Physiological and Physical Seed Dormancy of Some Hypericum Species Growing in Turkey

Cüneyt Çirak, Ali Kemal Ayan, Kudret Kevseroglu Phys i o log i cal and phys i cal seed dor mancy of some hypericum species grow ing in Turkey Andrzej Anio³

PLANT BREEDING AND SEED SCIENCE Volume 53 2006

Cüneyt Çýrak1*, Ali Kemal Ayan2 and Kudret Kevseroglu1

1Univer sity of Ondokuz Mayis, Faculty of Agri cul ture , De part ment of Agronom y, Samsun/Tur key 2Univer sity of Ondokuz Mayýs, the High School of Profes sion of Bafra, Samsun/Tur key *Cor re spond ing au thor: Ad dress: Uni ver sity of Ondokuz Mayis, Fac ulty of Ag ri cul ture, Depart ment of Agronom y, 55139-Samsun, Turkey; Tel: +90-362-4576020; Fax: +90-362-4576034, e-mail: [email protected]

PHYSIOLOGICAL AND PHYSICAL SEED DORMANCY OF SOME HYPERICUM SPECIES GROWING IN TURKEY

ABSTRACT

To inves ti gate the nature of dorm ancy in Hypericum androsaemum, H. scabrum, H. lydum and H. tetrapterum seeds which did not show ger mina tion under nor mal labo ra tory condi tions. The 15 day long germ ina tion exper i - ment under the constant light preceded by 30 min soaking in wa ter (tap water , 40, 50 and 60°C hot water ), GA3 (50, 100 and 150 ppm) and H2SO4 (1, 2 and 3%) was perform ed. The ger mina tion response to the pre-soaking treatments was variable and discussed as a possi ble result of double dor mancy involv ing par tially dor mant em bryo and a chem i cal in hib i tor for H. androsaemum and H. scabrum. Seed ger mi na tion was re stricted only by hard seed coat in H. lydum, and H. tetrapterum indicating the presence of physical dormancy.

Key words: dou ble dor mancy, ger mi na tion, Hypericum androsaemum, H. lydum, H. scabrum, H. tetrapterum,, pre-soak ing treat ments

INTRODUCTION Hypericum specie s are of great inter est for mankind throughout many centu ries and have been used as tradi tional medic i nal plants due to their wound-healing (Yazaki and Okuda, 1990), bactericide (Ishiguro et al., 1998), anti-in flam ma tory (Dias et al., 1998), diureti c and seda tive proper ties (Holz and Ostrowiski, 1987) for last two hundred years. The Hypericum ge nus of Guttiferae is repre sent ed in Tur - key by 89 specie s of which 43 are endem ic; the most abundant and well known is H. perforatum (Da vis, 1988). Hypericum androsaemum L. grows in West Europe (Flora Europaea, 1968) and in damp or shady places of Turkey (Davis, 1988). Infu sion of H. androsaemum leaves has been used in tradi tional medi cine for its diureti c and anti-hepatotoxic ac - tivi ties (Costa, 1994). Pheno lic com pounds, such as flavonoids, pheno lic acids and xanthones, are known to be present in this specie s (Schmidt et al., 2000) and have Communicated by Andrzej Anio³ 4 Cüneyt Çirak, Ali Kemal Ayan, Kudret Kevseroglu been re ported to be re spon si ble for its phar ma co log i cal prop er ties (Valentao et al., 2002). Hypericum scabrum L. is an herba ceous peren nial plant which is distrib uted in dry rocky slopes and open woodland of Turkey (Davis, 1988) and has been used by Turk ish folk for its antispasmodic, seda tive and anti-inflam matory proper ties (Tanker, 1971). In Turkey, a skin rem edy is pre pared from its aerial parts and it is used as an oint ment against pso ri a sis in folk med i cine. An ti bac te rial ac tiv i ties of Hypericum scabrumhave been re ported by severa l in vitro studies (Azýrak and Erdoðrul, 2003). Although there are sev eral in vesti gations on the phytochemistry and bi o log i cal ac tiv ity of H. androsaemum and H. scabrum, lit tle ef fort has been ded i cated to the study of other as pects of these species and there is, so far, no report re lated to H. lydum Boiss and H. tetrapterum Fries growing wild in rocky slopes and stream sides in Tur key. These spe cies of Hypericum have not been dom esti cat ed yet in Tur key and due to uncon trolle d harvest ing as well as seasonal, clima tic, and geo graphic restric tions, the avail ability of wild plants from their native pop u lations is strongly limited. Ger mina tion is a crit i cal stage in the life cy cle of weeds and crop plants, and of ten con trols pop u la tion dy nam ics, with ma jor prac ti cal im pli ca tions (Keller and Kollmann, 1999). Gen er ally ger mi na tion ca pac ity of Hypericum spe cies is very low due to seed dorm ancy (Macchia et al., 1983) which is caused by a chem ical in- hibi tor exudate from the capsule in H. perforatum (Campbell, 1985) and absenc e of light has a nega tive effec t on germ ina tion in Hypericum spe cies (Çýrak et al., 2004a). Plant growth regu la tors such as GA3 (gibberellic acid) and IAA (indoleacetic acid) (Iglesias and Babiano, 1997); chem i cal sub stances such as H2SO4 (sulphuric acid) (Baes et al., 2002) and hot water treatm ents (Hermansen et al., 1999) have been recom mended in breaking dorm ancy and to enhance germ ina - tion. The objec tives of this study were to de term ine the effec t of exog e nously ap- plied GA3, H2SO4, hot and tap water on germ ina tion and to find an effec tive method for breaking seed dorm ancy of H. androsaemum, H.scabrum, H. lydum and H. tetrapterum.

