Seeds of Pulsatilla turczaninovii were categorized to full and empty seeds based on observations under a light microscope and on X-ray images. A germination test for full or empty seeds was evaluated as affected by the duration of gibberellin GA3 and the moist 5 °C cold stratification (CS) treatment. The morphological and elemental components of P. turczaninovii and P. cernua var. koreana seeds were analyzed using low-temperature scanning electron microscopy (LT-SEM) and energy dispersive X-ray diffraction analysis. The results showed that 64% of full and semifull P. turczaninovii seeds 10 weeks after harvesting germinated in 17 to 19 days; however, the germination rate, including empty and semiempty seeds, was lower (52.6%). Full seeds with damaged or dried vegetative organs (embryo or endosperm) and semifull seeds with severely damaged vegetative organs were observed, and this result could be related to low germination rates. Germination patterns of seeds stored dry at 5 °C for 44 weeks that showed a sigmoid pattern were increased by immersing seeds in a GA3 solution for 8 hours and treating seeds with 16 or 32 days of CS. More seeds germinated between 12 and 17 days (as compared with 17 to 29 days), especially when they were treated with GA3 and received 32 days of CS. Comparison of germination rates of visually full seeds upon harvest (52.6%) with those that had been stored dry for 44 weeks (26.3% to 29.7%) suggested that the viability of seeds may have decreased. Dormancy could be a factor that decreased germination and can be removed by low temperature and GA3 treatment. LT-SEM revealed a valley-like, sunken streak in empty seeds of P. cernua var. koreana. The nickel content in the trichome and seedcoat of full and empty seeds of both taxa ranged from 2.98 to 4.62 (weight %), as determined on X-ray images. Our study suggested that the low germination rate was due to either the presence of dormancy, damage to either embryo or endosperm, a loss in viability, or the presence of nickel in the seeds.

Pulsatilla cernua var. koreana seeds were harvested at six different dates between 16 days after flowering (DAF) (8 Apr.) and 43 DAF (5 May) in 2018 and categorized into six groups based on X-ray images. Germination tests were performed without and with 2 weeks of moist 5 °C treatment [cold stratification (CS)]. Seeds harvested at 38 DAF (30 Apr.) with well-developed vegetative organs (embryo and endosperm) in seeds categorized as A and B (seed A and seed B, respectively) based on the X-ray images were considered fully developed, and 80% of seeds were considered mature. However, the germination rates were less than 26% or 28% when full seeds harvested at 43 DAF received no or 14 days of CS treatment, respectively. Our study suggests that the low germination rate of fully developed seeds as judged by X-ray images showing well-developed embryo and endosperm could result from the presence of dormancy that was not broken effectively by 14 days of CS coupled with the loss of viability caused by 8 months of dry storage at 5 °C.

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