Increased sensitivity to desiccation in germinating embryos of orthodox seeds coincides with an increase in the rate and spectrum of metabolic processes following imbibition (Farrant et al., 1988). The presence of growth regulators such as gibberellic acid (GA3) in the culture medium has been reported to enhance the recovery of creep-reserved axes (Abdelnour-Esquivel et al., 1992). Therefore, the effects of drying rate on survival of embryonic axes deserve attention (Wesley-Smith et al., 2001 a; Waiters et al., 2001; Chapter 3).
The low water content required to avoid freezing damage can be compared to that achieved during slow freezing as described above (e.g., Sakai et al., 1968; reviewed by Mazur, 1990). Rehydration of plant cells in the dry state can occur within a matter of seconds or minutes after immersion in aqueous media (Swift and Buttrose, 1972; Buttrose, 1973; Tiwari et al., 1990). Finally, inner leaves of the resurrection plant, Eragrostis nindensis (Ficalho & Hiem; [Poaceae]) tolerate extreme desiccation (Vander Willigen et al., 2001) and illustrate the third category.
Immersion of leaves in distilled water for 30 min before conventional fixation had a similar effect and very frequently resulted in mesophyll cells showing disrupted cell walls (Fig. 2.3 c; Vander Willigen et al., 2001). A disadvantage associated with freeze-substitution is the likely extraction of lipids in organic solvents (Lancelle et al., 1985; Lancelle and Hepler, 1992; Thomson and Platt, 1997). Second, studies comparing drying rates of embryonic axes of recalcitrant seeds have reported that rapid drying is either beneficial (Waiters et al., 2002), detrimental (Fu et al., 1993), or unrelated to the extent of tolerated water loss (Leprince et al., 1999).
Rehydration artifacts were avoided by processing partially hydrated axes anhydrously for light and electron microscopy using freeze-substitution as described in the previous chapter (Ding et al., 1991; Wesley-Smith, 2001).
1I!l!!.t-iT FAN
Rehydration of slowly dried ashes often resulted in damaged tonoplasts, uneven distribution of ribosomal subunits in the cytoplasm, and local expansion of the nuclear envelope (Fig. 3.16). Other irregularities included clearing of the cytoplasm (asterisks) and local expansion of the nuclear envelope (arrows). The presence of freezeable water in embryonic axes of Landolphia kirkii was not necessarily related to chilling damage when the water content of the axes was less than 0.58 g g-1 (Vertucci 1989a,b; Vertucci et al., 1991).
The calorimetric properties of the water present in the wasps were recorded by differential scanning calorimetry. In this way, it was possible to determine the distribution of water in the main melting passage and in the sharp tip as a function of tissue water content. The initial temperature of the broad endothermic peak was similar for axes with water content greater than 0.6 9 g-1 (Figure 4.4).
There appeared to be no differences in the transition temperature for shafts of similar water content cooled at different rates. Initial temperature of melting of water in tea axes dried at different levels and cooled at different rates. The relationship between water content and enthalpy of melting transition of tea axes dried to the different levels and cooled at three rates.
There was no sharp peak in the melting endotherm of axes that cooled rapidly, regardless of water content. Electron microscopy of freeze-fractured replicas showed the importance of cooling rates on the ultrastructural preservation, as well as the crucial role played by the water content of the axes. Regardless of water content, axes cooled to 10°C min-1 were necrotic unless dried to the lowest water content tolerated, where only 5% survival was recorded.
140°C or supercooled nitrogen (SN2; approx. - 210°C). The horizontal scale bar represents the duration of the cryogen dive. The current aspect of the study examined the potential of using rapid cooling to increase the survival of exposed Aesculus hippocastanum ashes. At a water content of 0.5 g g-1, the differences in the efficiency of the two cryogens were highlighted by the normal development of all ashes cooled in isopentane (Fig.
This uncertainty was addressed in the present aspect of the investigation using embryonic axes of recalcitrant Poncirus trifoliata (L.) seeds. The lowest temperatures reached by fully hydrated axes at the end of immersion ranged between -40 and·.
TOTAL SURVIVAL NORMAL DEVELOPMENT 1.7 9 g-1
Franks, 1980]), large frost transitions were observed at the end of the submergence (Figure 7.3), and evidence of survival suggested that the resulting ice crystals were detrimental. In contrast, the apparent absence of freezing transitions from the temperature profiles of dip-cooled axes between 0.8 and 0.3 g g-1 (Figure 7.3) suggests that their reduced heat load was efficiently dissipated by convective cooling during the dip. Hierarchical log-linear analysis of the effects of cooling (C), warming (W) and water content (H) on survival (S) of the wasp Poncirus trifoliata.
On the other hand, axes cooled within the AI sheet or at 3.3°C S·1 appeared to be more tolerant to slow warming than immersion-cooled axes, with damage predominating in shoots after heating at 67°C S·1 ( Fig. 8.1 e and f, respectively). At the light microscopic level, median longitudinal sections through the radicle of control axes showed well-defined regions of meristem initials (MI), which gave rise to the root cap (RC) distally and in the ground meristem (GM), elements of the vascular system. (VS) and procambium (PC) proximally (Fig. 8.2 a; anatomical terminology after Raven et al., 1992). Radially located around the central mother cells and tunic were the mitotically active initials of pit (PTM) and peripheral meristems (PM; Fig. 8.2 c), which gave rise to the procambium and shoot ground meristem (Fig. 8.2 b) .
Accordingly, the differences in the size of ice crystals in the root and shoot cells between the treatments were assessed separately using one-way ANOVA. While fully hydrated axes, submerged cooled at 97°e S-1, appeared normal in FF replicates at low magnification (Fig. 8.5 a), closer inspection revealed that ice crystals had formed throughout the cytoplasm and most cellular compartments (Fig. 8.5 b). The vacuolar contents (V) often appeared uniform, and it is likely that this represented a solid mass of ice (Fig. 8.5f).
Plot of the frequency of ice crystals formed in cells from the root and shoot in shafts cooled at the indicated rates versus stacks of membrane fragments were occasionally observed attached to the outer leaflet of the nuclear envelope (arrows; Figure 8.6 d), suggesting that ice crystals or organelles pressing against the nucleus may have disrupted the regular fault plane. Ice crystals also formed on either side of the nuclear envelope, which could be clearly resolved in these cells (Fig. 8.7 d and e).
Ice sometimes appeared to penetrate the nuclear envelope, apparently through nuclear pores (arrow; Fig. 8.7 d), while at other times growth occurred within the inner and outer leaflets of the nuclear boundary (arrow; Fig. 8.7 e). Significantly, the matrices of organelles such as plastids (Fig. 8.7 d) and mitochondria (Fig. 8.7 f and g) appeared to be ice-free in approximately 80% of the cells investigated. Large areas of unfrozen cytoplasm were also observed, containing condensed - but apparently intact - ribosomal subunits and organelles (Fig. 8.8f).