MATERIALS AND METHODS The 10 month-old seeds of H. androsaemum, H.scabrum, H. lydum and H. tetrapterum wild plants growing in differ ent places of Black Sea geograph ica l re- gion of Turkey were used as a mate rial . In pre lim inary test, the seeds did not show ger mi na tion un der nor mal lab o ra tory con di tions (at 20 °C dur ing 21 days (Anon y- mous, 2005)), so they were soaked in 50, 100 and 150 ppm GA3; 1, 2 and 3% H2SO4 solu tions, tap water, 40, 50 and 60°C hot wa ter for 30 minutes and ger minate d on Petri dishes under contin u ous illu m ina tion (1200 lux white light) in growth cham - bers at 20°C. Tap wa ter ap pli cation was treated as con trol and the ger mina tion ca - pac i ties were cal cu lated as “per cent age”. Ger mi na tion of the seeds ex posed to dif fer ent pre-treat ments was eval u ated 15 days later. Three rep li cation s (100 seeds each) of each pre-treat ment were ob jected to ANOVA and dif fer ences among treat - ments were tested Duncan Mul ti ple Range Test (Level of sig nif i cance P<0.01). Physi o logi cal and phys ical seed dormancy of some hypericum species growing in Turkey 5

RESULTS The pre-soak ing treatm ents tested in the present study had a sig nifi cant (P<0.01) effe ct on germ ina ti on depend i ng on spec ies (Figure 1). )

% 35,00 ( a s

e 30,00 i t

i b

c 25,00

a a c b

p d 20,00 a c d c n 15,00 ee aa a o

i g f b

t b 10,00 c a h

n i i c dc 5,00 e m f f f f f eee e dddddd d r

e 0,00

G H. H. scabrum H. lydum H. tetrapterum androsaemum

40°C hot water 50°C ot hwater 60°C ot hwater 50 ppm GA3 100 ppm GA3 150 ppm GA3 0.5 % H2SO4 1.0 % H2SO4 1.5 % H2SO4 Control

Fig. 1. The ger mi na tion ca pac i ties of H. androsaemum, H. scabrum, H. lydum and H. tetrapterum seeds exposed to dif ferent pre-soaking treatm ents. (Values with differ ent letter s within colum ns for each species dif fer sig nif i cantly at the level of P<0.01).

In H. androsaemum, GA3 treatm ents were found to be most effec tive to im prove seed ger mi na tion while ger mi na tion was de te ri o rated by hot wa ter and H2SO4 when com pared to control. 150 and 100 ppm GA3 treatm ents gave the highest germ ina - tion for this specie s with 29 and 24% respec tively. These num bers were followe d by con trol with 20%. In contras t to the other Hypericum specie s evalu ate d in this study, germ ina tion re sponse of H. scabrum seeds to hot wa ter treatme nts was posi tive and sig nif icant. The high est ger mi na tion for H. scabrum was obtaine d from 40 °C hot water treat- ment (21%) and this was followe d by 150 ppm GA3 and wa ter-soaked control with 19 and 14% respec tively. Also, when com pared to control, H2SO4 treat ments were in ef fec tive to en hance ger mi na tion. H2SO4 treatm ents were the only effec tive ones in prom oting germ ina tion for H. lydum and H. tetrapterum seeds. 2 and 3% H2SO4 treatm ents resulte d in 11% germi na tion in H. lydum and 11 and 8.37% germ ina tion in H. tetrapterum re spec - tively. The other treatme nts were all gener ally in effec tive in improv ing ger mi na tion for both spe cies.

