Comparative Germination Analysis of Isolated Seeds and Indehiscent Fruits

The dispersal units of the related invasive, noxious, and weedy Brassicaceae species L. draba and L. appelianum are indehiscent fruits (Figure 1). The aim of the present study was to comparatively investigate the roles of the fruit coat (pericarp) in the dormancy mechanisms and germination biology of these species. To achieve this, the germination of isolated seeds (i.e., seeds manually removed from fruits by opening the pericarp; Figure 1A and B) was compared with germination of indehiscent fruits (Figure 1C and D). Visible emergence of the radicle through all covering layers (endosperm, testa, pericarp) was used as the criterion to score the completion of germination of seed or fruit populations. For isolated seeds, germination was accompanied by testa and endosperm rupture (Figure 1G and H) as described for the seeds of L. sativum (Müller et al. Reference Müller, Tintelnot and Leubner-Metzger2006). For the indehiscent fruits of L. draba, the tight encasement of the seeds by the pericarp did not allow the seeds to germinate within the fruits. Instead the fruit itself eventually completed germination with visible radicle emergence through all the covering layers of the seed (endosperm, testa) and fruit (pericarp) and visible pericarp rupture (Figure 1I). The pericarp rupture and visible radicle protrusion next to the tip of the L. draba fruit is therefore mechanically very similar to the fruit germination of L. didymum (Sperber et al. Reference Sperber, Steinbrecher, Graeber, Scherer, Clausing, Wiegand, Hourston, Kurre, Leubner-Metzger and Mummenhoff2017). For L. appelianum fruits, the seeds germinated within the fruits and subsequent embryo expansion eventually led to radicle emergence through the pericarp (Figure 1J).

The objectives of the first set of experiments were to identify the seed dormancy class (Baskin and Baskin Reference Baskin and Baskin2004; Finch-Savage and Leubner-Metzger Reference Finch-Savage and Leubner-Metzger2006; Willis et al. Reference Willis, Baskin, Baskin, Auld, Venable, Cavender-Bares, Donohue and Rubio de Casas2014) and to reveal the role of the pericarp. Therefore, the germination of freshly harvested mature fruit and isolated seed populations of L. draba and L. appelianum were compared with their germination responses in the afterripened state (Figure 2; Supplementary Figure S2) and their responses to cold stratification (Figure 2) and to GA treatment (Figure 3). Fresh isolated seed populations germinated at a high percentage (ca. 90%) in both species. The high percentage of germination of isolated seeds was not appreciatively affected by dry afterripening storage (Figure 2A; F(4, 20) = 0.834, P = 0.433), cold stratification (Figure 2B; F(4, 20) = 1.371, P = 0.274), and treatment of isolated seeds with GA₃ (Figure 3A; F(4, 10) = 0.06, P = 0.992). It is well established that these treatments release physiological dormancy (Baskin and Baskin Reference Baskin and Baskin2014; Finch-Savage and Leubner-Metzger Reference Finch-Savage and Leubner-Metzger2006; Willis et al. Reference Willis, Baskin, Baskin, Auld, Venable, Cavender-Bares, Donohue and Rubio de Casas2014). The isolated seeds of both the U.S. (KM 1269) and the Austrian (KM 1568) L. draba accessions germinated to a high percentage (ca. 90%) independent of the harvest year (2014 to 2015, 2015 to 2016, or 2016 to 2017) and state (fresh vs. afterripened) (Supplementary Figure S2 and corresponding statistics in Supplementary Table S1). Because both freshly harvested and afterripened isolated seed populations of L. draba andL. appelianum germinated at a high percentage and were not affected by these treatments, we conclude that these seeds are physiologically ND at maturity.

Figure 2.

The effect of afterripening and cold stratification on the germination of Lepidium draba and Lepidium appelianum isolated seeds and indehiscent fruits (seeds within pericarp). (A) The effect of afterripening (dry) storage at room temperature and humidity. (B) The effect of cold stratification in the imbibed state under dark conditions in a refrigerator (4 C). Mean values ± SE (N = 3 × 25) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest) at optimal germination assay conditions (12/12-h light regime at 25/15 C day/night for 28 d) are presented.

Figure 3.

