A method to isolate male gametic nuclei from pear pollen tubes

SummaryThe nucleus plays an essential role in regulating most biological activities in most living organisms. Pear (Pyrus bretshneideri) pollen is binucleate, possessing a vegetative nucleus and a generative nucleus. To reveal the nuclear proteome and to produce accurate physical maps of DNA sequences using fluorescence in situ hybridisation (FISH) analysis, it is essential to obtain high quality nuclei. Protocols to isolate intact nuclei usually involve liberating them from centrifuged cells, then filtering them using a nylon mesh. Releasing the nuclei is the most critical step to obtain high quality material from small amounts of tissue. Here, we describe an easy and efficient method to isolate and purify both the generative and vegetative nuclei from pear pollen tubes. The nuclei were liberated by grinding pollen tubes, filtered on a cell strainer with a pore size of 40 µm, then passing them through a second cell strainer with a mesh size of 25 µm. After these procedures, and purification through a 28%...