Dormancy and germination in Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee seeds: Effects of short-time moist chilling and plant growth regulators

Flora 206 (2011) 943–948 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.de/flora Dormancy and germination in Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee seeds: Effects of short-time moist chilling and plant growth regulators Gürcan Güleryüz a,∗ , Serap Kırmızı b , Hülya Arslan a , F. Selcen Sakar a a b Faculty of Science and Arts, Department of Biology, Uludağ University, 16059 Görükle/Bursa, Turkey Gemlik Asım Kocabıyık Graduate Vocational School, Horticulture Programme, Uludağ University, 16600 Gemlik/Bursa, Turkey a r t i c l e i n f o Article history: Received 18 December 2010 Accepted 11 April 2011 Keywords: Stachys germanica ssp. bithynica Moist chilling Germination Dormancy Gibberellic acid Kinetin a b s t r a c t We investigated the germination requirements of the species Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee (Lamiaceae). We studied the effects of scarification, short-time moist chilling (+4 ◦ C) for 15 and 30 days, and various doses of gibberellic acid (GA3 ; 0, 100, 150 and 250 ppm), Kinetin (KIN; 50 ppm) and a combination of 250 ppm GA3 and 50 ppm KIN. The hormone and moist chilling treatments were carried out under both continuous darkness (20 ◦ C) and photoperiodic (20/10 ◦ C; 12/12 h, respectively) conditions. Seeds failed to germinate in response to short-time moist chilling treatments with distilled water under both continuous darkness and photoperiodic conditions. Seeds were found to have dormancy. Treatments with GA3 or a combination of GA3 and KIN were successful at breaking seed dormancy. A maximum of 37% of the seeds germinated after GA3 application in all series. When only KIN was applied at a 50 ppm concentration, germination (12%) was found only with moist chilling for 30 days under continuous darkness. The highest germination rates were found in seeds treated with combination of 250 ppm GA3 and 50 ppm KIN. In the combination treatments, while the moist chilling treatments for 15 days resulted in 68 and 73% germination, respectively, these rates were up to 95% in the moist chilling treatments for 30 days under continuous darkness and photoperiodic conditions. Mean germination time (MGT) in GA3 and KIN combinations was lower than in other treatments. Scarification with 80% sulphuric acid did not promote germination. The characteristics of physiological dormancy of S. germanica ssp. bithynica seeds are consistent with conditions of existence in the in alpine habitat of this species. © 2011 Elsevier GmbH. All rights reserved. Introduction One of the critical early stages in the life cycle of plants controlling their reproductive success and persistence of their populations is seed germination and seedling establishment (Bu et al., 2008; Grubb, 1977; Harper, 1979). Germination response patterns can vary depending on habitat, life history traits, phylogenetic relationships and geographic distribution. The timing of germination plays a critical role in alpine habitats because the growing season is very brief. Seedlings emerging in spring will have greater fitness than those emerging in other seasons (Baskin and Baskin, 1998; Grime et al., 1981; Washitani and Masuda, 1990). From an ecological perspective, dormancy can be defined as the prevention of germination even when suitable conditions are prevalent. The ∗ Corresponding author. E-mail address: gurcan@uludag.edu.tr (G. Güleryüz). 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2011.07.003 dormancy mechanism allows a species to synchronise its germination with favourable environmental conditions, which increases its probability of survival and establishment (Baskin and Baskin, 1998). High-altitude ecosystems are largely controlled by climatic constraints, and many plants are found in environmental conditions that are close to their climatic limits of survival. Some alpine taxa and alpine vegetation types might become extinct (Grabherr et al., 1995), and rare, local species might disappear from some mountains if their refugial habitats are lost (Gottfried et al., 1999). Alpine and subalpine regions can provide a great opportunity for the study of germination timing and characteristics because their environmental conditions change temporally with season and spatially due to their complicated microtopographies (Billings and Bliss, 1959; Körner, 1999). S. germanica “Lamb’s Ears” is a widely distributed species throughout western, central and southern Europe, and its habitat extends from the Mediterranean to North Africa in the south-west 944 G. Güleryüz et al. / Flora 206 (2011) 943–948 and southern central