HORTSCIENCE 56(4):506–510. 2021. https://doi.org/10.21273/HORTSCI15683-20 Because of the presence of numerous trichomes developed on the seedcoat of Pulsatilla cernua Pulsatilla seeds (Yuan et al., 2019), Pulsa- Germination of var. tilla seeds immersed in water to separate full, viable seeds with developed vegetative or- koreana Seeds as Influenced by Harvest gans (full seed) from empty, nonviable seeds, as demonstrated in Corylopsis coreana Uyeki Dates and Development of Seeds seeds (Kim et al., 2017, 2018), could not be evaluated. X-ray imaging may be useful to distinguish full seeds from empty seeds on a Analyzed by X-ray Imaging large scale, to evaluate images of seeds, to Tao Yuan and Qiuying Wei assess seed development, and to improve School of Landscape Architecture, Beijing Forestry University; Beijing seed lot quality and germination (Carvalho et al., 2010; Kim et al., 2017, 2018; Yuan Laboratory of Urban and Rural Ecological Environment; Beijing Key et al., 2019). Low germination rates could Laboratory of Ornamental Plants Germplasm Innovation and Molecular result from harvesting immature seeds that Breeding; and National Engineering Research Center for Floriculture, No. cannot be verified by visual observation but 35 Qinghua East Road, Haidian District, Beijing 100083, People’s Republic can be detected by X-ray imaging (Kim et al., of China 2017, 2018; Yuan et al., 2019) and magnetic resonance imaging, which can be used to Pablo Jourdan study the internal seed structure to determine Department of Horticulture and Crop Science, Ornamental Plant Germplasm the time of harvest of mature seeds as re- ported for Styrax japonicus (Roh et al., 2004). Center, Ohio State University, Columbus, OH 43210 Seed dormancy could be a factor for low Yong Kwon Yoo germination rates if dormancy is present in the mature seeds and is not released by CS or Department of Horticultural Science, College of Natural Science, Mokpo plant growth regulator treatments, especially National University, Muan-Gun, Jeonnam 58854, Korea with gibberellin (Baskin and Baskin, 2004; Cadman et al., 2006; Yuan et al., 2019). Additional index words. cold treatment, dormancy, empty seeds, full seeds, maturity, seed Beyond the presence of dormancy in seeds, viability, X-ray scanning low germination rates could result from the Abstract. Pulsatilla cernua var. koreana seeds were harvested at six different dates immaturity of seeds that were harvested pre- between 16 days after flowering (DAF) (8 Apr.) and 43 DAF (5 May) in 2018 and maturely after flowering that are not viable categorized into six groups based on X-ray images. Germination tests were performed and fail to germinate. without and with 2 weeks of moist 5 8C treatment [cold stratification (CS)]. Seeds The objectives of this research were as harvested at 38 DAF (30 Apr.) with well-developed vegetative organs (embryo and follows: 1) to harvest Pulsatilla cernua var. endosperm) in seeds categorized as A and B (seed A and seed B, respectively) based on the koreana seeds at different development X-ray images were considered fully developed, and 80% of seeds were considered stages after flowering; 2) to document the mature. However, the germination rates were less than 26% or 28% when full seeds proportion of empty and full seeds to under- harvested at 43 DAF received no or 14 days of CS treatment, respectively. Our study stand the proper stages for harvesting seeds suggests that the low germination rate of fully developed seeds as judged by X-ray images using X-ray images to categorize seeds as showing well-developed embryo and endosperm could result from the presence of empty or full; and 3) to examine the germi- dormancy that was not broken effectively by 14 days of CS coupled with the loss of nation of these seeds with and without CS viability caused by 8 months of dry storage at 5 8C. treatment. Materials and Methods Pulsatilla cernua var. koreana (Y. Yabe approximately mid to late March, the flower ex) Nakai ex T. Mori (http://www.ipni.org/ head dries out, seed heads are developed, and Plant material and seed germination test. ipni/idPlantNameSearch.do?id=711999-1; seeds with filaments are formed; then, seeds P. cernua var. koreana seeds were collected accessed 17 Dec. 2020) is a perennial grow- are easily detached from the seed head, at the nursery located at Koheung-Kun, ing in sandy and nonfertile soil in Korea and expelled by wind, and dispersed. Jeonnam Province, Korea, from a group of northern parts of China. After flowering in Pulsatilla cernua var. koreana seeds ger- plants that reached flowering within a 3- to 5- minate within 14 d at 25 °C; however, ger- d period on 23 Mar. 2018. Seeds harvested on mination rates decrease after 6 to 8 weeks, 8, 15, 20, 25, and 30 Apr. 2018 and on 5 May