos of Juglans regia during stratification. J. Amer. Soc. Hort. Sci. 94, (1969). Miller, C. O. : Evidence for the natural occurrence of zeatin and derivatives: Compounds from maize which promote cell division. Proc. nat. Acad. Sci. (Wash.) 54, (1965). Ross, J. D., Bradbeer, J. W. : Concentrations of gibberellin in chilled hazel seeds. Nature (Lond.) 22, (1968). Rndnicki, R.: Studies on abscisic acid in apple seeds. Planta (Berl.) 86, (1969). Sifska,., Lewak, S. : Apple seed gibberellins. Physiol. Veg. 8, (197). Sitton, D., tai, C., Kende, H. : Decreased cytokinin prodnction in the roots as a factor in shoot senescence. Planta (Berl.) 73, (1967). Villiers, T.A., Wareing, P.F.: nteraction of a growth inhibitor and a natural germination stimulator in the dormancy of Fraxinus excelsior L. Nature (Lond.) 185, (196). Webb, D.P., Dumbroff, E.B.: Factors influencing the stratification process in seeds of Acer saccharum. Canad. J. Bot. 47, (1969). Dr. J. van Staden Botany Department University of ~atal Pietermaritzburg Rep. of South Africa