However, it was clear that the effects of these procedures were magnified when all steps of the cryo procedure were applied sequentially. Stages of cotyledon body protrusion are shown from the seed in the center to the right.
Introduction 1
Biodiversity in South Africa 1
Available approaches for plant germplasm conservation 2
Ex situ conservation can be achieved using plant material in various forms: whole plants, seeds, pollen, vegetative propagules and tissue or cell cultures (Engelmann and Engels, 2002; Paunescu, 2009; Reed et al., 2011). Of the variety of ex situ conservation methods developed to date, resource cultivation in botanical gardens, seed storage, and in vitro cultivation are the most commonly used (Kaviani, 2011).
Conventional conservation 3
- The importance of seed storage 4
- Seed storage behaviour and categorisation 4
- The significance of events during seed development in post-harvest
However, it should be noted that seeds from the majority of non-cultivated species have not yet been categorized (Hong and Ellis, 1996; Liu et al., 2008). Seed development is generally divided into three stages (Kermode and Finch-Savage, 2002; . Bewley et al., 2006).
Desiccation Tolerance in Seeds 7
- Mechanisms involved in desiccation tolerance 9
- Recalcitrance in relation to mechanisms conferring desiccation tolerance 11
- Explant type and size 16
- Explant developmental status 17
- Water content and drying rate 18
- Cryoprotection 23
- Cooling 24
Intracellular vitrification is thought to maintain subcellular integrity in the dry state (Illing et al., 2005). Desiccation damage is said to occur sensu stricto at low water content (Walters et al., 2001).
The problem of oxidative stress during cryopreservation 27
- The use of cathodic protection to quench unregulated ROS 28
- Assessing ROS production resulting from the cryopreservation process 29
Recently, Varghese et al. 2011) showed that ROS-mediated damage in slowly dried, recalcitrant seeds of T. Ammocharis coranica (commonly known as the Karoo lily) used for the treatment of mentally ill patients (Koorbanally et al., 2000 ).
Materials and Methods 36
- Seed collection and storage 36
- Embryo excision, cryoprotection and dehydration 37
- Cathodic protection during dehydration 40
- Rehydration and recovery 41
- Cooling 42
- Thawing and assessment of survival 42
- Localisation of hydrogen peroxide (H 2 O 2 ) as a representative of ROS, using
- Data analysis 44
The collected embryos were then separated into batches that were rapidly dehydrated (flash-dried [FD]; Pammenter et al., 2002). Embryos were spread evenly on the grid of a flash dryer (as originally designed by Berjak et al., 1989; see the modified apparatus shown in Figure 2.5) where they were exposed to a flow of silica gel-dried air from below. , created by the CPU fan. Therefore, in this study, an attempt was made to limit the production of ROS accompanying the dehydration stress of amaryllid embryos by providing cathodic protection (Pammenter et al., 1974) during the flash drying process.
These embryos were then plated for further development (five embryos per 65 mm diameter Petri dish) on a full-strength MS-based medium (Murashige and Skoog, 1962) containing 3% sucrose (w/v) and solidified with 8% agar (Agar Bacteriological ; Merck, South Africa). The embryos were subjected to each of the procedural steps of cryopreservation (as described above) and then exposed to cerium chloride (CeCl3), which reacts with H2O2 to produce electron-dense, insoluble precipitates of cerium perhydroxides, Ce(OH)2OOH and Ce(OH)3OOH , which can be detected visually as an indication of the presence of H2O2.
Results and Discussion 45
Response to cryoprotection 45
The reduction in WC after exposure to glycerol was probably due to the combination of glycerol diffusion into cells, which ultimately increased DM (Volk and Walters, 2006), and tissue osmotic dehydration, as discussed below (Sershen, 2006; Sershen et al. ., 2010). Glycerin is a chemical to which the cell membrane is permeable and has been reported to be highly penetrant (Gao et al., 1995). 49 increased cell dry mass, but an increased water flux would have ultimately led to an increase in WC (Anchordoguy et al., 1991; Gao et al., 1995).
