Chapter 3: Results and discussion 59
3.7 Cryoprotection 76
dishes and incubated at 25°C with 16 h light/8 h dark photoperiod) conditions, other species contained TAGs with higher melting temperatures that germinated only when seeds were warmed to above 35°C, following low temperature (-80°C) exposure.
However, the generally low levels of these fatty acids within the embryos of L. kirkii [11% collectively (Table 3.3)] suggest that lipid body crystallisation within the embryos is unlikely. Nevertheless, to investigate possible TAG crystallisation and whether warming at higher temperatures prevented possible subsequent fatal lipid body explosions, fresh and flash-dried embryonic axes of L. kirkii were subjected to rapid cooling and various thawing and rehydration treatments (see section 3.8.1). These results were corroborated by TEM images (see section 3.10).
flash-dried and rehydrated did not stain at all, indicating a lack of any respiratory activity (Figure 3.7).
Table 3.4:
Table 3.4:
Table 3.4:
Table 3.4: The effect of chemical cryoprotection on the viability of fresh and flash- dried embryonic axes of Landolphia kirkii excised with attached cotyledonary segments (n=30)
Cryoprotectant (5% for 1 h followed by 10%
for another 1 h)
Germination following cryoprotection treatment
only (%)
Germination following cryoprotection and dehydration to
c. 0.28±0.044 g g-1 (%)
Sucrose 100 0
Glycerol 100 0
DMSO 100 0
Sucrose + Glycerol 100 0
Sucrose + DMSO 100 0
Glycerol + DMSO 100 0
Sucrose + Glycerol + DMSO
100 0
Figure 3.7:
Figure 3.7:
Figure 3.7:
Figure 3.7: TTZ test results for Landolphia kirkii (a) fresh axes (2.24±0.05 g g-1) (dmb), (b) axes cryoprotected with sucrose and flash-dried to 0.28±0.04 g g-1 (dmb), (c) axes cryoprotected with glycerol and flash-dried to 0.28±0.04 g g-1 (dmb) and (d) axes cryoprotected with DMSO and flash-dried to 0.28±0.04 g g-1 (dmb). (major grid scale in cm, minor gridlines in mm)
Row a: Red to orange staining of embryonic axes and cotyledonary segments (refer to bars for comparison) – fully viable axes;
Rows b, c and d: No reaction with TTZ – axes non-viable
Despite the successful use of chemical cryoprotectants for cryopreservation in studies conducted on embryos/axes of recalcitrant seeds of other species (e.g. de Boucaud et al., 1991; Assy-Bah and Engelmann, 1992; Kioko et al., 1998; Walters et al., 2002b;
Martínez et al., 2003; Sershen et al., 2007), their use was precluded for embryonic axes of L. kirkii, proving lethal to all when cryoprotection was followed by rapid dehydration.
Mycock et al. (1995) suggested that although cryopreservation pretreatments such as dehydration and chemical cryoprotection have proved successful for zygotic embryos, this is not always the case, and optimum pretreatments need to be determined on a species-specific basis. Furthermore, Fuller (2004) conceded that cryoprotectants are chemicals that are not normally encountered by living organisms, and although cryoprotectant concentrations between 5 and 10% have been shown to be tolerable, most
a c d
b
chemical cryoprotectants are toxic to some degree (Mycock et al., 1995; Gui et al., 2004;
Fuller, 2004). Berjak and Pammenter (2004b) also observed that although the use of cryoprotectants has been effective when applied to somatic embryos, they appear highly injurious to excised axes. Therefore, it has been suggested that the major limitation of cryoprotectants is imposed by their phytotoxicity (Bajaj, 1985; Finkle et al., 1985;
Canavate and Lubain, 1994), which may cause irreversible cell structure injury, both before and after exposure to cryogenic temperatures (Farrant et al., 1977; Mycock et al., 1991). Furthermore, chemical cryoprotectants may induce reversible structural alterations to cell organelles including lipid components of the membranes (Costello and Gulik- Krzywicki, 1976) and membrane particles (Kirk and Tosteson, 1973; Farrant et al., 1977;
Stolinski and Breathnach, 1977), or retard explant growth processes (Mycock et al., 1991). The cytotoxicity of chemical cryoprotectants varies with the type and concentration of the cryoprotectant and most importantly, could be species- and even cell-type-specific (Kartha, 1985; Mycock et al., 1991; Fuller, 2004; Benson, 2008;
Walters et al., 2008). Additionally, it has been reported that the cytotoxic effects of certain individual cryoprotectants may be alleviated only by combining them with another cryoprotectant (Bajaj, 1985; Withers, 1985).