and seeds do not germinate after 14 weeks of 2018 (16, 23, 28, 33, 38, and 43 DAH, Received for publication 5 Jan. 2021. Accepted for storage under unspecified room conditions respectively) (Fig. 1) were packed in a paper publication 31 Jan. 2021. (Sang et al., 1993). Germination rates of the bag filled with silica gels and placed in plastic Published online 16 March 2021. fresh P. cernua var. koreana seeds have been bags with locking seals; then, they were This research was funded by the Special Fund ° for Beijing Common Construction Project, the reported as 63.5% (Sang et al., 1993) and stored dry at 5 C. Seeds were mailed to World-Class Discipline Construction and Charac- 94.8% (Sang et al., 1996); however, the rates Beijing Forestry University (Beijing, China) teristic Development Guidance Funds for Beijing continued to decrease to 0.3% within 14 on 23 Aug. and stored dry at 5 °C in silica gels Forestry University (2019XKJS0324), and the Bei- weeks (Sang et al., 1993) and 1.0% within until 26 Nov. 2018. jing Landscaping Color Extension Green Science 24 weeks (Sang et al., 1996) of storage under Seeds were stored either dry or after and Technology Innovation Project: Study on the room conditions. Seeds stored in moist ver- mixing with 50% moisture sands at 5 °C for technology of efficient propagation and cultivation miculite did not germinate at all after 24 weeks 14 d in CS. All seeds were sown on open trays and maintenance of garden plants in Beijing of storage (Sang et al., 1996). Low germina- filled with peat:vermiculite (1:1 by volume) (2019-KJC-02-10). tion rates may also result from the inclusion of on 12 Dec. 2018, and watered every other T.Y. is the corresponding author. E-mail: yuantao@ ° bjfu.edu.cn. nonviable seeds, incomplete development of day. Germination was recorded daily at 20 C This is an open access article distributed under the vegetative organs [embryo and endosperm under cool white fluorescent tubes with a CC BY-NC-ND license (https://creativecommons. (empty seed)] (Esau, 1965), and damaged photosynthetic photon flux density of 75 org/licenses/by-nc-nd/4.0/). vegetative organs (Yuan et al., 2019). mmol·m–2·s–1. There were 100 seeds per 506 HORTSCIENCE VOL. 56(4) APRIL 2021 Fig. 1. Development of flower head after flowering on 23 Mar. 2018, with numerous filaments of P. cernua var. koreana.(A) Most flower heads were still bent. (B) The tips of the filaments started to split. (C) Filaments were cut off, leaving 2–3 cm at the proximal end. (D) Flower heads are pointing upward, with some filaments turning white. (E) All filaments of most flower heads became mature and were ready to be separated from the flower head. replication and three replications per harvest SPSS Statistics for Windows (version 20.0 are considered full when harvested on or date, which were all randomized during the software; IBM Corp., 2011). before 28 DAF, when flower heads are bent seed germination test. and pointing down (Table 1). Two sets of 50 randomly selected seeds Results and Discussion When flower heads started to point up- for each harvest date were glued to the card ward and the tip of the filaments became (5 · 7.5 cm) and mailed to the Ornamental Development of inflorescence and visual puffy at 33 DAF (Fig. 2D), 57% of seeds Plant Germplasm Center (Ohio State Univer- observation of seeds. Fifty plants reached were evaluated and considered full. When sity, Columbus, OH) for X-ray scanning on flowering on 23 Mar. 2018, and seeds were harvested on or after 38 DAF, more than 75% 17 Feb. 2019, as described previously (Kim collected from 10 to 15 inflorescences at of seeds were full. This phenomenon (i.e., et al., 2018). After X-ray scanning, seeds each harvest date. On 8 Apr. (16 DAF), flower head pointing upward, which was were mailed to Beijing Forestry University inflorescences pointed downward (pendu- observed between 28 and 33 DAF in our and germinated as described. During the lous) (Fig. 1) and with most of flower stalk study) could result from pollination followed period when seeds were in the mail (7 d when supporting floral organ were still bent; how- by the development of seeds (Huang et al., mailed to Beijing Forestry University and ever, some started to point up 23 DAF 2002). 11 d when they mailed from the Ornamental (Fig. 1A). On 20 Apr. (28 DAF), the flower Full seeds with well-developed vegetative Plant Germplasm Center), temperature and stalk pointing upward straightened upward organs (Esau, 1965; Yuan et al., 2019) had other environment factors could not be mon- and all petals were dehisced from the recep- higher germination rates than empty seeds. itored. tacle; the remaining filaments with a tip Using light microscopy or the naked eye to Classification of seeds based on X-ray started to split (Fig.