DISCUSSION Light has been rec ognize d since the mid-nine-tenth century as a germ ina - tion-con trol ling fac tor. In gen eral, Hypericum spe cies need light to ger minate at the 6 Cüneyt Çirak, Ali Kemal Ayan, Kudret Kevseroglu highest level (Ash et al., 1998; Çýrak et al., 2004a). There fore, this study was con - ducted out un der con tin u ous il lu mi na tion to sup ply this light re quire ment. Seeds of many wild plants have hard seed coats which re strict water absorp tion by the em bryo. Perm eabil ity may be im proved by scari fy ing the seed coat by mechan i cal means (e.g. clip ping, abra sion or im mersion in hot water) or chem ical ly with strong oxi da tive agents (e.g. sulphuric acid or sodium hypochlorite) (Abdallah et al., 1989). In the present study, H2SO4 treatm ents were found to be the most effec tive to in duce seed ger mi na tion for H. lydum and H. tetrapterum. The results in - di cate the pres ence of phys i cal dor mancy in both species re lated to hard seed coat and over came by only acid scarification. However , it should be noted that the acid pre-treatm ent had a strong dam age on seed coat in H. androsaemum. The high germi na tion ob tained with H2SO4 treatm ents agree with those obtaine d for other spe - cies like: Prosopis ferox (Baes et al., 2002), Aca cia origena, Aca cia pilispina and Pterolobium stellatum (Teketay, 1998). Studies of genet ics and physi ol ogy have shown the im portant roles of the plant horm ones like abscisic acid and gibberellin in the regu la tion of dorm ancy and germi na tion (Koornneef et al., 2002). Gibber el lins com prise the class of horm ones most direc tly im plicat ed in the control and prom otion of seed germ ina tion. These com pounds oc cur at rel a tively high con cen tra tions in de vel op ing seeds but usu ally drop to a lower level in mature dor mant seeds (Schwachtje and Baldwin, 2004). En- dog e nously ap plied gib ber el lins can re lieve cer tain types of dor mancy, in clud ing phys io log i cal dorm ancy, photodormancy and thermodormancy acting as a substi - tute for low tem pera tures , long days, or red light (Seiller, 1998). In this study, GA3 in creased ger mi na tion sig nif i cantly de pend ing on doses in H. androsaemum and H. scabrum in di cat ing the pres ence of phys i o log i cal dor mancy re lated to par tially dor- mant em bryo for both spe cies. The germ ina tion enhanc ing effec t of GA3 was re - ported from the studies car ried on other specie s such as Sesamum indicum (Kyauk et al., 1995), Zea mays and Glycine max (Wang et al., 1996). Previ ous re searches with hot wa ter treatm ents to induce germ ina tion were incon - sis tent and variable . Some of them have reported hot water treatm ents to enhance germ ina tion of hard coated seeds by ele vat ing wa ter and O2 per me abil ity of testa. Hermansen et al. (1999) re ported that germ ina tion was enhance d by presoking in 44, 49 and 54°C hot wa ter for 5-40 minutes in carrot seeds and dipping seeds in boiling wa ter for 1–60 s. was very effec tive in prom oting germ ina tion for Dodonea viscosa (Davis et al., 2004). On the contrar y, no signif i cant seed germ ina tion re - sponse to hot wa ter treatm ents has been re ported for some Aca cia spe cies (Masamba, 1994) and Myrtus communis (Khosh-Khui and Bassiri 1976). Sim i- larly, in the present study the ef fects of hot water treatm ents on seed germ ina tion of the Hypericum spe cies tested were change able and H. scabrum was the only spe - cies show ing pos i tive ger mi na tion re sponse to hot wa ter treat ments in di cat ing the pres ence of phys i cal dor mancy. Chem i cals that ac cu mu late in fruit and seed cov er ing tis sues dur ing de vel op ment and re main with the seed after harvest can be shown to act germ ina tion inhib i tors. Some of the sub stances as so ci ated with in hi bi tion are var i ous phe nols, coumarin and abscisic acid and can be leached out of the seeds by soaking in wa ter. For exam - ple, in a pre vious study on Hypericum perforatum we ob served that the chem i cal Physi o logi cal and phys ical seed dormancy of some hypericum species growing in Turkey 7 inhib i tor in exudate from capsule could be elim inate d effectively by a sim ple soak ing in tap wa ter (Çýrak et al., 2004b). In this study, sim ilarl y, the colour of the water in which both seeds were soaked turned yel lowish and water- soaked control re sulted in higher germ ina tion in H. androsaemum and H. scabrum. The re sults suggest ed that seeds of both spe cies include some chem ical inhib i tors can be leached by soaking in tap water.