Russia and the Orient in the east. The species is believed to be native in Britain, where it is very late and endangered or vulnerable (Dunn, 1997). The species is rare in Southwest Anatolia. Davis (1982) has recorded ssp. bithynica (Boiss.) Bhattacharjee from Turkey and Greece. In northern Turkey, S. germanica ssp. bithynica can be found in the following locations: Uludağ Mountain (Bithynian Olympos) 2200 m, Bursa; Bolu (Aladağ) 2100–2200 m and Çorum (14 km from Iskilip to Tosya) 1050 m, Kırklareli (Demirköy); and Samsun (Akdağ Mount) 1600–1900 m, Isparta (Davros Mount) 1830 m and Dedegöl Mount 1600 m, Kastamonu (Davis, 1982). Dunn (1997) reported that seeds of this species have not germinated immediately on shedding under natural conditions, and germination is almost exclusively under the parent plant and extends over several months, from late April or May. Viable seeds may remain dormant for many years. In addition, he gave some germination characteristics (for example, germination responses to light and continuous darkness, different temperatures) according to R.S. Band’s personal communications (unpublished data). We aimed to obtain additional information on the germination protocol of Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee using several treatments under laboratory conditions. The treatments included moist short-time chilling and various doses of gibberellic acid alone and in combination with Kinetin. Hormone and short-time chilling treatments were compared to control treatments under both continuous darkness and short-day photoperiod conditions. Germination requirements of this subspecies have not been previously studied, and data from this study can be used in ex situ conservation management. Materials and methods Species description Stachys germanica L. subsp. bithynica (Boiss.) Bhattacharjee is a perennial mesophytic herb that rather often has developed only a basal rosette of leaves. Cauline leaves of flowering shoots are ovate to ovate-lanceolate or more rarely crenate to crenatedentate. Fruits are nutlet and obovoid (Davis, 1982). Seeds are faintly trigonous, slightly winged at base, black when dry. On average they are 2.25 mm long, 1.82 mm broad, 1.27 mm thick, and the mean air dried dry mass of a nutlet is 1.43 mg. Seeds are not readily shed from the tight whorls of calyces, even when completely dry. S. germanica L. subsp. bithynica is distributed on limestone rocks and moist or dry slopes at altitudes over 1600 m. The species prefers dry grasslands, wood margins and open vegetation on calcareous soils (Dunn, 1997). Flowering specimens can be observed between June and August. Seed collection and germination tests Mature seeds of S. germanica subsp. bithynica were collected from the alpine belt of Uludağ Mountain between the altitudes of 2100 and 2200 m in September 2009. Seeds, although mature and dry, were further dried in air immediately upon collection, for 1 week, and then stored dry in a paper bag at room temperature for about 1 month (18–20 ◦ C) until they were used in the germination tests. We studied the effects of scarification, short-time moist chilling (+4 ◦ C) for 15 and 30 days, and various doses of gibberellic acid [GA3 , C19 H22 O6 (Merck 44916373); 0, 100, 150 and 250 ppm], Kinetin [KIN (Sigma, K0753); 50 ppm] and a combination of 250 ppm GA3 and 50 ppm KIN and distilled water as a control (0 ppm hormone). The hormone and short-time moist chilling (+4 ◦ C) for 15 and 30 days treatments were carried out under both continuous darkness (20 ◦ C) and photoperiod (20/10 ◦ C; 12/12 h, respectively) conditions. Photoperiod treatment was conducted in a growth chamber (Nüve GC 400) irradiated by cool white fluorescent 18-W tubes that provided an irradiance (400–700 nm) of ca. 40 ␮mol m−2 s−1 . For scarification of testa, seeds were treated with 80% sulphuric acid for three different durations (5, 10 and 15 min) and then rinsed with tap water. In addition, in early June of the next spring (06/06/2010), verticillasters with seeds on dried branches were collected from sites of snow melt and near snow patches. Same hormone treatments were also applied to these collected seeds. Four replicates of 25 seeds per Petri dish were prepared. Germinated seeds were counted and removed every day for up to 25 days. Seeds were recorded as having germinated when the radicle emerged from the testa. Mean germination times (MGT) were calculated from the germination counts and used to determine the speed of germination. Final germination percentages and mean germination times were determined. Statistical analyses Final percentage values were arcsine square-root transformed for statistical analysis, but non-transformed data are shown in figures and