The combination of glycerol with DMSO was chosen based on their beneficial effects on other antagonistic seed materials (Sershen, 2006 [glycerol] and Naidoo et al., 2011 [DMSO]) and to test whether they could provide an interactive effect useful in the embryos of One such example is that of ethylene glycol and DMSO when used in combination with glycerol (Volk et al., 2006) and propanediol used with DMSO (Boutron and Kaufmann, 1979; Szurek and Eroglu, 2011).
Response to dehydration 52
- Rapid dehydration of explants not exposed to cryoprotectants 52
- Rapid dehydration coupled with cryoprotection 59
The rapidity of the initial decrease in water content is consistent with the time-related minimization of any effects of imbalanced metabolism and, consequently, a limitation of metabolism-linked desiccation damage (Pammenter et al., 1998; Walters et al., 2001). This loss of viability is thought to be due to purturbation or actual removal of structure associated/bound water in cells, which is known to cause desiccation damage sensu stricto (Berjak and Pammenter, 2001; Walters et al., 2001). Non-orthodox tissues tend to undergo desiccation damage at WCs lower than 0.45 g g-1 (Vertucci and Farrant, 1995), culminating in significant viability losses below 0.25 g g-1 (Pammenter et al., 1993 ).
Based on suggestions that longer, as opposed to shorter, drying times predispose recalcitrant zygotic embryos to higher levels of post-cryo viability loss (Sershen et al., 2012b), it was hypothesized that B. Dehydration has been reported to increase the concentration of cryoprotective means to increase molecules inside the cells (Reed, 1995; Al-Ababneh et al., 2002), thus placing an osmotic tension.
Attempts to improve survival after partial dehydration 64
- Cathodic protection 64
- Vacuum-supplemented flash drying 67
65 Figure 3.8: Comparison of the effect of cathodic flash drying (Cat FD) and conventional flash drying (FD) on the viability of zygotic embryos (n=10) of selected species. Complete vacuum drying was accomplished by applying a gentle vacuum to the explants during flash drying. The results show that the application of vacuum during rapid drying had no significant effect (p>0.05) on the rate of water loss in the embryos of A.
68 Figure 3.10: Comparison of the effects of cathodic flash drying (Cat FD) and vacuum-supplemented flash drying (VFD) with unmodified flash drying (FD) on the water content and viability (G= germination) of the zygotic embryos of A. Therefore, cathodic protection applied in the partial quick-drying of the zygotic embryos used in subsequent cryopreservation studies.
Response to exposure to cryogenic temperatures 68
For these reasons, all results described hereafter refer to embryos rapidly cooled by exposure to nitrogen slurry (hundreds of ˚C min-1; Wesley-Smith et al., 2004b). Although the reduction in explant size allows for faster heat dissipation, promoting relatively faster cooling rates (Wesley-Smith et al., 2004a; b) than in whole embryos, the water content in the range of c. High tissue WCs imply lower intracellular viscosity, which requires ultra-rapid cooling rates to prevent/limit ice crystal damage to cells (Wesley-Smith et al., 2004a).
However, the ability to rapidly cool to high water content is limited by the tissue's ability to dissipate heat quickly enough to prevent severe damage (Wesley-Smith et al., 2004a). Furthermore, vitrification agents can structure the water remaining in the cells so that it is less likely to freeze (Wolfe et al., 2002).
Hydrogen peroxide can be used as a marker for changes in the oxidative status of plant tissues (Oracz et al., 2007), since higher concentrations of non-dismutated H2O2 are particularly damaging intracellularly (Riley, 1994) and can induce cell death (Gechev and Hille, 2005). In addition, its small size enables it to cross cellular membranes and migrate to different compartments, facilitating its signaling functions (Bienert et al., 2006). Consequently, incorporating cathodic protection (by providing cathodic water as a reducing solvent [Berjak et al., 2011b]) into the other cryopreservation procedures known to contribute to ROS accumulation.