CONCLUSION Conse quently, the results from the present study showed that the effec ts of differ - ent pre-soaking treatm ents on seed germ ina tion of tested Hypericum spe cies were sig nif i cant de pend ing on spe cies. GA3 and wa ter-soaked control were the most effec tive treat ments to im prove ger mi na tion of H. androsaemum, suggest ing that the seeds ex hibit both phys i o log i cal dor mancy in volv ing a par tially dor mant em bryo and chem i cal dor mancy as so ci ated with a chem i cal in hib i tor. For H. scabrum, all treat ments were gen er ally ef fec tive in pro mot ing ger mi na tion. But hot water and GA3 treat ments in creased ger mina tion mainly. It can be con cluded that H. scabrum seeds, like those of H. androsaemum, exhibit double dorm ancy: the phys ical dormancy involv ing a hard seed coat and the phys io log i cal dorm ancy re lated to a par- tially dorma nt embry o. H2SO4 treatm ents were the only effec tive ones in prom oting ger mi na tion for H. lydum and H. tetrapterum seeds. In contras t to H. androsaemum and H. scabrum, seed ger mina tion was re stricted only by hard seed coat in H. lydum and H. tetrapterum in di cat ing the pres ence of phys i cal dor mancy.

REFERENCES

Abdallah M. M. F., Jones R. A., El-Beltagy A. S. (1989). An ef ficient method to overcome seed dormancy in Scotch broom (Cytisus scoparius). En vi ron men tal and Ex per i men tal Bot any. 29, 499-501. Anon ymous, (2005). Inter na tional Rules for Seed Testing (ISTA), Chap ter 76: Germi na tion test envi ron - ments and dormancy-break ing treatments for species in other fami lies. Available on the internet: http://www.ipgri.cgiar.org/publications/HTMLPublications/52/ch61.htm. Ash J. E., Groves R. H., Wil lis A. J. (1998). Seed Ecology of Hypericum gramineum, an Aus tralian forb. Aus tra lian Jour nal of Bot any. 45,1009-1022. Azýrak S., Erdoðrul, Ö.T. (2003). An ti bac te rial ac tiv i ties of Hypericum scabrum. ISOPS-7, pro- ceedings and abstracts , June 23-26, An kara, Turkey. Baes P. O., Marta L. V., Silvia S. (2002). Ger mi na tion in Prosopis ferox seeds, effects of mechan i cal, chem- i cal and bi o log i cal scari fi ca tors. Journal of Arid Envi ron ments. 1, 185-189. Campbell M. H. (1985). Germi na tion, emergence and seedling growth of Hypericum perforatum. Weed-Research. 25, 259-266. Costa A. F. (1994). Fundação Calouste Gulbenkian, Lisboa. Farmacognosia. 2,1022–1023. Çýrak C., Ayan A., Kevseroðlu K., Çalýþkan Ö. (2004a). Germi na tion rate of St. John’s wort (Hypericum perforatum l.) seeds ex posed to dif fer ent light in ten si ties and il lu mi na tion pe ri ods. Jour nal of Bi o log i cal Sci ences. 4, 279-282. Çýrak C., Ayan A., Kevseroðlu K. (2004b). The effects of light and some presoaking treatments on ger mina tion rate of St. John’s worth (Hypericum perforatum L.) seeds. Paki stan Journal of Bio log i - cal Sciences. 7, 182-186. Da vis B. H., Baskin C. C., Gleason S. M., Cordell, S. (2004). Physi cal dormancy in seeds of Dodonaea viscosa (Sapindales, Sapindaceae) from Ha waii. Seed Science Re search. 14,81-90. Da vis P. H. (1988). Flora of Turkey and the East Aegean Islands. Edin burgh, Ed inburgh Univer sity Press, pp 230-262. Dias A.C.P., Fran cisco A., Barberan T., Ferreria F., Ferreres F. (1998). Unusual flavanoids produced by cal- lus of Hypericum perforatum. Phytochemistry. 48,1165-1168. Flora Europaea (1968). London, Cam bridge Univer sity Press. 8 Cüneyt Çirak, Ali Kemal Ayan, Kudret Kevseroglu