tables. Final germination (arcsine transformed) and MGT were analyzed by two-way ANOVAs for the species and the two germination test environments. All tests were performed at the significance level of ˛; 0.05 with SPSS ver 16.0 for Windows (SPSS Inc. 2007) packet program. Results Seeds of Stachys germanica ssp. bithynica failed to germinate in the control treatment, in which neither GA3 nor moist chilling was applied. Seeds also failed to germinate in moist chilling treatments with distilled water under both, continuous darkness and photoperiodic conditions (Fig. 1). Hormones were added in the form of GA3 , KIN, and a combination of GA3 and KIN under both continuous darkness and photoperiod conditions in order to test whether the hormones can replace the cold treatment. Among the tested GA3 concentrations (100, 150 and 250 ppm), a maximum of 37% of the seeds germinated without a need for chilling (Table 1). When only KIN was applied at a 50 ppm concentration, germination (12%) was found only with moist chilling for 30 days under continuous darkness. Seed germination did not occur in KIN (50 ppm) treatments in other series. However, when GA3 and KIN were applied in combination (250 ppm GA3 and 50 ppm KIN), in 15 days stratified seeds, germination rates increased under both, continuous darkness and photoperiodic conditions (68 and 73%, respectively) (Fig. 1; Table 1). With 15 days stratification, while both GA3 treatment and stratification were significantly effective in darkness, only GA3 treatment was significantly effective in photoperiod. Germination rates were reached of 93% for darkness and 95% for photoperiod after 30 days of stratification in KIN + GA3 treatment. The decrease in the MGT values after 30 days of stratification in KIN+ GA3 treatment support the effectiveness of the combination. GA3 , chilling and their interaction were all of significant importance increasing germination percentage and MGT in 30 days stratified seeds (Table 3). Naturally stratified seeds collected in spring failed to germinate in control treatments under darkness and photoperiod (Fig. 2). Among the GA3 doses applied, the highest was 250 ppm GA3 , which favoured 20% germination rate in darkness and 16% with photoperiod. Application of KIN alone did not result in germination. However, germination percentages influenced by KIN and GA3 in G. Güleryüz et al. / Flora 206 (2011) 943–948 945 Fig. 1. Cumulative germination percentage diagrams for Stachys germanica ssp. bithynica seeds in different treatments under dark (20 ◦ C) and photoperiod (20/10 ◦ C; 12 h dark/12 h light, respectively) conditions. combination reached only 23% in darkness and 43% under photoperiod treatment (Table 2). As an additional test to break seed dormancy, S. germanica ssp. bithynica seeds were treated with 80% H2 SO4 . However, this treatment did not permit germination (Table 1). Discussion Cold stratification of seeds is an effective way to alleviate the dormancy in many species, especially those from temperate regions (Baskin and Baskin, 1998, 2004), and its main function is to pre- Fig. 2. Cumulative germination percentage diagrams for Stachys germanica ssp. bithynica seeds after natural stratification under dark (20 ◦ C) and photoperiod (20/10 ◦ C; 12 h dark/12 h light, respectively) conditions. 946 G. Güleryüz et al. / Flora 206 (2011) 943–948 Table 1 Final germination (%) and mean germination times (MGT; days) of Stachys germanica seeds under different treatments [values are means followed by standard error (n = 4)]. Treatment series Germination % ± SE Darkness (20 ◦ C) Photoperiod (20/10 ◦ C) Darkness (20 ◦ C) 15 days stratification Photoperiod (20/10 ◦ C) Darkness (20 ◦ C) 30 days stratification Photoperiod (20/10 ◦ C) vent seed germination under inappropriate seasonal conditions (Baskin and Baskin, 1998; Finkelstein et al., 2008; Vleeshouwers et al., 1995). Cytokinins are reported to be involved in mobilization of storage reserves for utilization during germination in cereals (Fincher, 1989; Hocart and Letham, 1990). Moreover, it has been proposed that during germination they may play a role in offsetting the effect of germination inhibitors (Khan, 1975). The results of this study show that the best dormancy breaking treatment is a combination of cytokinin and gibberellin. There may be a high content of ABA in S. germanica seeds that can only be overcome by a combination of the two plant growth regulators. Phartyal et al. (2003) reported also that a combination of GA3 and cytokinin is Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 + 50 ppm KIN Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 + 50 ppm KIN Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 + 50 ppm