Histological comparison between wheat embryos developing in vitro from isolated zygotes and those developing in vivo.

There is currently great interest shown in understanding the process of embryogenesis and, due to the relative inaccessibility of these structures in planta, extended studies are carried out in various in vitro systems. The culture of isolated zygotes in particular provides an excellent platform to study the process of in planta embryogenesis. However, very few comparisons have been made between zygotic embryos grown entirely in cultures and those grown in vivo. The present study analyses the differences and similarities between the in vitro and in vivo development of wheat zygotic embryos at the level of morphology and histology. The study was possible thanks to an efficient culture system and an appropriate method of preparing isolated wheat zygotes for microscopy. The in vitro embryos were fixed, embedded and sectioned in the two-celled, globular, club-shaped and fully differentiated stages. Embryos developing in vitro closely followed the morphology of their in planta counterparts and their cell types and tissues were also similar, demonstrating the applicability of the present culture system for studying the process of zygotic embryogenesis. However, some important differences were also detected in the case of in vitro development: the disturbance of or lack of initial polarity led to changes in the division symmetry of the zygotes and subsequently to the formation of uniform cells in the globular structures. Presumably, differences between the in vitro and in planta environments resulted in a lower level of differentiation and maturation in in vitro embryos and in abundant starch and protein accumulation in the scutellum.

HvPG1 and ECA1: two genes activated transcriptionally in the transition of barley microspores from the gametophytic to the embryogenic pathway.

Microspores genetically programmed to produce male gametes can be switched to the embryogenic pathway to give rise to haploid embryos. Microspore embryogenesis is usually induced in barley by stress pre-treatment applied to vacuolated microspores. We studied the expression of two genes during the early stages of microspore embryogenesis to gain further insight into the microspore transition from the gametophytic to the embryogenic pathway. RT-PCR together with in situ hybridization on sections (ISH) and whole-mount in situ hybridization (WISH) were used to analyse the expression of the early-culture abundant gene (ECA1), which is expressed in barley during microspore embryogenesis, and a polygalacturonase gene (HvPG1), a late pollen gene expressed during gametogenesis only after microspore division. Both ECA1 and HvPG1 genes were transcriptionally active after stress pre-treatment in the same populations of microspore-derived structures, representing the sporophytically induced ones. ECA1 transcripts were also detected after 3 days' culture. Our results point to the possibility of using ECA1 gene expression as a marker for the induction of microspore embryogenesis and the earliest stages of this process. Finally, we demonstrate that WISH is a suitable technique for studying gene expression in embryogenic microspore populations and, because different structures can be examined individually, is an appropriate complement to transcriptomic profile analyses in the study of early microspore embryogenesis.

Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development.

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.