Farrant et al. (1977) and Fuller (2004) further proposed that the damage caused to tissue by chemical cryoprotection is a direct function of concentration, time and temperature of exposure, as well as the rate of addition. Numerous studies support this, generally showing that higher concentrations of, and prolonged exposure to chemical cryoprotectants adversely affect viability of various biological tissues (e.g. Nowshari et al., 1995; Gui et al., 2004; Volk et al., 2006b; reviewed by Fahy, 1986; Fuller, 2004). A review on cryoprotectant toxicity by Fahy (1986) also showed that the use of high concentrations of cryoprotectants resulted in more damage that could be accounted for on the basis of the calculated increase in solute concentrations, thus suggesting cryoprotectant toxicity. Studies have also shown that chemical cryoprotectants may cause the disassembly of microtubules and microfilaments that could be reversed only if the cryoprotectant exposure times and temperatures were reduced (Fuller, 2004) (although specific details were not provided by the author). Furthermore Volk et al. (2006b)
showed that when mint shoot tips were treated with glycerol, damage was significantly less when the exposure temperature was 0°C rather than at room temperature (22°C).
This is possibly due to lower temperatures reducing the permeability of cryoprotectants (McGann, 1978). However, Fuller (2004) argued that lowering exposure temperatures, and thus the passive permeation of cryoprotectants, in itself may cause problems as it takes longer to achieve the sufficient concentrations required to facilitate cryoprotection.
Ashwood-Smith (1987) warned that in addition to chemical, osmotic toxicity will also be detected if cryoprotectant exposure is not optimised. When chemical cryoprotectants are added to biological explants, they traverse cellular membranes relatively slowly compared with water. This results in a rapid efflux of water from the cells, with associated volume collapse, and is referred to as osmotic toxicity (Leibo et al., 1978).
With regard to the present investigation, embryonic axes survived exposure to cryoprotectants but viability decreased drastically when explants were flash-dried (Figure 3.8). Prior to culture on germination medium or treatment with TTZ, axes were rehydrated in a CaMg solution and it is therefore possible that the opposite of osmotic toxicity may have occurred. Rehydration of embryonic axes, whilst they were still loaded with cryoprotectants, may have led to rapid over-swelling of the cells (Fuller, 2004). It has been stated that cells can tolerate only moderate repeated changes in cell volume without significant damage (Pegg, 2002) and therefore over-swelling of cells within axes of L. kirkii may have led to their death.
In the present study, four of the seven cryoprotectant treatments tested (alone or in combination), contained the penetrating cryoprotectant DMSO. This cryoprotectant has been shown to be highly phytotoxic (Canavate and Lubain, 1994), although this effect appears to be species-specific with regard to exposure time, as well as being dependent on the size of the explant (Sakai, 2004). Fahy (1986) suggested that the toxicity of cryoprotectants may in fact manifest itself in the form of extra freezing-injury (termed
‘cryoprotectant-associated freezing-injury’) over and beyond the freezing-injury due to classic, well-known causes. Simply stated, cryoprotectants may cause added injury that are not as a result of the cryobiological properties of the cryoprotectant, but rather are due
to direct cytotoxic effects (Fahy, 1986; Fuller, 2004). Fahy (1986) also reported an increase in cryoprotectant-associated freezing-injury with increasing concentrations of DMSO. Furthermore, Kim et al. (2009) showed a decrease in growth recovery when concentrations of DMSO were increased. Dimethyl sulphoxide is also known to bind to monomers of actin thus reducing the extent of the actin filaments within cells (Morriset et al., 1993) and therefore disrupting the cytoskeleton. Kioko (2003), working on embryonic axes of Trichilia dregeana, recorded similar reductions in viability when cryoprotected axes were dehydrated, and suggested that this cytoskeleton disruption may have further reduced the percentage of axes developing into plantlets.