KIN Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 + 50 ppm KIN Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 + 50 ppm KIN Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 + 50 ppm KIN 0.0 24.5 30.0 37.0 0.0 68.0 0.0 9.0 22.0 24.0 0.0 73.0 0.0 9.0 29.0 28.0 0.0 53.0 0.0 16.0 21.0 29.0 0.0 74.0 0.0 15.0 13.0 21.0 12.0 93.0 0.0 12.0 12.2 17.0 0.0 95.0 MGT ± SE ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.0 2.1 3.9 5.2 0.0 2.3 0.0 1.9 2.6 2.5 0.0 4.5 0.0 1.9 3.4 2.3 0.0 5.3 0.0 2.9 1.9 3.8 0.0 1.2 0.0 1.0 3.0 1.0 1.6 2.3 0.0 2.3 1.6 3.0 0.0 1.0 0.0 ± 10.4 ± 7.4 ± 10.7 ± 0.0 ± 7.2 ± 0.0 ± 16.6 ± 12.4 ± 13.7 ± 0.0 ± 10.3 ± 0.0 ± 9.3 ± 12.1 ± 12.5 ± 0.0 ± 7.8 ± 0.0 ± 9.0 ± 8.0 ± 7.4 ± 0.0 ± 7.4 ± – 7.1 ± 7.8 ± 9.4 ± 11.5 ± 5.9 ± – 10.5 ± 8.5 ± 9.1 ± – 4.5 ± 0.0 1.0 1.3 1.2 0.0 0.2 0.0 1.3 1.0 0.8 0.0 0.7 0.0 1.4 1.0 1.2 0.0 0.2 0.0 1.0 0.4 0.7 0.0 0.5 1.3 0.4 0.8 2.3 1.7 2.4 1.6 1.8 1.0 the best dormancy breaking treatment in Acer caesium seeds. In addition, Khan (1971) suggested that gibberellin-induced germination cannot occur in the presence of abscisic acid (ABA) unless a sufficient amount of cytokinin is present. Furthermore, cytokinins promote cell division by accelerating rates of protein synthesis and decreasing the duration of the cellular interphase (Davies, 1995). In seeds that undergo dormancy cytokinins are most abundant during early tissue differentiation of the embryo, a period of rapid cell division and growth, and later in embryogeny, when they may influence seed germination through complementary signal transduction pathways that are sensitive to light (Thomas et al., 1997). Cytokinins are present in relatively high concentrations, e.g., in Table 2 Final germination (%) and mean germination times (MGT; days) of Stachys germanica seeds after stratification under natural conditions (seeds collected in spring from overwintered plants) [values are means followed by standard error (n = 4)]. Treatment series Germination % ± SE Darkness (20 ◦ C) Natural stratification Photoperiod (20/10 ◦ C) Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 +50 ppm KIN Control 100 ppm GA3 150 ppm GA3 250 ppm GA3 50 ppm KIN 250 ppm GA3 +50 ppm KIN 0.0 16.0 8.0 20.0 0.0 23.0 0.0 0.0 10.0 16.0 0.0 43.0 ± ± ± ± ± ± ± ± ± ± ± ± MGT ± SE 0.0 1.6 1.6 2.8 0.0 4.1 0.0 0.0 2.6 1.6 0.0 5.5 – 10.3 ± 10.9 ± 12.5 ± – 13.9 ± – – 15.6 ± 17.9 ± – 15.1 ± 1.4 0.5 1.1 0.6 1.9 1.4 1.0 G. Güleryüz et al. / Flora 206 (2011) 943–948 947 Table 3 Two-way ANOVA results for arcsine transformed germination percentage of Stachys germanica and mean germination time (MGT) in darkness and under photoperiod conditions [means of germination percentage and MGT were analyzed for GA3 × chilling interaction in level of ˛; 0.05]. Factor Germination percentage df Stachys germanica Photoperiod Chilling GA3 Chilling × GA3 Intercept Darkness Chilling GA3 Chilling × GA3 Intercept Stachys germanica Photoperiod Chilling GA3 Chilling × GA3 Intercept Darkness Chilling GA3 Chilling × GA3 Intercept * F MGT P df P* F 15 days stratification 2 3 3 1 0.94 134.1 1.891 925.4 0.404 0.000 0.159 0.000 2 3 3 1 43.4 8.6 3.4 35.4 0.000 0.001 0.035 0.000 2 3 3 1 11.8 52.7 1.8 884.1 0.000 0.000 0.182 0.000 2 3 3 1 1.8 6.3 2.2 389.2 0.192 0.003 0.110 0.000 30 days stratification 1 4 3 1 78.911 362.853 8.106 1772.272 0.000 0.000 0.001 0.000 1 4 3 1 363.295 30.869 9.527 1167.412 0.000 0.000 0.000 0.000 1 4 3 1 4.779 144.510 23.799 2543.203 0.038 0.000 0.000 0.000 1 4 3 1 50.903 11.763 5.831 650.720 0.000 0.000 0.003 0.000 P < 0.05 indicate the significant difference among treatment series. the recalcitrant seeds of Citrus spp. (Elotmani et al., 1995), Avicennia marina (Farrant et al., 1993), and several other viviparous species of mangroves (Farnsworth, 1997), indicating that they may be involved in maintaining continuous cellular activity. In addition, cytokinins may also help halophytes to overcome salt-induced inhibition of germination (Gul and Weber, 1998). Therefore, maintenance and release of seed dormancy is controlled by a balance between inhibitor and promoter hormones (Ross, 1984; Taiz and Zeiger, 2002). But responses of plants to cytokinins and gibberellins may differ between ecotypes, species and different higher-ranked systematic units (Kabar, 1998). Germination behaviour may even vary within a single species from one population to another, from year to year and among individuals (Urbanska and Schütz, 1986). However, it seems that there is a general synergistic relationship between cytokinins and gibberellins in the regulation of dormancy and germination. Dunn (1997) reported that nutlets of S. germanica have been found unshed for up to 12 months in nature and our results suggest that S. germanica seeds may persist also for