This article describes the usage of non-commercial environmental scanning electron microscope (ESEM) for the visualization of plant extracellular matrix in Abies alba and Abies numidica. Non sputter-coated samples free of using the common fixation technique observed at the relatively low humidity of the air environment with the pressure 550 Pa and the low temperature of the sample from -18 to -22 °C give surprisingly very good results that show the natural structure of the tissues. This seems to be generally applicable. Moreover, a specially designed ionization detector of secondary electrons and a YAG:Ce 3+ detector of backscattered electrons were used for better comparison.Additional key words: Abies alba, A. numidica, extracellular matrix. ⎯⎯⎯⎯In the environmental scanning electron microscope (ESEM) the specimens can be observed across a wide range of pressure from the vacuum (comparable with the SEM) to the high pressure of various gases (over a thousand Pa) in the specimen chamber. In high pressure conditions very wet non-conductive samples can be observed free of charging artefacts without a conductive coating covering their surface (Danilatos 1988). If the pressure of the gas is sufficiently increased or the sample temperature reduced, their natural and fully hydrated surface structure is preserved (Stokes 2003, Neděla 2007. Thus, in situ observation could be performed on non-fixed zygotic and somatic embryos. Using ESEM, the surface changes in white spruce somatic and zygotic embryos under various regimes were studied by (Reid et al. 1999). The equipment AQUASEM (Tescan, Brno, Czech Republic) was used by Hřib (2005) for observation of zygotic and somatic embryos of Algerian fir.The early stages leading to somatic cell regeneration into a completely new plant seem to be very important. In several in vitro cultured plants, the SEM analysis revealed that induction of morphogenesis is linked to the appearance of a fibrillar network, referred to as the extracellular matrix (ECM) or extracellular matrix surface network (ECMSN) (Sondahl et al. 1979, Dubois et al. 1992, Šamaj et al. 1995. Bobák et al. (2003/4) suggested that ECM formation can be a stress response of explants, elicited by specific in vitro conditions. Previous authors used the SEM to describe the extracellular matrix on fixed biological material in buffered glutaraldehyde, dehydrated, dried by the CO 2 critical point drying system and sputtered with gold-palladium, etc. PopielarskaKonieczna et al. (2010) showed the ultrastructure of the ECM in Actinidia deliciosa endosperm-derived callus culture using SEM and ESEM.Here we presented detailed ESEM studies of native ECM structure of early conifer embryogenic tissues in specific conditions of observation (gas type, gas humidity and temperature of sample) and using the BSE YAG detector of high energy electrons and a specially designed ionization detector of low energy secondary electrons.The embryogenic tissue of silver fir (Abies alba Mill.) and Algerian fir (Abies numidica De Lann.) was initiated ...
Defence reactions of embryonal suspensor mass (ESM), precotyledonary, cotyledonary and desiccated cotyledonary somatic embryos of Abies numidica were tested by dual cultures with Phaeolus schweinitzii. Defence reactions were expressed at a very early stage of somatic embryo development. Both ESM and early somatic embryos inhibited mycelial growth, but the strongest defence was shown by the precotyledonary somatic embryos. The cotyledonary and desiccated cotyledonary embryos also showed defence reactions but with less intensity. Eight major components of soluble proteins, already present in the ESM, increased in concentration during subsequent developmental stages. Synthesis of two low-molecular components of 6 and 3 kDa appeared in desiccated embryos. Probable regulation of defence reactions by auxins in early somatic embryos, as well as by abscisic acid content and storage proteins in subsequent developmental stages, is discussed.
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