Isolation of gametes from Brachypodium distachyon

Abstract

Isolated gametes from maize, rice, Arabidopsis, wheat and tobacco have been used for investigations for mechanisms in reproductive or developmental processes, such as gamete differentiation, gamete fusion, and early zygotic development. In addition, the isolated gametes from maize and rice can also be applied for in vitro fertilization to analyze postfertilization events. In the last decade, Brachypodium distachyon (Brachypodium) has emerged as an effective model for wheat, since wheat with its hexaploid nature shows some analytical difficulties. In this study, to take advantages of this new model monocot plant for investigations using isolated gametes, procedures for the isolation of Brachypodium gametes were established as the first step. Ovaries were first harvested from mature and unpollinated Brachypodium flowers. Thereafter, a transverse incision was placed at the bottom region of the ovary, resulting in direct access to the embryo sac, and egg cells, which were released from the dissected ovaries, were isolated. For sperm cell isolation, when pollen grains were immersed in mannitol solution, sperm cells were successfully released from pollen grains.