long periods of time in the soil. A mechanism that delays germination until the next spring, when conditions become optimal for reproductive success, could be advantageous in harsh habitats (Dar et al., 2009). After seed dispersal, some species, like S. germanica, will experience moist chilling during the colder winter months and subsequently germinate in the next season. Naturally stratified seeds collected in spring failed to germinate in control treatments under darkness and photoperiod (Fig. 2). These findings suggest that naturally stratified seeds could have developed secondary dormancy in their habitat. Baskin and Baskin (1989) proposed that seeds not germinating in autumn are brought into secondary dormancy by low winter temperatures, being unable to germinate in following spring. Furthermore, if seeds mature in early autumn but do not germinate until the second spring, they may have physiological dormancy. In the first spring after dispersal seeds may not germinate because they are dispersed too late in autumn to be exposed to a long enough period of warm stratification needed to complete the first phase of dormancy loss (Baskin and Baskin, 2004). In addition, most seeds of perennials produced in autumn also require a cold stratification treatment for dormancy loss. Under such conditions non-dormant seeds germinate in spring and/or summer (Baskin and Baskin, 1998). However, the combination of kinetin, ethephon, and GA4+7 could remove secondary dormancy in Chenopodium bonus-henricus L. (Khan and Karssen, 1980). Exogenous gibberellin (GA3 , GA4+7 ) or Benzyladenine (BA) are able to release secondary dormancy in Amaranthus caudatus seeds and this release from dormancy involves both ethy˛ ński et al., 2006). In Prunus lene biosynthesis and action (Kepczy serotina equal volumes of cytokinin and GA3 resulted with only 21% germination after 120 days stratification (Phartyal et al., 2009). Pinfield et al. (1972) found that 104 ppm GA3 (3 × 10−4 M) was effective breaking the dormancy in Stachys alpina seeds. Afterripening up to 20 weeks under dry storage did not permitted germination. No germination occurred either with chilling less than 4 weeks; 12–20 weeks chilling gave 20–30% germination in S. alpina. In addition, we found that up to 10 months of chilling in nature also did not permit germination in S. germanica ssp. bithynica. Kinzel, 1926, found that for maximum germination of S. sylvatica chilling for two winters is required. After a single winter germination rate was only 28%. Karlsson and Milberg (2008) reported on seed dormancy and germination of four annual Lamium species (Lamiaceae), where cold stratification did not reduce dormancy, neither warm stratification or dry storage. Comparing the results from Scandinavian Lamium with germination behaviour of populations from Kentucky (Baskin and Baskin, 1984a,b) they concluded that the higher dormancy strength of Swedish Lamium populations could result from adaptation to the particular environment (both summers and winters are colder in Sweden). Since, naturally stratified seeds do not germinate in the following spring, but their germination is promoted by GA3 and by a combination of GA3 and KIN, we asume that the type of dormancy in S. germanica ssp. bithynica seeds is physiological as explained by Baskin and Baskin (2004). Gibberellic acid sensitivity is known to increase in seeds during the breaking of non-deep physiological dormancy (Finch-Savage and Leubner-Metzger, 2006). The results of the sulphuric acid scarification support this suggestion. The dormant status of the seeds is probably not related to the seed coat. Similar results were found by Brändel (2006) for Stachys palustris. 948 G. Güleryüz et al. / Flora 206 (2011) 943–948 In conclusion, an effective strategy for breaking seed dormancy and enhancing seed germination of S. germanica ssp. bithynica is low-temperature stratification and application of a combination of GA3 and KIN. To achieve this in nature, longer periods of seeds remaining on the mother plant and laying in soil are needed. This physiological dormancy of S. germanica ssp. bithynica seeds is consistent with conditions at its alpine habitat. These traits give the species a critical capacity to control the timing of reproduction and the establishment of a new offspring generation. Acknowledgement This study was part of a research project funded by the Turkish Scientific and Research Council (no. 107T494). References Baskin, J.M., Baskin, C.C., 1984a. Effect of temperature during burial on dormant and non-dormant seeds of Lamium amplexicaule L. and ecological implications. Weed Res. 24, 333–339. Baskin, J.M., Baskin, C.C., 1984b. 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