The effect of gibberellic acid (GA₃) treatment on the germination of Lepidium draba and Lepidium appelianum fresh and afterripened seeds and fruits and the levels of endogenous bioactive gibberellins (GA). (A) Dose response for the effects of exogenous GA₃ on germination responses of fresh isolated seeds and fruits (seeds within pericarp). Mean values ± SE (N = 3 × 25) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest) at optimal germination assay conditions (12/12-h light regime at 25/15 C day/night for 28 d) are presented. (B) Endogenous levels of bioactive gibberellins (GA₁, GA₃, GA₄, and GA₇) in fresh and afterripened seeds and pericarps of L. draba. N = 4 × 20 mg (dry weight, DW) of seed/pericarp are presented.

Analysis of endogenous GA metabolite levels in fresh and afterripened seed and pericarp tissues in L. draba revealed the presence of the bioactive gibberellins GA₁, GA₃, GA₄, and GA₇ (Figure 3B), as well as their precursors and inactive metabolites (unpublished data). The dry seed and pericarp contained nanogram quantities of these GA metabolites, with GA₁ being the dominant bioactive GA. No striking differences were evident between fresh and afterripened seeds (Figure 3B). The finding that fresh and afterripened seeds do not appreciably differ in the levels of bioactive GAs further supports the conclusion that L. draba seeds are ND.

Our study species, L. draba and L. appelianum, are highly invasive weeds throughout the Middle East, Asia, Australia, New Zealand, Canada, and the western United States (Chipping and Bossard Reference Chipping, Bossard, Bossard, Randall and Hoshovsky2000; Gaskin Reference Gaskin2006; Gaskin et al. Reference Gaskin, Zhang and Bon2005; McInnis et al. Reference McInnis, Kiemnec, Larson, Carr and Sharratt2003) and are dispersed in disturbed areas, including roadsides. Analysis of their global distribution using the Global Biodiversity Information Facility (https://www.gbif.org/) database suggests that their main occurrence in the western part of North America is expanding toward the eastern regions for both species (Supplementary Figure S1). Bani-Aameur and Sipple-Michmerhuizen (Reference Bani-Aameur and Sipple-Michmerhuizen2001) and Presotto et al. (Reference Presotto, Poverene and Catamutto2014) reported that the lack of dormancy in weedy species increases the survival ability of the species, because germination and early seedling growth are the most critical factors for species establishment. Therefore, higher germination and seedling recruitment have been recognized as being among the major factors promoting naturalization success of invasive species (Fernández-Pascual et al. Reference Fernández-Pascual, Jiménez-Alfaro, Caujapé-Castells, Jaén-Molina and Díaz2013; Mandák Reference Mandák2003; Udo et al. Reference Udo, Tarayre and Atlan2016; Walck et al. Reference Walck, Hidayati, Dixon, Thompson and Poschlod2011). Rezvani and Zaefarian (Reference Rezvani and Zaefarian2016) found that isolated seeds of L. draba are light requiring and that GA₃ treatment can replace the light requirement to trigger germination. Similar to L. draba and L. appelianum (Figures 1–3), other Brassicaceae species, including Noccaea papillosa (Boiss.) F. K. Mey (Kirmizi Reference Kirmizi2017), L. sativum (Graeber et al. Reference Graeber, Linkies, Steinbrecher, Mummenhoff, Tarkowská, Turečková, Ignatz, Sperber, Voegele, de Jong, Urbanová, Strnad and Leubner-Metzger2014), and L. didymum (Sperber et al. Reference Sperber, Steinbrecher, Graeber, Scherer, Clausing, Wiegand, Hourston, Kurre, Leubner-Metzger and Mummenhoff2017), produce ND seeds. In contrast to this, other Brassicaceae weedy species, for example, Coincya rupestris subsp. leptocarpa (Gonz. Albo) Leadlay, Coincya rupestris subsp. rupestris Porta & Rigo ex Rouy (Copete et al. Reference Copete, Herranz and Ferrandis2005), clasping pepperweed (Lepidium perfoliatum L.) (Tang et al. Reference Tang, Tian and Long2010), L. papillosum (Graeber et al. Reference Graeber, Voegele, Büttner-Mainik, Sperber, Mummenhoff and Leubner-Metzger2013), A. thaliana (Baskin and Baskin Reference Baskin and Baskin2014), Chorispora sibirica (L.) de Candolle, Syrian mustard [Euclidium syriacum (L.) W. T. Alton], Goldbachia laevigata (Marschall von Bieberstein) de Candole, Spirorhynchus sabulosus Karelin & Kirilov,Sterigmostemum fuhaiense H. L. Yang, Tauscheria lasiocarpa Fischer ex de Candolle (Lu et al. Reference Lu, Zhou, Tan, Baskin and Baskin2015), and Isatis violascens Bunge (Zhou et al. Reference Zhou, Lu, Tan, Baskin and Baskin2015) produce PD seeds (Baskin and Baskin Reference Baskin and Baskin2014).

