Abstract
Spores and pollen of plants were used as flow cytometric materials to efficiently infer genome sizes. Given this advantage, they hold great potential for various flow cytometric applications, particularly as plant genome size standards. To develop such novel standards, we investigated conditions of pretreatment (bead vortex), buffer, and reliable genome sizes of three fern spore collections-Cibotium taiwanense "Kuo4395", Sphaeropteris lepifera "Tang0001", and Alsophila metteniana "Lee s.n.". Additionally, up to 30 year-old spore collections were obtained from herbarium specimens and from samples stored at 4 °C; their spore nuclei were extracted, and the quality and quantity of these nucleus extractions through storage ages were examined. Nuclear extractions with a longer bead vortex duration or lower spore/bead ratio generally resulted in a higher recovered quantity but a lower quality or purity. For each spore standard, the protocol optimization was determined by their performance in bead vortex conditions, and a 1C genome size was further inferred by linear regression (C. taiwanense "Kuo4395" = 5.058 pg; S. lepifera "Tang0001" = 7.117 pg; and A. metteniana "Lee s.n." = 19.379 pg). Spore nucleus quality and quantity are significantly negatively correlated with storage ages. Nuclear extractions of 10-year-old refrigerated spores remained qualified as a genome size standard; however, none of the herbarium spore collections fit such criteria. Our study is the first to develop and apply dried and refrigerated spores for genome size standards. These standards are ready to use, easy to manipulate, and feature long-term storage in comparison with traditionally used standards of fresh leaves.