These observations should be complemented by further work to compare the amount of H2O2 accumulated in the embryos during recovery after each step with biochemical quantification of this ROS (Oracz et al., 2007) to determine the oxidative status of the cells. . It is suggested that oxidative stress was one of the (probably major) factors contributing to the poor post-cryo survival obtained for the species investigated in this study, particularly for embryos exposed to DMSO prior to further treatment , and gives even greater motivation for studies. on the improvement (especially by cathodic protection) of oxidative stress during cryopreservation of recalcitrant zygotic germplasm (Berjak et al., 2011b).
Overview and recommendations 86
The influence of drying rates and explant size 87
However, at this stage other factors (eg cuticle characteristics) limiting the drying speed of A. This prompted the modification of the rapid drying apparatus by Sisunandar et al. 2010) to improve the dehydration rate of coconut embryos, which increased their cryo-survival, but such positive effects need to be confirmed, especially since the response to dehydration-induced stress appears to be largely species-specific. Reducing the size of explants also deserves further investigation as smaller explants would allow for faster and possibly more homogeneous drying (Muldew et al., 2004; . Hor et al., 2005).
Uneven water distribution has been recorded for recalcitrant seed tissues (e.g. Pammenter et al., 1998; Tompsett and Pritchard, 1998; Wesley-Smith et al., 2001b), with meristems likely to be at higher WCs than other parts of the axes. A good starting point is suggested to characterize the embryo tissue architecture and composition in relation to the rate at which water is lost under the same flash drying conditions, of a number of recalcitrant seeded species as part of efforts to improve the drying rate; and, where possible, choose to use tissues of uniform (ie, meistematic regions) rather than those with varying cell architecture (ie, whole embryos).
The use of cryoprotectants 89
The effect of each cryoprotectant in a mixture must be determined as it may vary with species, cell type, temperature and other solution components (Fahy et al., 1984; Finkle et al., 1985). For example, the effects of glycerol and DMSO are known to differ (Bhandal et al., 1985) and their interactive effects are not fully understood, arguing for future studies on both the individual and interactive effects of cryoprotectants on various outplant types.
Cooling rates 90
91 embryos can successfully be cooled at somewhat higher toilets than those currently used, this may also limit the extent of possible freeze-induced dehydration (Engelmann, 2004). However, it is known that embryos/axes at higher WCs require faster cooling rates to limit ice crystallization and associated freeze damage (Walters et al., 2008), so it is important that the smallest possible explants are used to promote rapid cooling ( Muldew et al., 2004).
Cathodic protection 91
Possible pathways include removal of ROS (Shirahata et al., 1997), including selective quenching of H2O2 (Lee et al. 2006) and/or ascertaining whether endogenous antioxidant activity is enhanced (Hanaoka, 2001). Furthermore, the production of other potent ROS species (such as the hydroxyl radical and singlet oxygen [Sharma et al., 2012) along with their possible quenching should also be investigated.
Conclusions 92
Cathodic amelioration of the adverse effects of oxidative stress associated with procedures required for cryopreservation of embryonic axes of species with recalcitrant seeds. Preliminary observations on the cryopreservation of dried embryonic axes of tea seeds (Camelia sinensis (L.) O. Kuntze) for genetic conservation. Cryopreservation of embryonic axes of Quercus suber and Quercus ilex: dehydration and cooling factors of in vitro culture.
Seeds of the African pepperbark (Warburgia salutaris) can be cryopreserved after rapid dehydration in silica gel. ed.), Cryopreservation of tropical plant germplasm - current research progress and applications. Studies on factors affecting viability after cryopreservation of excised zygotic embryos from recalcitrant seeds of two amaryllid species. Cryopreservation of embryonic axes of a homoiofree (recalcitrant) seed in relation to calorimetric properties of tissue water.
Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and tissue water thermal properties.