Distinct Roles of the Pericarp in Lepidium draba and Lepidium appelianum Fruit Germination

Figure 2 shows that while seeds of both species are physiologically ND and germinate readily, the two species differ in the germination responses of their indehiscent fruits. In populations of L. appelianum fruits, the seeds germinated within the fruits and subsequent radicle expansion led to emergence through the pericarp (Figure 1J) of ca. 90% already in the fresh mature state (Figure 2A). In contrast to this, populations of L. draba fresh fruits exhibited pericarp rupture and visible radicle emergence (Figure 1I), with only ca. 50% of the fruits completing germination (Figure 2A). Interestingly, afterripening for 16 wk resulted in ca. 90% fruits germinating in L. draba with visible pericarp rupture (Figure 2A). This finding for L. draba was evident for both the U.S. (KM 1269) and the Austrian (KM 1568) accessions and consistent over the harvest years (2014 to 2015, 2015 to 2016, 2016 to 2017) (Supple-mentary Figure S2 and corresponding statistics in Supplementary Table S1). As with afterripening, treatment with GA₃ also increased the maximum fruit germination percentage of L. draba from ca. 50% to ca. 90% (Figure 3A). In contrast to this, cold stratification had no appreciable effect on the maximum germination percentage of L. draba (Figure 2B). When the germination responses of fruits and isolated seeds (Figures 2 and 3; Supplementary Figure S2) are compared, it is clear that in L. appelianum the pericarp has no effect on the maximum germination percentage of the population, but in L. draba it inhibited the germination of about half of the population. We conclude that while both species have ND seeds, the roles of the pericarps differ. In L. appelianum, the pericarp does not affect the germination capacity, while the indehiscent fruits of L. draba have pericarp-imposed dormancy. Interestingly, this pericarp-imposed dormancy of the L. draba indehiscent fruits can be released by afterripening and by GA₃ treatment (Figures 2 and 3).

To further investigate the finding that the pericarp confers coat dormancy in L. draba, while it does not affect the germination in L. appelianum, we compared the patterns of water uptake of fruits and seeds. Figure 4 shows that the water uptake patterns of isolated seeds of both species were very similar, with three typical phases: imbibition (phase 1), plateau (phase 2), and completion of germination by radicle emergence and subsequent growth (phase 3) (Finch-Savage and Leubner-Metzger Reference Finch-Savage and Leubner-Metzger2006). In Figure 4A a comparison of fresh mature fruits of L. draba with fresh mature isolated seeds shows that the pericarp slowed down the water uptake during imbibition (phase 2) and delayed the onset and rate of phase 3 water uptake, which in seeds is associated with radicle emergence and embryo postemergence growth. While all L. draba seeds completed the germination process within ca. 6 d, even after 28 d, the L. draba fruit population had only reached ca. 50%. The results in Figure 4A demonstrate that the L. draba pericarp is water permeable, that is, the fruits do not have physical dormancy, but the pericarp confers coat dormancy in association with slowing down the water uptake and the transition to phase 3 and the completion of fruit germination. In contrast to this, Figure 4B shows that when fruits and isolated seeds of L. appelianum were compared, the pericarp did not appreciably affect the water uptake patterns and permitted maximum germination. We conclude that the pericarp-imposed dormancy of L. draba fruits is caused by some mechanism that decreases the water uptake and inhibits the transition from phase 2 to phase 3 required for the completion of germination.

Figure 4.

The effects of the pericarp (fruit coat) on the water uptake of (A) Lepidium draba and (B) Lepidium appelianum seeds. A single asterisk refers to the time of full (>90%) completion of germination of fresh isolated seeds or fruits (seeds within pericarp), whereas a double asterisk refers to the maximum germination (52%) due to the pericarp-mediated dormancy of L. draba (see Figure 2A). Isolated seeds and fruits exhibit a typical three-phase pattern of water uptake by seeds: phase 1 (imbibition) is followed by the plateau phase 2 (metabolic activation), and upon endosperm rupture, the radicle emergence is associated with phase 3 (water uptake indicative for the completion of germination). N = 3 × 20 (fresh seeds) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest); N = 3 × 10 for each time point measured (fresh seeds within pericarp).

