PROPAGATION AND TISSUE CULTURE
HORTSCIENCE 48(3):347–351. 2013. sp. seed germination. Mesle´ard and Lepart (1991) reported seed germination of A. unedo with alternating 15 and 20 C without any Seed Germination of Arbutus unedo, pretreatment and concluded that seeds do not possess dormancy. In contrast, the recent A. andrachne and Their Natural years, Demirsoy et al. (2010), Ertekin and Kirdar (2010), Hammami et al. (2005), Kose Hybrid A. andrachnoides in Relation (1998), and Tilki (2004) reported physio- logical dormancy in seeds stored for zero to five months, which was successfully overcome to Temperature and Period of Storage and germination occurred only after pretreat- Konstantinos F. Bertsouklis and Maria Papafotiou1 ments with cold stratification or gibberellic Laboratory of Floriculture and Landscape Architecture, Department of acid (GA3). Similarly, Karam and Al-Salem (2001), Kose (1998), Mostafa et al. (2010), Crop Sciences, Agricultural University of Athens, Iera Odos 75, 11855 Olmez et al. (2007), and Tilki and Guner Athens, Greece (2007) reported dormancy in A. andrachne seeds stored for zero to five months, which Additional index words. dormancy, cardinal temperatures, cold stratification, woody orna- was broken by cold stratification or GA3 mental, fruiting cut foliage pretreatments; hot water scarification was Abstract. Arbutus unedo L., A. andrachne L., and their natural hybrid, A. 3andrachnoides also tested rather unsuccessfully (Karam and Link, are the three Arbutus species of the Eastern Mediterranean Macchia. A. unedo is Al-Salem, 2001; Mostafa et al., 2010; Tilki used as an ornamental plant and as cut foliage, whereas the other two species have the and Guner, 2007). Concerning seed germi- potential to be introduced to the floricultural industry. This study was carried out to nation of other Arbutus species, cold strati- clarify whether the seeds of these Arbutus species possess dormancy to determine the fication was also suggested for the pacific temperature range for their germination and the effects of storage period on germi- madrone A. menziesii (Harrington and Kraft, nation. Seeds of the three species, which were stored dry at 25 8C for either three or 2004). All these researchers, which reported 11 months, germinated at very high percentages (82% to 99%) and in a short period of seed dormancy of Arbutus species, placed time (24 to 46 days) when incubated at 15 or 10 8C without any pretreatment proving that seeds to germinate at high temperatures they do not possess dormancy; germination was faster at 15 8C. At 20 8C only seeds of (20 to 30 C), whereas cardinal temperatures A. unedo and A. andrachne stored for three months germinated (29% to 34%), whereas used for germination of Arbutus are not at 25 8C, there was practically no germination. Seeds stored for three months germi- determined. To the possession of dormancy nated (61% to 75%) when incubated at 5 8C, but the germination was delayed and the in A. andrachne seed contrasts, preliminary seedlings did not grow further than the appearance of the radicle unless transferred at work of ours showed germination at 15 C a higher temperature (10 to 25 8C). After 27 months of dry storage at 25 8C, seeds did without any pretreatment of seeds stored for not germinate when incubated at the range of 10 to 25 8C even after pretreatment with either three or 11 months (Bertsouklis and cold stratification. Papafotiou, 2010). There are no reports in the literature for A. 3andrachnoides seed germination. The evergreen woody shrubs Arbutus production, because its growth speed is higher The objective of this study was to clarify unedo (Strawberry tree), A. andrachne (Greek than A. unedo, the production of flowers and whether seeds of A. unedo, A. andrachne, and or Eastern strawberry tree) and their natural fruits is low, and has 90-d vase life (Cervelli A. 3andrachnoides possess dormancy and to hybrid, A. 3andrachnoides (Ericaceae fam- et al., 2012). All three species could be used in determine cardinal temperatures for germi- ily), are the three Arbutus species found in the Mediterranean parks and gardens contribut- nation. In this way wrong practices in the sclerophyllous Macchia zone of the Eastern ing to high biodiversity and sustainability and nurseries that lead to low seed germination Mediterranean (Torres et al., 2002). In certain emphasizing the traditional character of the rates will be avoided (Olmez et al., 2007). The countries they are listed as endangered plants area (Diekelmann and Schuster, 2002). They effectofstorageperiodonseedgermination (Garzuglia, 2006; Sˇatovic´, 2002). A. unedo is could also be used for reforestation in Medi- of these three species was also