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<I>Pinus Dabeshanensis</I>

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<I>Pinus Dabeshanensis</I> Xiang, Zhang and Wu (2019). Seed Science and Technology, 47, 2, 229-235. https://doi.org/10.15258/sst.2019.47.2.09 Research Note Effects of seed storage conditions on seed water uptake, germination and vigour in Pinus dabeshanensis, an endangered pine endemic to China Xiaoyan Xiang, Zhongxin Zhang and Ganlin Wu* Anqing Normal University, China *Author for correspondence (E-mail: [email protected]) (Submitted January 2019; Accepted May 2019; Published online August 2019) Abstract Assessing seed vigour and germination in rare endangered species is critical for developing effective in situ and ex situ conservation strategies. Pinus dabeshanensis is an extremely endangered pine endemic to eastern China. To obtain fundamental propagation information for this species, its seed traits and germination were studied. The viability of fresh filled seeds was 100% based on the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) method, and viability was maintained for 10 months at both 4°C and room temperature. Scarification resulted in rapid uptake of water, but it was still faster for seeds stored at room temperature than for seeds stored at 4°C. For seeds stored at room temperature, there were significant differences in the mean germination time among seeds germinated at 20/10, 25/15, 30/20 or 35/25°C, but final germination was similar (78.1-90%). Overall, the storage conditions greatly affected water uptake, seed vigour and germination. An understanding of these relationships is vital for the conservation and management of this endemic endangered species. Keywords: germination, Pinus dabeshanensis, seed storage conditions, seed vigour, water absorption Experimental and discussion The Chinese white pine, Pinus dabeshanensis W.C. Cheng & Y.W. Law, is an endangered species that is endemic to the Dabieshan Mountains of eastern China. According to Xiang et al. (2015a), the jeopardy of this species is affected by factors such as a lack of gaps and intense competition. Furthermore, sexual reproduction is the sole mode of population regeneration and expansion in this species. Because saplings of this species are very scarce, its populations have been decreasing rapidly (Xiang et al., 2016). Dawanggou (DWG) in Yuexi County, Anhui Province, hosts the largest population (approximately © 2019 Xiang 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/4.0 229 XIAOYAN XIANG, ZHONGXIN ZHANG AND GANLIN WU 200 individuals in total); it is extremely rare in other areas. For example, only one mature individual remains on a cliff in Yaoluoping Nature Reserve, Yuexi County, and two mature individuals are found in Yingshan County, Hubei Province. To preserve this extremely endangered species, an ex situ-conserved population (reserve base of Miaodaoshan) was established at Miaodaoshan Farm, Yuexi County. All the individuals were transplanted from the DWG field population, but only approximately 100 individuals survived. Studies have considered pollen dispersal, interspecific competition, genetic diversity and dynamics of the P. dabeshanensis population ( Xiang et al., 2014, 2015a, b, 2016), but its seed traits and germination remain unclear. This lack of information makes the implementation of conservation measures difficult. In the present study, seed traits and germination were investigated to develop an optimised reproduction protocol. We focused on three major objectives: (1) seed characteristics; (2) water uptake with and without scarification; and (3) the effect of temperature and storage conditions on seed germination. These important data will provide valuable guidelines for the conservation and management of both in situ and ex situ populations. A total of 240 healthy mature cones of P. dabeshanensis were collected in September 2013 and 2014 from eight wild individuals in DWG. The cones were stored individually and dried in a ventilated area in the laboratory. Five cones were randomly selected from each individual, and seeds were extracted manually from these dried cones and visually divided into two groups: one group of seeds was normal in appearance, and the other group was shrunken in appearance, with developed wings but no seeds. Seeds of the former group were immersed in water for 20 minutes to separate floating, empty seeds from filled ones (Ramírez-Valiente and Robledo-Arnuncio, 2015). Then, the numbers of shrunken, empty and filled seeds were counted. The shrunken and empty seeds were regarded as invalid. The proportion of invalid seeds (%) was calculated as follows: (A + B ) / (A + B + C) × 100, where A is the total number of shrunken seeds, B is the number of empty seeds and C is the number of filled seeds. The other