ABA-mediated Inhibition as Mechanism for the Pericarp-imposed Dormancy in Lepidium draba

To investigate the mechanisms through which the pericarp confers coat dormancy to seeds in L. draba fruits, we conducted scarification and leaching experiments. Figure 5A shows that neither pericarp scarification (i.e., removing a possible mechanical constraint by manually removing the part of the pericarp covering the seed’s radicle end with razor blade) nor pericarp surface sterilization to eliminate possible microbial activity removed the pericarp-imposed dormancy. The germination percentages of mechanically scarified fruits (58%) and surface-sterilized fruits (54%) did not differ significantly from nonsterilized fruits (control, 52%) (F(2, 120) = 3.8, P = 0.086) of L. draba. The treatments did not affect the ca. 90% germination of subsequently isolated L. draba seeds, and the comparison with L. appelianum shows that neither the scarification nor the sterilization treatment had negative effects (Figure 5A). The finding that neither pericarp scarification nor surface sterilization affected the pericarp-mediated dormancy of L. draba strongly suggests that lack of germination is not mechanical in nature and that microbial activity is not involved in its release. It is therefore different from the recent finding in L. didymum that the pericarp-imposed dormancy is caused by a mechanical constraint and the microbial activity of common fungi is involved in the release of the pericarp-imposed dormancy (Sperber et al. Reference Sperber, Steinbrecher, Graeber, Scherer, Clausing, Wiegand, Hourston, Kurre, Leubner-Metzger and Mummenhoff2017). Many other Brassicaceae species, including wild radish (Raphanus raphanistrum L.) (Cousens et al. Reference Cousens, Young and Tadayyon2010), Diptychocarpus strictus (Fischer ex Marschall von Bieberstein) Trautvetter (Lu et al. Reference Lu, Tan, Baskin and Baskin2010), turnipweed [Rapistrum rugosum (L.) All.] (Ohadi et al. Reference Ohadi, Mashhadi and Tavakol-Afshari2011), Lachnoloma lehmannii Bunge (Mamut et al. Reference Mamut, Tan, Baskin and Baskin2014), C. sibirica, E. syriacum, G. laevigata, S. sabulosus, S. fuhaiense, T. lasiocarpa (Lu et al. Reference Lu, Zhou, Tan, Baskin and Baskin2015), and I. violascens (Zhou et al. Reference Zhou, Lu, Tan, Baskin and Baskin2015) seem to have a coat dormancy with a mechanical component similar, at least in part, to the pericarp-imposed mechanical dormancy of L. didymum (Sperber et al. Reference Sperber, Steinbrecher, Graeber, Scherer, Clausing, Wiegand, Hourston, Kurre, Leubner-Metzger and Mummenhoff2017). In contrast to this, in L. appelianum, the pericarp does not confer a coat dormancy at all.

Figure 5.

The effects of pericarp scarification, sterilization, washing, and abscisic acid (ABA) treatment on the germination of Lepidium draba and Lepidium appelianum freshly harvested mature fruits. (A) Germination of fresh isolated seeds, untreated fresh fruits (seeds enclosed within untreated pericarp), scarified fresh fruits (seeds enclosed within scarified pericarp, that is, mechanical constraint of pericarp removed by scarification with razor blade), surface-sterilized fresh fruits (seeds enclosed within surface-sterilized pericarp to eliminate microbial activity), and washed fresh fruits (fruits washed for 24 h to remove water-soluble chemical inhibitors) of L. draba and L. appelianum. (B) Germination of fresh and afterripened indehiscent fruits and isolated seeds without (control) and with addition of 5 µM ABA. Mean values ± SE (N = 3 × 25) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest) at optimal germination assay conditions (12/12-h light regime at 25/15 C day/night for 28 d) are presented. Different letters (a, b) designate significantly different mean values as determined by Tukey’s pairwise multiple-comparison test (P < 0.05).

In contrast to pericarp scarification, washing of fresh L. draba fruits removed the pericarp-imposed dormancy and caused ca. 90% germination, as is the case for the isolated ND seeds of L. draba (Figure 5A). For the ND L. appelianum fruits, germination occurs without the washing treatment (Figure 5A). This finding strongly suggests that chemical inhibitors present in the pericarp of fresh L. draba fruits cause the pericarp-imposed dormancy, while such inhibitors are lacking in L. appelianum. Washing may have caused leaching out of the chemical inhibitors from L. draba pericarps, thereby releasing the dormancy. To further quantify the effect of the inhibitors, we added wash water of L. draba pericarps to the germination media of fresh (Figure 6A) and afterripened (Figure 6C) L. draba seeds. The wash water from fresh L. draba pericarps caused a significant delay in the onset of seed germination and in the maximum germination percentages of fresh (Figure 6A) and afterripened (Figure 6C) seeds. In contrast to this, no inhibition of seed germination was obtained with wash water from afterripened or wash water from previously washed fresh pericarps (Figure 6A and C). It is therefore clear that only the fresh L. draba pericarp contains water-soluble germination inhibitors that leach out and thereby inhibit germination.

