Morphology and protein pattern during microspore-derived embryogenesis of Brassica napus

Abstract

Developmental pattern of in vitro embryogenesis from microspore of Brassica napus L. cv. Topas was studied by histological analysis and two dimensional gel electrophoresis of protein. Developmental stage of responsive microspore for embryo induction was determined after staining the cells with 4,6-diamidino-2-phenylindole (DAPI). Initial viability of microspores was 63.9% and these different viabilities of microspores resulted in microspore dimorphism in the early stage of culture. The first cell division switching to embryogenic pathway took place to form two identical daughter cells inside the pollen wall followed by suspensor development. Rapid embryo development from globular to torpedo-shape was accompanied with various tissue differentiations. For two dimensional gel electrophoresis, 5 phases of the microspore embryogenesis were recognized as follows; 1) freshly isolated microspores, 2) 3-day cultured cells, 3) globular-/heart-shaped embryos, 4) torpedo-shaped embryos, and 5) mature cotyledonous embryos after 21-day culture. Twenty-three proetins disappeared in the early stage of embryogenesis. In 3-day cultured cells, eight proteins were induced specifically or lastingly. Forth-two proteins in the range of 20∼50 kD were induced rapidly or progressively in the course of the later embryo development.