The genus Pulsatilla includes 26 species native to North America, Europe, and Asia (USDA, Agricultural Research Service, National Plant Germplasm System, 2018). They are early flowering perennials ideal for border planting in gardens and thrive under conditions of well-drained soil and full sun. Pulsatilla turczaninovii Krylov and Serg. grows widely in Nei Mongol, China, and West Siberia, Russia (Wang and Bartholomew, 2001), while Pulsatilla cernua var. koreana (Yabe ex Nakai) Y. N. Lee grows in northeastern China and Korea.
Pulsatilla cernua var. koreana seeds germinate in 14 d at 25 °C; however, germination rates decrease after 6 to 8 weeks, and seeds do not germinate at all after 14 weeks of storage under unspecified room conditions (Sang et al., 1993). When stored dry in silica gel at 0 or 10 °C, however, the seeds have longevity of up to 24 weeks, with germination rates >66.3% (Sang et al., 1996). If good seeds are stored in moist vermiculite at room temperature under conditions of natural humidity, germination rates can dropped to 5.3% (Sang et al., 1996). However, no descriptions on the criteria of good seeds were provided.
Low germination rates might reflect improper storage conditions and for long storage duration of seeds, resulting in low viability (Sang et al., 1993, 1996) or poor seed quality, which cannot be identified visually (Baskin et al., 2006). The viability of Swertia chirayita (Roxb. Ex. Fleming) H. Karst seeds declined as dry storage duration increased over 24 months, even at 4 °C (Pradhan and Badola, 2012). In Pulsatilla, low germination rates may also result from the inclusion of both nonviable seeds, lacking a developed embryo (EM) or endosperm (EN) (vegetative organs) (Esau, 1965). Due to trichomes developed on the seedcoat, soaking Pulsatilla seeds in water to separate full, viable seeds from empty, nonviable seeds, as demonstrated with Corylopsis coreana Uyeki seeds (Kim et al., 2017), could not be performed.
Germination of viable seeds may be delayed or failed to germinate even at favorable temperatures due to dormancy (Baskin and Baskin, 2004). Seed dormancy could be a factor in low germination rates if dormancy is not released by low temperature or plant growth regulator treatments, especially those including gibberellin (Cadman et al., 2006). Germination of P. cernua seeds can increase to >92% if they are treated with kinetin, gibberellin GA3, and 2, 4-D for 24 h compared with 18% in untreated controls (Gu et al., 2014). The germination of fresh P. turczaninovii seeds is significantly improved by treatment with 100 mg·L−1 gibberellin GA3 (64.0% in 32 d vs. 15.0% by the control in 60 d) (Shi et al., 2005). Li and Piao (2010) also reported that soaking seeds in 100 mg·L−1 GA3 for 12 h increased germination. Germination of P. turczaninovii also increased compared with the control when treated with 100 mg·L−1 GA at 25 to 30 °C (Wang et al., 2013). However, in those studies, neither the age of the seeds nor their storage conditions were indicated.
Visual observations along with SEM to examine the surface of seeds may be useful to distinguish full from empty seeds. Therefore, X-ray scanning technology can be applied to evaluate images of seeds on a large scale, to assess seed development, and to improve seed lot quality and germination (Carvalho et al., 2010). X-ray diffraction techniques could be used to detect various elements in vivo, such as magnesium, silicon, and nickel (Bolton et al., 2014; Roh et al., 2012). Another factor influencing low seed germination rates could be the occurrence of nickel, as this factor has been shown to inhibit the germination of radish (Raphnus sativus cv. Early Menu) seeds (Yadav et al., 2009).
The objectives of this research were to 1) categorize Pulsatilla turczaninovii seeds as full or empty using X-ray imaging and to study the germination of these seeds; 2) investigate the germination of dry stored seeds at 5 °C as influenced by gibberellin GA3 and durations of CS; 3) compare the morphologies of full and empty seeds of P. turczaninovii and P. cernua var. koreana using low-temperature SEM (LT-SEM) and visual observation under a light microscope; and 4) analyze the elemental components of P. turczaninovii and P. cernua var. koreana seeds using energy dispersive X-ray diffraction analysis.
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