Proembryo culture: In vitro development of early globular-stage zygotic embryos from Brassica juncea

Abstract

We have systematically investigated the nutritional requirements for in vitro culture of zygotic proembryos of Brassica juncea. Normal embryo development in vitro was achieved in a new embryo culture medium (ECM) which contains mineral salts, sugars, amino acids, organic acids and coconut water. The culture system is comprised of two agar layers, with the top layer containing a higher osmolarity than the bottom layer. Proembryos were embedded in the top layer in which the osmotic pressure decreased gradually during culture because of the diffusion of osmotically active compounds into the bottom layer. Using such a double-layer culture system and the ECM, proembryos as small as 35 μm (8-36 cells) could be cultured and developed into normal, mature embryos with an efficiency of at least 75%. In contrast to previous . findings, we found that the removal of the suspensor had only a small effect on the development of embryos 55 μm or smaller, but no effect on larger proembryos. We expect this system to be very useful for investigations of the mechanism of plant embryogenesis.