Application of confocal microscopy to the assessment of pollen development stage in Keteleeria fortunei

Keteleeria fortunei (Murr.) Carr. is a vulnerable endemic species to China. Using laser scanning confocal microscope(LSCM) technique, this study examined the whole development stages of pollen in K. fortunei. Pollen samples were treated with double fluorescence staining with Eosin and Hoechst33342, followed by methyl salicylate clearing. The results reveal that pollen development consists of several distinct stages. Each uninucleate microspore undergoes four times asymmetric mitotic division (the microspore mitosis) to give rise to a mature pollen grain containing two degenerated prothallial cells, a body cell, a stalk cell, and a vegetative cell. The application of confocal microscopy in conjugation with fluorescence staining produces superior images to the traditional acetocarmine staining of pollen and highlights the nuclei at all stages of pollen development. This technique represents an efficient tool for the assessment of nuclear status of pollen grains.