Isolation of sperm cells from mature pollen grains of Spinacia oleracea L.

Abstract

When mature pollen grains of Spinacia oleracea were squashed in a 25% sucrose solution and subsequently centrifuged on a percoll layer, sperm cells were isolated in high numbers. All steps were carried out at 4 ~ C. Isolated sperm cells could be kept alive for several hours.

Fifty years of sperm cell isolations: from structural to omic studies

“…In this technique, pollen grains are placed on an abraded surface and sperm cells are released by the pressure applied on top of a cover glass or a glass roller. This method has been successfully used in monocots such as Lolium perenne ( van der Maas et al , 1993 ) and dicots such as S. oleracea ( Theunis et al ., 1988 ; Theunis and Van Went, 1989 ) or G. jamesonii ( Southworth and Knox, 1989 ) due to the fact that sperm cells are comparably small and therefore difficult to crush by physical techniques.…”

“…Large amounts of P. zeylanica sperm cells were isolated by sugar gradient ( Russell, 1986 ). Percoll gradients were used for Z. mays ( Dupuis et al , 1987 ) or S. oleracea ( Theunis and Van Went, 1989 ), and are more popular because of their low osmotic potential, biocompatibility, and lack of cytotoxicity. The downside of this method is the substantial amount of starting material needed, limiting its applicability to species with an abundance of pollen.…”

“…Determination of the size, shape, and organellar content of sperm cells was the main goal of many isolation studies, especially the early ones. Sperm cells isolated from monocots such as H. vulgare ( Cass, 1973 ) and Z. mays ( Dupuis et al , 1987 ) or dicots such as S. oleracea ( Theunis and Van Went, 1989 ) and B. oleracea ( Matthys-Rochon et al ., 1987 ) show shape transitions from spindle-shaped to more spherical morphology, whereas others remain elongated such as those of G. jamesonii ( Southworth and Knox, 1989 ) or ellipsoidal such as those of Rhododendron sp. ( Shivanna et al , 1988 ).…”

Current Methods Used to Isolate and Purify Generative and Sperm Cells from Pollen Grains and Tubes of Flowering Plants

“…细胞(vegetative cell, VC)和1个存在于营养细胞内 的、较小的生殖细胞(generative cell, GC)。随后, 营 养细胞不再进行细胞分裂 (Twell et al, 1998;Twell, 2011) 成功应用 (Southworth and Knox, 1989;Theunis and Van Went, 1989;Theunis et al, 1992;Borges et al, 2008) (Zhou, 1988;Taylor et al, 1991;Zhao et al, 2013) 2) 过滤网孔径的选择。花粉粒的直径比GCs 或SCs大, 细胞碎片的直径通常比GCs或SCs小, 选 择合适的滤网可以将它们分开 (Zhou, 1988) Harrison and Heslopha-Harrison, 1970)。FDA染色 法因具有快速、操作便捷、易于观察等优点而被广泛 应用于GCs和SCs的活性检测 (Zhou et al, 1986;莫 永胜和杨弘远, 1991;Borges et al, 2008;Abiko et al, 2013;Anderson et al, 2013)。Evans blue染色也 是一种基于质膜半透性检测细胞活性的方法, 该染料 不能透过活细胞的质膜, 不能使活细胞着色, 但可以 通 过死细 胞的 质膜进 入死 细胞中 , 使 死细胞 着色 (Gaff and Okong'o-Ogola, 1971)。相对于FDA染色 法, Evans blue染色法效果不明显, 不容易区分染料 的蓝色与背景的蓝色 (Huang et al, 1986), 因此仅有 少 数 的 应 用 案 例 (Russell, 1986;Southworth and Knox, 1989 究采用的缓冲液是单纯的蔗糖溶液 (Russell, 1986;Zhou, 1988), 主要是提供维持细胞完整性的适当的 渗透势。有尝试利用含有多种无机盐离子的液体培养 基BKS (Brewbaker and Kwack, 1963)作为分离玉米 SCs的缓冲系统, 并获得成功 (Dupuis et al, 1987…”

Untitled

“…Wagner, Kardolus & Van Went, 1989(7;Wagner, Dumas & Mogensen, 1989f;Van Went & Kwee, 1990;Faure et ai, 1993; reviewed by Theunis, Pierson & Cresti, 1991;Huang & Russell, 1992), sperm cell isolation (e.g. Theunis & Van Went, 1989;Wagner et al, ]989£-;Mogensen, Wagner& Dumas, 1990;Wagner e? a/., 1990;Zhang et ah, 1992, reviewed by Theunis et ai., 1991 and the ability to perform fertilization in vitro (Kranz & Lorz.…”