Hermansen A., Brodal G., Balvoll G. (1999). Hot wa ter treat ments of car rot seeds, effects on seed-borne fungi, germi na tion, emergence and yield. Seed Science and Technol ogy. 27, 599-613. Holz J., Ostrowiski E. (1987). St. John’s worth HPLC analy sis of main compo nents and their variabil ity in the popu la tions, Deutschland Apothekertz. 127, 1227-1230. Iglesias R. G., Babiano M.J. (1997). En dog e nous abscisic acid dur ing the ger mi na tion of chick pea seed. Phys i ol ogy Plant. 100, 500-504. Ishiguro K., Nagareya N., Fukomoto H.A. (1998). Phloroglucinol deriv a tive from cell suspen sion cultures of Hypericum perforatum. Phytochemistry. 47, 347-369. Keller M., Kollmann J. (1999). Effects of seed prove nance on germi na tion of herbs for agri cul tural com pen- sa tion sites. Ag ri cul ture, Eco sys tems and En vi ron ment. 72, 87-99. Khosh-Khui M., Bassiri, A. (1976). Physi cal dormancy in myr tle seed, Scientia Horticulturae. 5, 363-366. Koornneef, M., Bentsink L., Hilhorst, H. (2002). Seed dormancy and germi na tion. Cur rent Opinion in Plant Bi ol ogy. 5, 33-36. Kyauk H., Hop per N.W., Brigham R.D. (1995). Ef fects of temper a ture and pre-soaking on germi na tion, root length and shoot length of sesame (Sesamum indicum L.). En vi ron men tal and Ex per i men tal Bot- any. 35, 345-351. Macchia N., Benvenuti A., Angelini, L. (1983). Ger mi na tion char ac ter is tics of some seeds of me dic i nal plants, Rastitel’nye-Resursy, 21, 461-463. Masamba C. (1994). Presowing seed treatments on four Af rican Aca cia spe cies, ap pro pri ate tech nol ogy for use in forestry for rural devel op ment. Forest Ecology and Manage ment. 64, 105-109. Schmidt W., El-Mawla A., Wolfender J.L., Hostettmann K., Beerhues, L. (2000). Xanthones in cell cultures of Hypericum androsaemum. Planta Medica. 66, 380–381. Schwachtje, J., Baldwin I.T. (2004). Smoke expo sure alters endog enous gibberellin and abscisic acid pools and gibberellin sensi tiv ity while elicit ing germi na tion in the post-fire annual, Nicotiana attenuata. Seed Science Re search. 14,51-60. Seiller, G.J. (1998). Seed matu ri ty, storage time and temper a t ure, and treatm ent effects on germi na t ion of two wild sunflow ers. Agron omy Journal. 90, 221-226. Tanker N. (1971). Stud ies on Hypericum scabrum L. Jour nal of Faculty of Pharmacy of An kara Univer sity. 1,10-15. Teketay D. (1998). Germi na ti on of Acacia origena, A. pilispina and Pterolobium stellatumin response to dif fer ent pre-sow ing seed treat ments, tem per a ture and light. Jour nal of Arid En vi ron ments. 38, 551-560. Valentao P., Fernandes E., Carvalho F., Andrade P. B., Seabra R.M., Bastos M.L. (2002). An tiox i dant ac- tiv ity of Hypericum androsaemum in fu sion, scav eng ing ac tiv ity against superoxide rad i cal, hydroxyl rad i cal and hypochlorous acid. Bi o log i cal and Phar ma ceu ti cal Bul le tin. 25,1320–1323. Wang Q., Feng Z., Smith D. (1996). Ap pli ca tion of GA3 and kinetin to improve corn and soybean seedling emer gence at low tem per a ture. En vi ron men tal and Ex per i men tal Bot any. 36, 377-383. Yazaki K., Okuda T. (1990). Procyanins in cal lus and mul tiple shoots of Hypericum erectum, Planta Medica. 56, 490-491.