Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph