Desiccation tolerance of somatic embryoids = [Uitdroogtolerantie van somatische embryoiden]

This thesis describes the research performed on the subject "Desiccation tolerance in somatic embryoids". Somatic embryoids are bipolar structures formed in tissue culture, with both a shoot and a root apex, which resemble very much zygotic embryos found in seeds. Through simultaneous development of root and shoot, these embryoids can grow out into complete plantlets.In Chapter 2 we describe an optimized method to produce completely desiccation tolerant carrot ( <em>Daucus carota</em> ) embryoids. Using this method at least four different factors are important: developmental stage, abscisic acid (ABA) concentration, drying rate and rehydration mode. Embryoids may become desiccation tolerant when they have reached the torpedo stage of development. In contrast, at the earlier globular and heart stages, embryoids; never germinated after any drying treatment. Addition of at least 20 μM ABA to the pro-embryogenic masses after 7 days of culture in 2,4-dichlorophenoxyacetic acid-free B _{<font size="-2">5</font>} medium was necessary to induce complete desiccation tolerance. Less ABA resulted in desiccation tolerance of the roots only, whereas high ABA (>80 μM) concentrations retarded developmental growth. Slow drying is essential for the acquisition of complete desiccation tolerance. Slowly dried embryoids (moisture content 0.05 g H _{<font size="-2">2</font>} O.g <sup><font size="-2">1</font></SUP>dry weight) germinated for 100%. However, rapidly dried ones germinated for only 0-30%. Initially viable dry embryoids may suffer injury when they are imbibed in water without prehydration in water vapour. Hundred percent germination was reached by prehydration of the embryoids in moist air for 4 to 8 hours at 24°C before imbibition in B _{<font size="-2">5</font>} medium. With the optimized protocol we were able to producedesiccation tolerant embryoids in two genotypes having completely different genetic backgrounds.With this manipulable protocol at hand, we have assessed damage associated with desiccation (Chapter 3). Fast drying caused loss of viability, and all K <sup><font size="-2">+</font></SUP>and soluble carbohydrates leached from the embryoids within 5 min of imbibition. The phospholipid content decreased by about 20% and the free fatty acid content increased, which was not observed after slow drying. However, the extent of acyl chain unsaturation of the phospholipids was unaltered, irrespective of the drying rate. These results indicate that during rapid drying irreversible changes occur in the membranes which are associated with extensive leakage and loss of germinability. The status of membranes after 2 h of imbibition was analyzed in a freeze-fraction study and by Fourier transform infrared spectroscopy (FTIR). Rapidly dried embryoids had clusters of intramembraneous particles in their plasma membranes and the transition temperature (T _{<font size="-2">m</font>} ) of isolated membranes was above room temperature. Membrane proteins were irreversibly aggregated in an extended β-sheet conformation and had a reduced proportion of α-helical structures. In contrast, the slowly dried embryoids had irregularly distributed, but nonclustered, intramembraneous particles, T _{<font size="-2">m</font>} was below room temperature and the membrane proteins were not aggregated in a β-sheet conformation. We suggest that desiccation sensitivity of rapidly dried carrot embryoids is indirectly caused by an irreversible phase separation in the membranes due to deesterification of phospholipids and accumulation of free fatty acids.In Chapter 4 and 5 we have studied the role of the endogenous soluble carbohydrates during acquisition of desiccation tolerance. For carrot embryoids we demonstrated an apparent minimum requirement of umbelliferose plus sucrose for surviving severe dehydration, suggesting that these sugars play an important role in anhydrobiosis. We show with FT-IR spectroscopy, that both sucrose and umbelliferose depress the transition temperature (T _{<font size="-2">m</font>} ) of dry liposomal membranes, which is evidence for their interaction with the phospholipid polar headgroups. Furthermore, both sugars prevent leakage from dry liposomes during drying and subsequent rehydration. We interpret this in the sense that both sugars are able to form a stable glass in the dry state. Fructose and glucose were lacking in dry viable embryoids. In the light of the plasticizing effect of these monosaccharides on sugar glasses, a stable glassy state seems important during anhydrobiosis of carrot somatic embryoids. We show that umbelliferose can protect a protein that is desiccation sensitive.To characterize desiccation tolerance we studied not only membranes and carbohydrate metabolism, but also the role of repiration (Chapter 6) and DNA replication (Chapter 7). Addition of ABA to developing carrot embryoids affected respiration and carbohydrate metabolism. Non- treated embryoids had a high level of respiration expressed per gram protein and consumed almost completely their endogenous carbohydrates during the ten day culture period. In contrast, embryoids grown with either 1.9 or 38 μM ABA, had a reduced respiration rate and maintained their carbohydrate contents at 20% of the DW. Embryoids acquired complete desiccation tolerance, when they were treated with 38 /AM ABA, whereas only 65% of the embryoids survived desiccation with 1.9 μM ABA. The reduced respiration of the developing embryoids might result in reduced free radical levels after dehydration, in this way preventing a subsequent viability loss. We suggest that there is a relation between viability loss due to desiccation and respiration rate, although the latter is not the only limiting factor involved.Employing flow cytometry we investigated the effect of ABA addition and slow drying on DNA replication and cell cycle activity of developing carrot embryoids. DNA replication was determined as percentage of 4C nuclei. Addition of ABA did not alter DNA replication and cell cycle during embryoid development in vitro, in spite of the putative quiescent state of the torpedo- shaped embryoids. In contrast, during slow drying the nuclei were preferentially arrested in the presynthetic G _{<font size="-2">0</font>} /G _{<font size="-2">1</font>} -phase, and the amount of G _{<font size="-2">2</font>} nuclei decreased. Dry zygotic carrot embryos that are completely desiccation tolerant, did not contain any G _{<font size="-2">2</font>} nuclei. The decline of G _{<font size="-2">2</font>} nuclei in dry somatic embryoids seems to coincide with the increase in desiccation tolerance, which is incomplete compared to zygotic embryos. Our results suggest that in order to withstand anhydrobiosis, DNA replication may be controlled by the embryoid developmental program and by slow dehydration, but not by the plant growth regulator ABA.Finally, we performed scanning electron microscopy studies to establish the possible changes during desiccation (Chapter 8). Cryofixation and analysis by low temperature scanning electron microscopy (LTSEM) are excellently suitable to compare the morphology of specimens having different moisture contents. Using LTSEM we examined dry and hydrated carrot zygotic embryos and compared them with fresh, rapidly dried, and slowly dried carrot somatic embryoids, also after rehydration. Rapidly dried somatic embryoids were not able to germinate, whereas approximately 100% of slowly dried embryoids germinated. Somatic embryoids always had reduced and abnormal cotyledons, mostly fused, and the surface was irregular. The surface of the dry somatic embryoids was also more wrinkled than that of zygotic embryos. No morphological differences were detected between tolerant (slowly dried) and intolerant (rapidly dried) somatic embryoids before and shortly after rehydration. However, clear morphological differences were detected after imbibition for three days. Tolerant embryoids showed clear cell expansion, whereas intolerant ones did not. It is concluded that LTSEM is a very powerful technique to study plant materials in their native state.