seeds from each individual, which were normal in appearance, were mixed and assigned to two groups. For group I, six replicates of 100 seeds were randomly selected and weighed using a digital balance. Three replicates of 20 seeds were randomly chosen, and the seed length, width and thickness were individually measured using callipers. Seeds in group II, which only included filled seeds, were kept at 4°C or room temperature (between 15 and 20°C). For the following experiments, all the tested seeds were collected in 2013. The viability of filled seeds kept at 4°C or room temperature was tested at 0, 6, 10 and 12 months. Three replicates of 15 seeds for each regime were treated as follows: after removal of the coats and endosperms, the embryos were immersed in a 0.1% 2,3,5-triphenyl-2H- tetrazolium chloride (TTC) aqueous solution at 25°C for 12 hours. Embryos with red or pink staining were considered viable, and those that did not stain were considered nonviable (Baskin and Baskin, 2014). The filled seeds stored for six months at 4°C or room temperature were used to test for water absorption of scarified and non-scarified seeds. Sixty seeds were chosen from each storage condition and their coats were partially scarified near the radicle with a scalpel, without damaging the endopleura. Three replicates of 20 seeds per group were weighed, 230 SEED VIGOUR, ABSORPTION AND GERMINATION IN PINUS DABESHANENSIS sown on two layers of moistened filter paper (Whatman No. 1) in 90 mm-diameter Petri dishes and incubated at 35/25°C. At the same time, 120 seeds with intact seed coats stored under the conditions described above were tested under the same conditions as the control. Seeds were quickly surface-dried with filter paper and reweighed daily to the nearest 0.0001 g using a digital balance. The percentage increase in seed mass ( Wp) was calculated as Wp = [(Wi − Wd) /Wd] × 100, where Wd is the initial seed weight and Wi is the weight after water absorption each time. The effect of storage c onditions and alternating temperatures on seed germination was examined. For the storage conditions, filled seeds were kept at 4°C or room temperature for six months before they were sown. To simulate field conditions, each storage protocol was combined with four a lternating temperature levels (20/10, 25/15, 30/20 and 35/25°C). Three replicates of 20 seeds were used for each combination. The seeds were surface- sterilised with 0.5% KMnO4 for 30 minutes, washed thoroughly with sterile distilled water and placed in 90 mm-diameter Petri dishes on two layers of pre-moistened filter paper ( Whatman No. 1). Dishes were stored in programmed incubators under a 10-hour:14- hour light:dark cycle. Light was provided by cool white fluorescent bulbs. Germination was checked daily and the germinated seeds were removed from the Petri dishes. The filter paper was moistened regularly with distilled water. A seed was considered to have germinated when the radicle exceeded 2 mm in length (Ramírez-Valiente and Robledo- Arnuncio, 2015). The experiment was terminated when no germinated seeds were observed over the course of one week. The mean germination time (MGT) was determined as follows: MGT = Σ(ti × ni ) / N, where ti is the number of days since the beginning of germination, ni is the number of seeds germinated on day t, and N is the total number of germinated seeds (Ellis and Roberts, 1981). Statistical analysis was conducted using SPSS 19.0. The results were subjected to one- way ANOVA and Duncan’s test was performed to identify significant differences among treatments. All statements of significance correspond to P ≤ 0.05 unless otherwise stated. The length and thickness of seeds and 100-seed weight were significantly different between 2013 and 2014 (table 1). The 100-seed weight in 2013 was much greater (87.1 g) than that in 2014 (28.6 g). There was no sig nificant difference (P > 0.05) in the per centage of filled seeds per cone between 201 3 and 2014. Be cause the seeds are scarce, the initial water contents were not measured. Seed quality is one of the key factors affecting future population dynamics (Xie and Li, 2000). Heavier seeds often contain larger embryos and more energy reserves and have higher resistance to environmental hazards (Parker et al., 2006; Castoldi and Molina, 2014). In pines, unpollinated ovules develop wings but not Table 1. Seed traits of Pinus dabeshanensis from Dawanggou in Yuexi County, Anhui Province, China. Filled seeds Seed length Seed width Seed thickness 100-seed weight Year (%) (mm) (mm) (mm) (g) 2013 45.5 ± 12.7 a 12.4 ± 0.9 b 7.3 ± 0.4 a 5.6 ± 0.0 b 87.1 ± 11.4 a 2014 32.3 ± 11.4 a 13.8 ± 0.9 a 7.8 ± 0.6 a 6.2 ± 0.0 a 28.6 ± 6.8 b Note: The same letter within each column means no signifi cant diff erence (P > 0.05).
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