Figure 6.

The effect of treatment with abscisic acid (ABA), wash water from fresh pericarp, wash water of washed fresh pericarp, and wash water of afterripened pericarp on the germination kinetics of Lepidium draba isolated seeds. (A) The effect of wash water from L. draba pericarp on the germination of L. draba fresh seeds. (B) Germination dose response of L. draba fresh seeds incubated with different ABA concentrations applied. (C) The effect of wash water from L. draba pericarp on the germination of L. draba afterripened seeds. (D) Germination dose response of L. draba afterripened seeds incubated with different ABA concentrations applied. Mean values ± SE (N = 3 × 25) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest) at optimal germination assay conditions (12/12-h light regime at 25/15 C day/night for 28 d) are presented. Pericarp tissues weighing 300 mg were washed with 3 ml of distilled water using a shaker at 100 rpm for 6 h to obtain the pericarp wash water applied in the germination assays.

To investigate whether pericarp-released ABA is involved in mediating the pericarp-imposed dormancy of L. draba, we initially compared the germination responses to treatment with ABA of fresh and afterripened fruits and isolated seeds of both species. Treatment with 5 µM ABA inhibited the germination of fruits and seeds of both species in both physiological stages (Figure 5B). Fresh and afterripened seeds of both species seem to be equally sensitive to inhibition by ABA. Interestingly, seeds in fresh L. draba fruits were more sensitive to the inhibition compared with those in fresh L. appelianum fruits (Figure 5B). To precisely quantify the seed sensitivities, we conducted dose–response experiments for the ABA treatment with fresh (Figure 6B) and afterripened (Figure 6D) L. draba seeds. These results demonstrated that increasing ABA concentrations caused a similar delay in the onset of the completion of seed germination of fresh and afterripened L. draba seeds (Figure 6). Increasing ABA concentrations also caused a decrease in the maximum germination percentages reached after ca. 1 mo of incubation of the seed populations. This quantification demonstrated that the fresh and afterripened L. draba seeds had the same ABA sensitivity (Figure 7A). A comparison of these ABA sensitivity values with the results obtained with the wash water of fresh pericarp (Figure 7B) revealed that its inhibitory effect corresponds to a ca. 0.3 µM ABA concentration in the incubation medium (Figure 7).

Figure 7.

The effect of exogenous abscisic acid (ABA), wash water from Lepidium draba pericarp (fresh, fresh-washed, and afterripened) on the germination of L. draba fresh and afterripened isolated seeds. (A) Germination dose-response of L. draba seeds incubated with different ABA concentrations. (B) The effect of wash water from pericarp on the germination of L. draba seeds. Wash water of fresh L. draba pericarp inhibits at a level similar to 0.3 µM ABA. Lepidium appelianum pericarp does not contain ABA or other water-soluble compounds that may inhibit germination. Mean values ± SE (N = 3 × 25) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest) at optimal germination assay conditions (12/12-h light regime at 25/15 C day/night for 28 d) are presented. Pericarp tissues of 300 mg were washed with 3 ml of distilled water using a shaker at 100 rpm for 6 h to obtain the pericarp wash water applied in the germination assays.

