Peng, Hu, Sun and Li (2019). Seed Science and Technology, 47, 3, 301-306. https://doi.org/10.15258/sst.2019.47.3.05 Research Note Dry after-ripening, light, cold stratification and temperature effects on seed germination of Primula poissonii from Yunnan, China Deli Peng 1, Xiaojian Hu3, Hang Sun2 and Zhimin Li1* 1 School of Life Science, Yunnan Normal University, Kunming, 650500, Yunnan, China 2 Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China 3 National Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China * Authors for correspondence (E-mail: [email protected]) (Submitted June 2019; Accepted August 2019; Published online October 2019) Abstract Primula poissonii, an attractive wild plant growing in the subalpine/alpine region of southwest China, has low seed germination in cultivation. This study attempted to improve seed germination by testing the effect of several treatments including dry after-ripening (DAR), light, cold stratification (CS) and temperature gradient treatments. DAR increased germination at 15/5 and 25/15°C, as compared with fresh seeds. DAR seeds germinated significantly better (˃ 80%) at higher temperatures (20-28°C) than at lower (10°C, < 20%; 15°C, < 30%) and extreme high temperatures (30°C, < 55%; 32°C, 0%). Incubation at alternating temperature (25/15°C) did not significantly improve germination; whereas at 15/5°C germination increased significantly, compared with the corresponding constant temperature (20 and 10°C, respectively). DAR seeds had a strict light requirement at all temperatures. As DAR and CS are sufficient to break seed dormancy, the seeds of P. poissonii appear to have non-deep PD. For non-dormant cold-stratified seeds, the estimated Tb and thermal time (θ50) were 2.3°C and 74.1°Cd, respectively. Keywords: alpine plant, alternating temperature, germination, light requirement, seed dormancy Experimental and discussion Primula L. is the largest and most widespread genus in Primulaceae, comprising about 500 species (Hu and Kelso, 1996). This genus is distributed throughout the moister and cooler regions of the Northern Hemisphere, and the alpine region of the e astern Himalayas and western China has the greatest concentration of species and the greatest © 2019 Peng et al. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/licenses/by-nc/4.0 301 DELI PENG , XIAOJIAN HU, HANG SUN AND ZHIMIN LI diversity (Hu and Kelso, 1996). Plants of Primula are attractive, colourful plants due to their rosette growth form and/or bright flowers, and so have great potential as ornamental garden plants (Hu and Kelso, 1996). However, the seeds of many Primula species possess physiological dormancy (PD), and require light for germination (Hitchmough et al., 2011; Baskin and Baskin, 2014), resulting in slow, non-uniform and low germination. Therefore, effective methods must be used to break PD (e.g. cold stratification, dry after-ripening) and obtain uniform and rapid germination with high germination percentage. Primula poissonii Franch. is an attractive wild plant growing at middle altitude (2100-3700 m a.s.l.) in southwest China. The present study was undertaken to determine the effects of dry after-ripening (DAR), cold stratification (CS) and temperature on seed germination for P. poissonii, and to find effective methods to break seed dormancy for this species. P. poissonii flowers between early May and early July, and the fruits mature between late August and September. In late September 2014, f reshly-mature fruits were collected from Shangri-La Alpine Botanical Garden, where this species grows naturally in a subalpine moist meadow (27°54'29.20'', 99°38'21.63'', 3308 m a.s.l., Yunnan Province, China). Fruits were collected from at least 30 individuals. Non-seed structures were removed by hand in the laboratory. Some of the fresh seeds were used in germination experiments, whilst the remaining seeds were air-dried and stored in a paper bag at room temperature for six months (DAR) until the onset of the experiments. To de termine the level of PD (if present) of mature seeds, fresh seeds were incubated one week after collection, on 1% agar/water substrate, in plastic Petri dishes (90 mm-diameter) in incubators (MMM: friocell 404) under two alternating temperatures: 15/5 and 25/15°C (day/night). The two alternating temperatures were selected to approximate daily temperature regimes in the germination season (late April to late August). The daily photoperiod was -2 -1 12-hours light with 22.2 μmol m s illumination from cool white fl uorescent bulbs and 12-hours dark. To test the effect of constant versus alternating temperatures