investigated; growing wild in all the Mediterranean and up terranean regions because of their potential for there was no reference on this in the litera- to the coasts of south England (Villar, 1993) sprouting after fires, because they may have ture, except a preliminary work of ours on and is found as a nursery plant as north as the their aboveground burnt to ground level with- A. andrachne (Bertsouklis and Papafotiou, United Kingdom. It is used as a landscape out major damage to the roots (Konstantinidis 2010). plant and as a complement in the cut flower et al., 2006; Mesle´ard and Lepart, 1991). industry, mainly during Christmas time when Apart from ornamental value, A. unedo has Materials and Methods it bears flowers and fruits (Metaxas et al., edible fruits rich in vitamin C and there is in- 2004). It could also be introduced as a pot creased demand for A. unedo and A. andrachne Ripe fruits were collected late Dec. 2008 plant bearing simultaneously white flowers for medicinal purposes (Issa et al., 2008; from one wild adult tree of each species, A. and orange–red fruits. A. andrachne has po- Mostafa et al., 2010; Pavlovic et al., 2009). unedo, A. andrachne,andA. 3andrachnoides, tential use as a landscape plant because of its Clonal propagation of these three species located on the southeast side of Mount ornamental features, which include attrac- by cuttings is difficult (Al-Salem and Karam, Parnitha (lat. 38 8#19$ N, long. 23 47#44$ E, tive plant architecture, winter fruiting, and 2001; Cervelli et al., 2012; Metaxas et al., 300 to 400 m altitude, Varympompi, Attiki). smooth, cinnamon-red bark, peeling in long 2004). There is a number of reports on in Fruits were collected when they became soft paper strips, revealing the inner gray–green vitro propagation, most of them concerning and had orange–red color (eight months after bark (The European Garden Flora, 1997). A. A. unedo (El-Sayed El-Mahrouk et al., 2010; anthesis), at the stage of full maturity, but be- 3andrachnoides, apart from its potential value Gomes and Canhoto, 2009; Mereti et al., 2002) fore they turned completely red. as a landscape plant, is suitable for cut foliage and a few A. andrachne and A. 3andrachnoides Immediately after collection, the fruits (Bertsouklis and Papafotiou, 2011, 2009; were immersed in water and the seeds manu- Mostafa et al., 2010). ally separated from the fruit flesh. Any floating Received for publication 5 Dec. 2012. Accepted for In the horticultural and forestry practice, seeds (10%) were discarded. Seeds were publication 22 Jan. 2013. the plants are propagated mainly by seed. lefttodryontissuepaperfor24hatroom 1To whom reprint requests should be addressed; However, there is rather confusing informa- temperature and then were placed in unsealed e-mail [email protected]. tion concerning the ecophysiology of Arbutus plastic petri dishes between filter paper. The
HORTSCIENCE VOL. 48(3) MARCH 2013 347 petri dishes were placed in Styrofoam boxes significance of the results was tested by germination. To the contrary, higher than (for darkness) and stored in a constant con- analysis of variance (JMP 6.0; SAS Institute, 20 C temperatures (24, 26, even 30 C) were dition chamber at 25 C and 30% relative Cary, NC). tested by many other researchers (Demirsoy humidity. et al., 2010; Ertekin and Kirdar, 2010; Karam Germination of Arbutus seed was con- Results and Discussion and Al-Salem, 2001; Olmez et al., 2007) that ducted after three, 11, and 27 months of also led to failure of germination and led storage. Storage periods were selected based The present work investigated for the first researchers to conclude seed dormancy. All on nursery practice, where seeds (collected time the temperature range for seed germina- these researchers suggested cold stratifi- in winter) are sown in spring or fall. Thus, tion of Arbutus species and cardinal tempera- cation, usually at 5 C for 40 to 90 d, and/ seeds could be sown in the next spring (three tures were defined for A. unedo, A. andrachne, orGA3 pretreatments for breaking the sup- months after collection), the next fall (11 and their natural hybrid, A. 3andrachnoides. posed dormancy. Like in these works, seeds months after collection), or in the spring two Seeds of the current year (three months stored) of the current year were used (stored for zero years after their collection (27 months after of all three species germinated at high per- to five months); cold stratification could have collection). Before initiating germination centages (84% to 99%) and in a short period possibly acted as an appropriate tempera- treatments, seeds were surface-sterilized with of time (24 to 30 d) when incubated at 15 and ture for germination, like the present work 20% (v/v) commercial bleach (4.6% w/v 10 C. A. unedo and A. andrachne seeds ger- revealed (Figs. 1B–C, 2B–C, and 3B–C). This sodium hypochlorite) for 10 min, rinsed four minated at low percentages at 20 Cand could explain why in Ertekin’s and Kirdar times (3 min each) with sterile distilled water, practically did not germinate at 25 C(1% (2010) work ‘‘cold stratification at 9 Cwas and sown in 9-cm plastic petri dishes with germination), whereas A. 3andrachnoides more efficient than at 4 CforA. unedo seed hormone-free, half-strength Murashige and seeds did not germinate either at 20 or at germination.’’ Concerning the use of GA3 Skoog (MS) medium (Murashige and Skoog, 25 C (Figs. 1A, 2A, and 3A). At 5 Cthe forbreakingsupposeddormancy(Demirsoy 1962) containing 2% (w/v) sucrose and solid- seeds of all species germinated at high per- et al., 2010; Karam and Al-Salem, 2001; ified with 0.8% (w/v) agar. The pH of the centages (90% to 100%), but the germination Kose, 1998; Olmez et al., 2007; Tilki, 2004; medium was adjusted to 5.6 to 5.7 before was delayed (40 d in A. andrachne and Tilki and Guner, 2007), it may have pro- autoclaving at 121 C min for 20 min. 60 d in A. unedo and A. 3andrachnoides) moted germination at supraoptimal high The following treatments were applied: and the seedlings did not grow further than the temperatures, as has been shown in lettuce 1) seeds directly after storage were surface- appearance of the radicle unless transferred at (Dong et al., 2012). Our work, being in sterilized, sown on MS medium, and placed a higher temperature (Figs. 1B–C, 2B–C, and agreement with Mesle´ard’s and Lepart for germination in a constant condition growth 3B–C). The optimum seed germination tem- (1991) who reported A. unedo seed germina- chamber under a 16-h photoperiod of 37.5 perature for all three species was 15 Cat tion with 12 h at 15 Cand12hat20 C, mmol·m–2·s–1 photosynthetic photon flux den- which the maximum germination percentage clearly indicated that A. unedo, A. andrachne, sity of cool fluorescent light at 10, 15, 20, or and germination rate were achieved without and A. 3andrachnoides do not possess any 25 C; 2) seeds directly after storage were any pretreatment indicating that there was no seed dormancy and germination percentages surface-sterilized, sown on MS medium, and dormancy either in current-year seeds (three are very high if the appropriate range of placed at 5 C in the dark, in a refrigerator months stored) or seeds stored for 11 months temperatures (10 to 15 C) are applied. Similar (cold stratification), for 40 d, and then trans- (Figs. 1A, 2A, and 3A). At 20 C, temperature low temperatures (10 to 15 C) were found ferred in the growth chamber (see previously) that is given as optimum for seed germination optimum for seed germination of other at 10, 15, 20, or 25 C; and 3) seeds were of sclerophyllous evergreen species in Baskin drought-tolerant shrubs of the Mediterranean, treated like in (2) with the difference that cold and Baskin (2001), germination percentage too, such as Lithodora zahnii (Papafotiou stratification was applied for 60 d. was low for A. unedo and A. andrachne and and Kalantzis, 2009), Dianthus fruticosus Germination was defined as the appear- zero for A. 3andrachnoides (Figs. 1A, 2A, (Papafotiou and Stragas, 2009), Thymelaea ance of a radicle at least 2 mm long according and 3A), which coincides with results of hirsuta (Shaltout and El-Shourbagy, 1989), to the rules of the International Seed Test- Hammami et al. (2005), Kose (1998), Tilki and Cistus incanus spp. creticus (Thanos ing Association (1999). Germination was re- (2004), and Tilki and Guner (2007) for A. and Georgiou, 1988). Avoiding germination corded every 2 d. One hundred twenty-five unedo and A. andrachne that led them to at high temperatures shows the adaptation of seeds were used per treatment (25 seeds per conclude that these species possess seed dor- these species to the Mediterranean Macchia petri dish, five petri dishes per treatment). The mancy without testing lower temperatures for climate, where high temperatures coincide
Fig. 1. (A–C) Germination time course curves of A. andrachne seeds as affected by storage period, cold stratification, and temperature at germination. Five replicates of 25 seeds were used for each treatment. SDs ranged from 0.00 to 8.00 and have been omitted for clarity.
348 HORTSCIENCE VOL. 48(3) MARCH 2013 Fig. 2. (A–C) Germination time course curves of Arbutus 3andrachnoides seeds as affected by storage period, cold stratification, and temperature at germination. Five replicates of 25 seeds were used for each treatment. SDs ranged from 0.00 to 3.90 and have been omitted for clarity.
