Germination of Styrax japonicus Seeds as Influenced by Storage and Sowing Conditions

This study evaluated the effect of storage and sowing conditions on seed germination of Styrax japonicus Sieb. et. Zucc, an ornamental tree with seeds that exhibit double dormancy. The germination of freshly harvested seeds was compared with seeds that had been stored dry at 20 C for a year before sowing. After sowing, potted seeds were placed in an air-refrigerated greenhouse maintained 18.5/18 C. Pots were kept for 0, 1, 2, 3, or 4 months in the greenhouse for warm stratification (WS), and then moved to a 5.5 C cooler for 3 months for cold stratification (CS). After CS, pots were moved back to the greenhouse. None of the fresh or dried seeds that received CS immediately after sowing germinated. Germination of fresh and dried seeds did not differ when the seeds received WS for at least 1 month. Dry seeds, that received 3 or 4 months of WS, had a lower germination percentage than fresh seeds. The effect of moisture after sowing on the germination of freshly harvested seeds was determined by either watering or not watering the seeds before they were placed in the cooler. In addition seeds under the moist treatment were watered as needed to keep the medium moist. More than 60% of the seeds that received CS for at least for 1 month germinated. A radicle did not emerge through the seed coat after warm stratification, or at the completion of cold stratification. It is recommended that Styrax seeds receive 1 month of WS, followed by 2 months of CS. INTRODUCTION Styrax japonicus is a small, low branched tree that produces white flowers with yellow stamens (Dirr, 1990) (Fig. 1). This species can be propagated from stem cuttings or seeds. Seeds that are sown in the fall without a sequential warm and cold stratification may germinate the second spring (Dirr, 1990; Kwon, 1995). Kwon (1995) suggested that a radicle might emerge after 3 months of WS, and then enter a dormancy. Stratification of seeds using warm and cold temperatures is required to break seed dormancy for many woody plants to improve seed germination (Young and Young, 1992). Seed dormancy is mediated, in part, by a hard seed coat or testa, or by a dormant embryonic axis and cotyledons (Bandyopadhyay et al., 1999). To improve germination Styrax seeds should be stored in a moist, warm environment for 3 (Kwon, 1995) or 5 months (Dirr, 1990), and then stored at low temperatures for 3 or 4 months (Dirr, 1990; Kwon, 1995). Seeds that are sown immediately after collection may germinate by the following spring, suggesting that non-fresh seeds may take longer to germinate (Dirr, 1990). Thus, this study aimed to determine (a) the effect of dry storage duration at 20 C, and (b) the effect of moisture during storage at 18.5/18 C on germination of Styrax seeds. MATERIALS AND METHODS Dry Storage Duration Seeds (NA 60191) from the U. S. National Arboretum, Woody Landscape Plants Germplasm Repository at Glenn Dale, MD, were harvested on Oct. 3, 2000. A group from these seeds was sown on Nov. 8 and held for 0, 1, 2, 3, or 4 months without watering (dry) in an air-refrigerated greenhouse maintained at 18.5/18 C. After dry storage, pots were watered and kept moist in the greenhouse for 2 months of warm stratification (WS) and moved to a 5 C cooler for cold stratification (CS). Another group of seeds was sown, watered, kept moist for 0, 1, 2, 3, or 4 months of WS and then moved Proc. XXVI IHC – Asian Plants Eds. Jung-Myung Lee and Donglin Zhang Acta Hort. 620, ISHS 2003 Publication supported by Can. Int. Dev. Agency (CIDA) 412 to a cooler for 3 months of CS. Forty seeds were sown per 15 cm pots filled with ProMix BM (Stamford, Conn). Three pots were used per treatment. After CS, pots were moved to the greenhouse where the number of seeds that germinated was recorded. Peak seed germination was defined as the maximum number of seeds that germinated during the test period. This number was calculated as a percentage, i.e., (no. of germinated seeds/ total number of seeds per pot)*100. Weeks for germination peak was defined as the number of weeks that elapsed from the start of the experiment, i.e., when put into the greenhouse, until reaching peak seed germination. These two dependent variables were analyzed using a 2-way analysis of variance (ANOVA), with interaction. Duration of storage (in months) and storage condition (moist vs. dry) were tested as main effects. The control group, i.e., those seeds that were sown without any WS were not included in the analysis to keep a balanced design. Means of the dependent variables and the standard error of the means (SEM) were obtained using the LSMEANS procedure within SAS (SAS Institute 1999). Means were compared using contrasts at P < 0.05. Because germination was recorded over time, germination was modeled as a polynomial function of time using a mixed model, repeated measures analysis with an autoregressive order one covariance structure. Parameter estimates were obtained using the Solution option in the Model statement. Moisture During Storage Seeds (NA 60191) were harvested on Sept. 24, 1999 and Oct. 3, 2000, and seeds were dry stored at 20 C until sowing Nov. 8, 2000 until sowing. After sowing, pots were watered and exposed to 0, 1, 2, 3, or 4 months of WS, followed by CS at 5 C for 3 months. Forty seeds were sown per 15 cm pots, filled with ProMix BM (Stamford, Conn). Three pots were used per treatment. Pots were moved to a greenhouse where the number of seeds that germinated were recorded weekly. Data were analyzed using a 2-way ANOVA, with interaction. The year when seeds were collected and months of WS were treated as main effects. Because germination was recorded over time, germination was modeled as a polynomial function of time using a 2-way, repeated measures analysis of variance with an autoregressive order one covariance structure. Parameter estimates were obtained using the Solution option in the Model statement. RESULTS Dry Storage Duration Control seeds that were kept in a 18.5/18 C greenhouse did not germinate by the time the experiment was completed on Sept. 10 (Table 1). Examination of nongerminated seeds revealed that the seed coat was intact in most of the seeds, yet a radicle was visible through the seed coat in less than 10% of the non-emerged seeds (Fig. 2). The mean peak germination was significantly influenced by the interaction between storage duration and moisture (F = 55.13; df = 3,16; P = <0.0001; N = 24). After 1 month of moist WS, more than 70% of the seeds had germinated. More than 53.3% of seeds germinated when receiving moist WS. Germination of seeds receiving dry WS decreased to 28.3% as the storage period increased to 4 months (Fig. 3). The mean number of weeks to reach the germination peak was not significantly influenced by the interaction between storage duration and moisture (F = 2.91; df = 3,16; P = 0.0666; N = 24). The number of weeks until germination peak was significantly influenced by storage moisture as a main effect (F = 5.40; df = 1,16; P = 0.0336; N = 24). Regardless of storage duration, seeds that received dry WS (6.33 ± 0.22 week) took longer to reach their peak germination than seeds that received moist WS (5.58 ± 0.22 week). Germination regressed over time was significantly influenced by the interaction between storage duration and storage moisture (F = 124.24; df = 8, 65.9; P < 0.0001; N = 168). The quadratic effect was significant, thus included in the model (F = 16.17; df = 8, 111; P < 0.0001; N = 168). Regression slopes were significantly different among treatment combinations.