Protoplasma (1990) 156:149-162 PROTOPLASMA 9 by Springer-Verlag1990 Structural changes during the first divisions of embryos resulting from anther and free microspore culture in Brassica napus M. A. M. Zaki* and H. G. Dickinson Department of Botany, School of Plant Sciences, University of Reading, Whiteknights, Reading Received March 1, 1990 Accepted May 20, 1990 Summary. Ultrastructural and cytochemicalfeatures of embryo de- velopment during anther and free microspore culture in Brassica napus have been followed" from the late uninucleate microspore stage through the first embryonic division. On transfer to culture, the microspore cytoplasm possesses a large vacuole, often containing electron opaque aggregates,and a peripheral nucleus. Mitochondria, endoplasmic reticulum and starch-flee plastids are distributed throughout the cytoplasm.The conditions of culture induce a number of major changes in the cytoplasmicorganisation of the microspore. First, the central vacuole becomes fragmented allowing the nucleus to assumea central position within the cell. Secondly,starch synthesis commencesin the plastidswhich, in turn, are seento occupya domain investing the nucleus. Thirdly, the cell develops a thick fibrillar wall, situated immediately adjacent to the intine of the immature pollen wall. Finally, the microspores develop large cytoplasmic aggregates of globular material. The nature of this substance remains unknown, but it remains present until the young embryos have reached the 30 cell stage. The first division of cultured microspores destined to become embryos is generally symmetrical, in contrast to the asym- metric division seen in normal development in vivo. Consideration is given to the differences observed between embryos developing from anthers and free microspores in culture. Keywords: Anther embryogenesis; Brassica napus; Electron micros- copy; Histochemistry; Microspore embryogenesis;Pollen division. Introduction The production of pollen derived plants from anther culture was first reported by Guha and Maheshwari (1964, 1967) for Datura innoxia, and haploid plants have subsequently been produced in this fashion from over 240 plant species from 85 genera belonging to 38 families (Srivastava and Johri 1988). Embryogenic pol- * Correspondence and reprints: Department of Botany, School of Plant Sciences, University of Reading, Whiteknights, Reading RC6 2AS, U.K. len from different species varies considerably in initial patterns of cell division. In Nicotiana tabacum, embryo- genesis commences via consecutive divisions of the veg- etative cell (Sunderland and Wicks 1969, 1971). This sequence, termed the A pathway, occurs in anthers explanted following the first pollen mitotic division. A second pathway (B) occurs in Nicotiana anthers cul- tured prior to these divisions (Nitsch 1972), and in- volves a modification of pollen mitosis, with two equal cells being formed, rather than the asymmetric division normally characteristic of this stage. The third, so- called C, pathway reported for Datura innoxia (Sunderland and Dunwell 1974, Sunderland et al. 1974) involves a normal asymmetric mitosis within the mi- crospore, but then a fusion of the daughter nuclei to form a single, central nucleus which then commences division to form the embryo. The final pathway re- ported so far (D) has been described in Hyoscyamus niger (Raghavan 1977) and resembles the A pathway until the asymmetric division has taken place, when the generative rather than the vegetative nucleus con- tinues to proliferate. Haploid plants of Brassica were first produced via an- ther culture of Brassica oleracea (Kameya and Hinata 1970). Subsequent studies have resulted in the devel- opment of protocols for the production of haploid plants in Brassica napus (Thomas and Wenzel 1975) and Brassica campestris (Keller et al. 1975). This early work has been supplemented by research aimed at im- proving the numbers of embryos yielded by these cul- tures and progress has been made with a range of spe- cies, including Brassica napus (Keller and Armstrong