Fig. 3. (A–C) Germination time course curves of Arbutus unedo seeds as affected by storage period, cold stratification, and temperature at germination. Five replicates of 25 seeds were used for each treatment. SDs ranged from 0.00 to 5.95 and have been omitted for clarity. or are shortly followed by the drought nature in comparison with its parental spe- et al., 2005; Kose, 1998; Tilki, 2004) and period that could be lethal for young seed- cies. The lower threshold germination tem- A. andrachne (Karam and Al-Salem, 2001; lings. Germination response to temperature of perature for the three Arbutus species was Kose, 1998; Olmez et al., 2007; Tilki and Watsonia species (South African plants) was not defined exactly, but we could put it at Guner 2007), this response of seeds that were found to exhibit clear trends that are depen- 5 C, because at 10 C, germination percent- put first at 5 C and then at 20 to 25 Cwas dent on the season in which rainfall occurs at ages were maximum and only the speed of interpreted wrongly as dormancy breaking the geographic region from which the species germination was reduced compared with 15 C, by cold stratification. At 5 C, the maximum originated; species from the winter rainfall re- whereas at 5 C, germination (as defined by germination percentage was reached after gion germinated optimally at 10 to 20 Cand the International Seed Testing Association, 40 d in A. andrachne and 60 d in A. unedo those from the summer rainfall region at 15 to 1999) reached very high percentages (90% and A. 3andrachnoides (Figs. 1B–C, 2B–C, 25 C (Ascough et al., 2007). to 100%) in all three species (Figs. 1C, 2C, and 3B–C). Seeds transferred from 5 to 25 C So, germination of A. andrachne, A. 3C), but the seedlings did not grow further before the appearance of the radicle (mainly 3andrachnoides, and A. unedo seeds is a than the appearance of the radicle, even after A. unedo and A. 3andrachnoides seeds in- matter of the right temperature and unfavor- 90 d (data not shown). Olmez et al. (2007) also cubated for 40 d at 5 C) did not germinate able environmental conditions for germina- observed germination in A. andrachne during (Figs. 1B, 2B, and 3B). At 5 C, the seeds were tion should not be perceived as seed dormancy the stratification treatment at 5 C and they kept in the dark, whereas in all the other (Thompson and Ooi, 2010). attributed this to the extremely low germina- temperatures, they were put in 16 h light/8 h The upper threshold germination temper- tion percentages that they got then under dark, but these should not have affected ature for A. unedo and A. andrachne was in- greenhouse or open field conditions in March. their ability for germination, because both dicated between 20 and 25 C, whereas their In our work, these seedlings grew in a very A. unedo and A. andrachne have been shown hybrid, A. 3andrachnoides, was between 15 short period of time (1 to 2 d) when transferred to germinate equally well in the light or dark and 20 C (Figs. 1A, 2A, and 3A). The narrower at 10, 15, 20, or 25 C (Figs. 1C, 2C, and 3C). (Bertsouklis et al., 2004; Karam and Al-Salem, temperature range for seed germination of In previous works on A. unedo (Demirsoy et al., 2001; Mesle´ard and Lepart, 1991; Tilki and A. 3andrachnoides may explain its rarity in 2010; Ertekin and Kirdar, 2010; Hammami Guner, 2007).
