Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb)

Abstract

Summary The isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6–8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

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

“…Additionally, the timing is highly dependent on the temperature and specific components in the germination media (e.g. reduced forms of nitrogen or polyethylene glycol) ( Read et al , 1993 ), or on the influence of female tissues such as in Allium tuberosum ( Lin et al , 2020 ). To overcome this bottleneck, pollen grains are germinated on the stigma of a cut style, allowing the pollen tube to grow under the influence of female tissues and thus triggering generative cell division.…”

Isolation and Transfection of Rice Egg Cells and Zygotes for Cellular Localization

“…Therefore, the great effort with significant progress has been made in the manual isolation of plant gametes, mainly sperm cells, egg cells, and zygotes during the past several decades (Lin et al 2020; Russell 1985; Russell et al 1990; Theunis 1991; Zhang et al 2010).…”