on germination after DAR, in addition to the two alternating temperatures (15/5 and 25/15°C), seeds were also incubated in the light at seven constant temperatures (10, 15, 20, 25, 28, 30 and 32°C). To determine the effect of darkness, seeds were sown on 1% agar/water substrate in Petri dishes that were wrapped in two layers of aluminum foil, and then incubated at 5, 10, 15, 20, 25, 25/15 and 15/5°C. After DAR, seed germination decreased suddenly at 10 and 15°C (< 30%), which showed that seeds might still remained dormancy or did not germinate at low temperatures. Therefore, cold stratification was applied to break dormancy and/or accelerate germination. For CS, DAR seeds were sown on 1% agar/water substrate in plastic Petri dishes and incubated in dark at 5°C for 16 weeks (according to winter duration in seed collection site). No germination occurred during stratification, and then seeds were incubated in the light at four constant temperatures (10, 15, 20 and 25°C) and two alternating temperatures (15/5 and 25/15°C). Three replicates of 20 seeds were used for each test condition. The Petri dishes were put into transparent plastic bags to prevent desiccation. Seeds incubated in the light were counted daily and germinated seeds were discarded, while dark-incubated seeds were counted only once at the end of the test to avoid any exposure to light. The criterion for 302 SEED DORANCY AND GERMINATION IN PRIMULA POISSONII germination was visible radicle protrusion. Experiments were terminated at 16 weeks, when no more seed germinated for two continuous weeks. The viability of ungerminated seeds was checked by a cut-test. Seeds with a plump, firm and white embryo were considered viable. The final germination percentage (GP) was calculated based on the total number of filled seeds. Germination rate (GR) was calculated as the inverse of the time taken to 50% germination (i.e. 1/t50). When normality and homogeneity of variance were satisfied for original data, we applied independent-sample t-tests to determine whether DAR had significantly increased GP, relative to control treatment (fresh seeds) at 15/5 and 25/15°C. After DAR, one- way ANOVA was employed to evaluate the effect of temperature on seed germination. A Tukey’s HSD test was performed for multiple comparisons to determine significant (P < 0.05) differences between treatments. To determine the differences of alternating versus constant temperatures on germination (10 vs. 15/5, 20 vs. 25/15°C), an independent- sample t-test was employed. We also applied an independent-sample t-test to determine whether CS had significantly increased GP and GR, relative to DAR seeds. When the assumptions were not satisfied, binary regression analysis was carried out. All data were analysed using the procedures in PASW Statistics 18 (PASW, 2009). A thermal-time model was applied to estimate the cardinal temperatures (Hardegree, 2006). Germination time courses for all three replicates at a given temperature were combined and fitted using the Weibull Function (Brown and Mayer, 1988) in OriginPro 9.5. The day to 50% germination (t50) was obtained by fitting cumulative germination progress curves. GR (1/t50) was plotted as a function of temperature and regressed using a linear model, to estimate the base temperature (Tb) below which GR was equal to zero. The slope of the linear regression line corresponded to the reciprocal of the thermal-time requirement at suboptimal temperatures (θ50). There was 6.7 and 31.5% germination of fresh seeds at 15/5 and 25/15°C, respectively. DAR increased seed germination, to 86.4% at 15/5°C and 93.0% at 25/15°C (figure 1). After DAR, no seeds germinated in dark at all the temperatures, while the effect of temperature was significant in light (figure 2). Seeds exhibited high GP within the suitable constant temperature ranges: 83.4-90.0% at 20-28°C; below 20°C or above 28°C, GP significantly decreased. Incubation at alternating 25/15°C did not significantly improve the germination compared with the corresponding constant temperature (20°C), whereas at 15/5°C germination increased significantly (P < 0.01) compared with the corresponding constant temperature (10°C). CS improved seed germination and widened the range of germination temperatures (figure 2). In particular, the effect of CS was 100% at 10°C and 94.7% at 15°C. CS also increased GR (1/t50) compared with untreated seeds at other temperatures where there was already high germination (figures 2 and 3). Seed germination in res ponse to temperature was well described by the thermal-time model at suboptimal temperatures (10-25°C) (figure 3).