HORTSCIENCE VOL. 48(3) MARCH 2013 349 In nature these three Arbutus species dis- of A. andrachne, A. 3andrachnoides, and El-Sayed El-Mahrouk, M., Y.H. Dewir, and perse their seeds during the winter and because A. unedo seem to have decreased longevity A.M.K. Omar. 2010. In vitro propagation there is no dormancy, almost all of the seeds at room temperature, because seeds stored for of adult strawberry tree (Arbutus unedo L.) germinate in the spring. So, as Mesle´ard and 27 months did not germinate in any treat- through adventitious shoots and somatic em- Lepart (1991) showed for A. unedo, there are ment. Seed storage temperature has not been bryogenesis. Propag. Ornam. Plants 10:93– 98. no seeds left in the soil to form a seed bank and investigated for these Arbutus species and in Ertekin, M. and E. Kirdar. 2010. Breaking seed after a summer fire, the survival of these some previous works on germination is not dormancy of strawberry tree (Arbutus unedo). species is based on their resprouting ability even reported (Kose, 1998; Tilki, 2004). Intl. J. Agr. Biol. 12:57–60. from the roots. Seed scarification by high Ertekin and Kirdar (2010) and Olmez et al. Garzuglia, M. 2006. Threatened, endangered and temperature did not affect or even prevented (2007) stored seeds of A. andrachne and vulnerable tree species: A comparison between germination of both A. unedo (Mesle´ard and A. unedo, respectively, at 4 to –6 Cuntil FRA 2005 and the IUCN Red List. Rome. Lepart, 1991) and A. andrachne (Karam and the next spring. The former had extremely Working Paper 108/E. Al-Salem, 2001; Mostafa et al., 2010; Olmez low germination (maximum 16%), whereas Gomes, F. and J. Canhoto. 2009. Micropropagation et al., 2007). the latter had 72% germination after 60 d of strawberry tree (Arbutus unedo L.) from The increase of storage period from three cold stratification and 18 to 24 Ctemperature adult plants. In Vitro Cell Dev. B. 45:72–82. Hammami, I., M. Jellali, M. Ksontini, and M.N. to 11 months decreased the range of germina- at germination vs. 90% to 95% in a similar Rejeb. 2005. Propagation of the strawberry tree tion temperatures in all three Arbutus species, treatment at the present study (Figs. 1A through seed (Arbutus unedo). Intl. J. Agr. Biol. restricting it between 10 and 15 C, without and 3A). In preliminary experiments with 7:457–459. affecting the total germination percentages, A. andrachne, we used seeds supplied from Harrington, C.A. and J.M. Kraft. 2004. Cold but reducing the speed of germination; seeds the Forestry of Parnitha that were stored stratification of pacific madrone seeds. Nativ. of the current year (three months’ storage) dry (inside cotton bags) in a refrigerator, Plant. J. 1:66–74. germinated faster than seeds of the previous and those seeds failed to germinate. So, for Immel, D.L. 2006. Pacific madrone Arbutus menziesii Pursh. United States Department year (11 months’ storage), as T50 (time to reach a short period of storage, dry-warm storage 50% germination) and germination curves seems most appropriate for Arbutus seeds of Agriculture-Natural Resources Conserva- show (Figs. 1A, 2A, and 3A). The delay of because dry-cold storage has rather negative tion Service. 7 May 2012.
350 HORTSCIENCE VOL. 48(3) MARCH 2013 Pavlovic, R.D., B. Lakusic, Z. Doslov-Kokorus, Thanos, C.A. and K. Georgiou. 1988. Ecophysiol- Tilki, F. and S. Guner. 2007. Seed germination of and N. Kovacevic. 2009. Arbutin content and ogy of fire-stimulated seed germination in Cistus three provenances of A. andrachne L. in re- antioxidant activity of some Ericaceae species. incanus ssp. Creticus (L) Heywood and C. sponse to different pretreatments, temperature Pharmazie 64:656–659. salvifolious L. Plant Cell Environ. 11:841–849. and light. Prop. Ornam. Plants 7:175–179. Sˇatovic´, Z. 2002. Legal protection, conservation The European Garden Flora. (1997). In: Cullen, Torres, J.A., F. Valle, C. Pinto, A. Garcı´a-Fuentes, and cultivation of medicinal and aromatic J., S.G. Knees, and H.S. Cubey (eds.). Vol. 5. C. Salazar, and E. Cano. 2002. Arbutus unedo p. 485–486. Cambridge University Press, L. communities in southern Iberian Peninsula plants in Croatia. In: Report of Working Group st Cambridge, UK. mountains. Plant Ecol. 160:207–223. on Medicinal and Aromatic Plants, 1 Meeting, Thompson, K. and M.K.J. Ooi. 2010. To germinate Villar,L.1993.Arbutus L., p. 79–90. In: Castroviejo, 12–14 Sept. 2002, Gozd martuljek, Slovenia. or not to germinate: More than just a question S., C. Aedo, C. Go´mez-Campo, M. Laı´nz, P. Shaltout, K.H. and M.N. El-Shourbagy. 1989. of dormancy. Seed Sci. Res. 20:209–211. Montserrat, R. Morales, F. Mun˜oz-Garmendia, Germination requirements and seedling growth Tilki, F. 2004. Improvement in seed germination G. Nieto-Feliner, E. Rico, S. Talavera, and L. of Thymelaea hirsute (L) Endl. Flora 183: of Arbutus unedo L. Pak. J. Biol. Sci. 7:1640– Villar (eds.). Flora iberica IV. CSIC, Madrid, 429–436. 1642. Spain.
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