Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-fre e, maltose-containing medium in the presence of ovaries serving as a condit ioning factor. Embryogenesis was followed in microspores isolated from imma ture anthers of freshly cut tillers or from heat- and starvation-treated, e xcised anthers. Three types of microspore were identified on the basis of t heir cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually op posite to the germ pore. This type was identical to the late microspore sta ge in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as typ e 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pock et in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type- 3 microspores. After a few more days, type-3 microspores absorbed their vac uoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as pr eviously assumed, distinct classes of responding and non-responding microsp ores. The first cell division of the embryogenic microspores was always sym metric. Cell tracking also revealed that the original microspore wall opene d opposite to a region in the multicellular microspore which consisted of c ells containing starch grains while the remaining cells were starch grain-f ree. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detect ed at the root pole of the embryo.