Determination of viability of regions of <i>Picea abies</i> embryos (vital staining).

<p>(<b>A</b>) A six week old cluster of ESEs was divided into six parts for subsequent cultivation. (<b>B</b>) Dependence of ESE viability on the cultivation time, which was detected by IA coupled with a fluorescence microscope. Embryos (~1 mg) used for the viability determination were prepared from six regions of the cluster. The cell material was mixed with fluorescein diacetate (FDA) and propidium iodide (PII), and the fluorescence was detected after 5 min by fluorescence microscopy. The percentage of green areas in the compact embryonal group of the embryos (marked by frames) was used to indicate live cells. Insets: upper inset–early somatic embryo (obtained from section 6b) stained with FDA/PI; bottom inset–early somatic embryo (obtained from section 1a) stained with FDA/PI. Average viability changes determined by (<b>C</b>) double FDA/PI staining and (<b>D</b>) esterase activity determination according to cultivation.</p

Schematic representation of the structure of the ESE cluster.

<p>Schematic representation of the structure of the ESE cluster.</p

Comparison of the water volume in ESE clusters by NMR.

<p>Volume of dry substances in ESE clusters over time. Nuclear magnetic resonance was used to calculate the number of ¹H nuclei (colour scale). This number corresponds with the volume of water in the ESE clusters (dry substance in clusters). * The formation of a cavity.</p

<i>Picea abies</i> embryogenic culture.

<p>Growth curve of the embryogenic culture over 42 and 56 days (<b>inset</b>). The average area of a cluster was 46 mm² and its fresh weight was 110 mg at the beginning of the experiment. The values represent an average of 10 clusters.</p

Determination of viability of early <i>Picea abies</i> somatic embryos in eight week old clusters (non-vital staining).

<p>(<b>A</b>) Schematic of the cluster structure of ESEs; (1) view from the top and (2) bottom, (a) inner part of cluster and (b) outer part of cluster. (<b>B</b>) Staining with 2,3,5-triphenyltetrazolium chloride (TTC). (<b>C</b>) Staining with acetocarmine; (a) embryos from the inner cluster, (b) embryos from the outer cluster.</p

The effect of spatial orientation of the clusters by transposition into new culture medium on the growth of the cluster area after 14 days of cultivation.

<p>(<b>A</b>) Six week old ESE clusters of clone 2/32 placed in the normal orientation or upside-down in the culture medium after 14 days of cultivation. The average area of a cluster was 190 mm² at the beginning of the experiment. The values represent the average of 5 clusters. (<b>B</b>) Two week old ESE clusters of clone 2/32 placed in the normal orientation or upside-down after 14 days of cultivation. The average area of a cluster was 22 mm² at the beginning of the experiment. The values represent the average of 5 clusters.</p