Germination and dormancy are controlled by the hormonal balance between promoting GAs and inhibiting ABA (Finch-Savage and Leubner-Metzger Reference Finch-Savage and Leubner-Metzger2006). To investigate whether ABA is involved in the pericarp-mediated dormancy of L. draba, we analyzed the endogenous hormone levels of fresh and afterripened L. draba seeds, pericarps, and fruits (Figure 8). In contrast to the low GA levels, fresh and afterripened dry seeds of L. draba contained equally high levels of ABA. Interestingly, the ABA content of L. draba fresh mature pericarp was more than 20-fold higher compared with the afterripened pericarp (Figure 8A). Remarkably, when we calculated this, the pericarp ABA content will upon pericarp washing lead to a ca. 0.1 µM ABA concentration in the wash water of fresh L. draba pericarps and to a ca. 0.005 µM ABA concentration in the wash water from afterripened pericarp. Compared with the ABA dose responses (Figure 7A), only the concentration from the fresh pericarp would inhibit germination, and this is exactly what we observed (Figure 7B). In agreement with the ABA in the pericarp playing a role in the chemical dormancy of L. draba fruits, the increased germination percentages of washed fruits were associated with a decrease of the ABA content during the washing process (Figure 8B). Analysis of the ABA catabolites demonstrates that these also decrease during the washing process (Supplementary Table S2) and that the classical 8′-hydroxylation pathway via phaseic acid and dihydrophaseic acid plays a major role in ABA degradation (Turečková et al. Reference Turečková, Novák and Strnad2009). Further, the high germination proportion of afterripened fruits was associated with a low ABA content (Figure 8B), and afterripened fruits did not germinate when imbibed in the presence of ABA (Figure 8B). We therefore conclude that fruit washing and afterripening released chemical dormancy by eliminating ABA from the pericarp by leaching and/or degradation.

Figure 8.

The effect of afterripening and washing on the abscisic acid (ABA) and gibberellin (GA) levels of Lepidium draba fruits. (A) Endogenous levels of ABA and bioactive GAs in fresh and afterripened dry seeds and pericarps. (B) ABA and bioactive GA levels during washing of fresh L. draba fruits, as compared with afterripened fruits, and with the resultant maximum germination responses presented. Mean values ± SE (N = 3 × 25) of accessions KM 1296 and KM 1754 (2014 to 2015 harvest) at optimal germination assay conditions (12/12-h light regime at 25/15 C day/night for 28 d) are presented. N = 4 × 20 mg (dry weight, DW) of seed/pericarp for ABA and bioactive GA analysis. For a detailed statistical analysis of the ABA contents and their catabolites, see Supplementary Table S2.

As for L. draba, but not L. appelianum, pericarp-mediated chemical dormancy is suggested to occur in crop and weed species of Brassicaceae, including R. raphanistrum (Cheam Reference Cheam1986; Mekenian and Willemsen Reference Mekenian and Willemsen1975), L. lehmannii (Mamut et al. Reference Mamut, Tan, Baskin and Baskin2014), and Leptaleum filifolium (Willd.) DC. (Lu et al. Reference Lu, Tan, Baskin and Baskin2017). Chemical inhibitors that leach out from the pericarp are thought to cause seed dormancy and can be removed by washing intact fruits with a large volume of water (Baskin and Baskin Reference Baskin and Baskin2014; Hu et al. Reference Hu, Yu, Yang, Wu, Wang and Boelt2010; Liu et al. Reference Liu, Liu, Ji, Chen and Xu2015; Mamut et al. Reference Mamut, Tan, Baskin and Baskin2014). In general, the pericarp can inhibit germination by (1) physical means, that is, inhibition of water imbibition (Cousens et al. Reference Cousens, Young and Tadayyon2010) and reduction of gas exchange (Adkins et al. Reference Adkins, Bellairs and Loch2002; Hu et al. Reference Hu, Wang and Wu2009); (2) mechanical means, that is, mechanical resistance on the radicle protrusion is imposed by the pericarp (Baskin and Baskin Reference Baskin and Baskin2014; Hu et al. Reference Hu, Yu, Yang, Wu, Wang and Boelt2010; Sperber et al. Reference Sperber, Steinbrecher, Graeber, Scherer, Clausing, Wiegand, Hourston, Kurre, Leubner-Metzger and Mummenhoff2017); and/or (3) chemical inhibitors residing inside the fruit coat inhibit germination (this study; Mamut et al. Reference Mamut, Tan, Baskin and Baskin2014; Sari et al. Reference Sari, Oguz and Bilgic2006). In the Brassicaceae, physical dormancy has not been recorded to date (Baskin and Baskin Reference Baskin and Baskin2014), and the possible dormancy types for fruits as dispersal units in this family are either nondormancy and/or pericarp-mediated mechanical (Cousens et al. Reference Cousens, Young and Tadayyon2010; Ohadi et al. Reference Ohadi, Mashhadi and Tavakol-Afshari2011; Sperber et al. Reference Sperber, Steinbrecher, Graeber, Scherer, Clausing, Wiegand, Hourston, Kurre, Leubner-Metzger and Mummenhoff2017) and chemical dormancy (this study; Cheam Reference Cheam1986; Mamut et al. Reference Mamut, Tan, Baskin and Baskin2014; Mekenian and Willemsen Reference Mekenian and Willemsen1975).