One possible approach to combat the toxicity of cryoprotectant solutions would be to decrease their concentration at application. As mentioned earlier, dehydration of explants will increase the concentration of penetrating cryoprotectants intracellularly. With regard to the present study, although 5 and 10% concentrations of cryoprotectant/cryoprotectant combinations were applied to embryonic axes, the intracellular concentration following flash-drying may in fact have been significantly higher. Therefore, if the concentrations of cryoprotectants/cryoprotectant combinations during application are reduced, after dehydration (which will cause an increase in intracellular cryoprotectant concentration) the final intracellular concentration may not be much higher than if there was initially no dehydration step. Although cryoprotectant concentrations of 5 – 15% are commonly employed for cryopreservation studies (discussed in section 1.7.1.3), it is possible that reducing concentrations to 1 and 2% may result in lower final concentrations after dehydration. As viability was maintained at 100% (when there was no dehydration step) (Table 3.4), it is therefore likely that such intracellular concentrations (5 and 10%) was not damaging to cells in embryonic axes of L. kirkii. Although studies using such low concentrations of cryoprotectant solutions are few, some success has been reported [e.g.
Chlamydomonas reinhardtii (Crutchfield et al., 1999)]. Nevertheless, with respect to the current investigation, testing of different concentrations of cryoprotectant solutions, exposure times and exposure temperatures were not feasible because of the high seed numbers required to carry out adequate drying curves such as those in Figure 3.8 (as discussed above).
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
Figure 3.8:
Figure 3.8:
Figure 3.8:
Figure 3.8: Drying time course and corresponding viability (root plus shoot protrusion) of axes excised with attached cotyledonary segments of Landolphia kirkii treated with cryoprotectants (singly and in combinations). Dashed line indicates drying time course for fresh, non-cryoprotected axes excised with attached cotyledonary segments. Bars indicate one standard deviation about the mean
sucrose
glycerol
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
Figure 3.8 Figure 3.8 Figure 3.8
Figure 3.8 continuedcontinuedcontinuedcontinued…:…:…:…: Drying time course and corresponding viability (root and shoot protrusion) of axes excised with attached cotyledonary segments of Landolphia kirkii treated with cryoprotectants (singly and in combinations).
Dashed line indicates drying time course for fresh, non-cryoprotected axes excised with attached cotyledonary segments. Bars indicate one standard deviation about the mean
DMSO
sucrose + glycerol
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
Figure 3.8 Figure 3.8 Figure 3.8
Figure 3.8 continuedcontinuedcontinuedcontinued…:…:…:…: Drying time course and corresponding viability (root and shoot protrusion) of axes excised with attached cotyledonary segments of Landolphia kirkii treated with cryoprotectants (singly and in combinations).
Dashed line indicates drying time course for fresh, non-cryoprotected axes excised with attached cotyledonary segments. Bars indicate one standard deviation about the mean
Sucrose + DMSO
glycerol + DMSO
0 1 2 3 4 5
0 5 10 15 20 25 30
Time (minutes)
Average water content g g-1
0 20 40 60 80 100
Viability (%)
Average WC Average WC (fresh) Viability
Figure 3.8 Figure 3.8 Figure 3.8
Figure 3.8 continuedcontinuedcontinuedcontinued…:…:…:…: Drying time course and corresponding viability (root and shoot protrusion) of axes excised with attached cotyledonary segments of Landolphia kirkii treated with cryoprotectants (singly and in combinations).
Dashed line indicates drying time course for fresh, non-cryoprotected axes excised with attached cotyledonary segments. Bars indicate one standard deviation about the mean
By exposing embryonic axes to penetrating cryoprotectants, the amount of solutes within the cells will increase (glycerol and DMSO are less volatile than water) and therefore the water content of axes should be lower after exposure to cryoprotectants. Surprisingly however, six of the seven cryoprotectant treatments tested resulted in an increase in the initial water content of embryonic axes of L. kirkii (Figure 3.8). Combinations of sucrose and glycerol, and glycerol and DMSO, showed the highest increases, with water contents of 3.41±0.34 and 3.98±0.28 g g-1 respectively. These values are significantly higher than that of non-cryoprotected axes that were shed at a water content of 2.24±0.04 g g-1. Unusually, only DMSO treatments resulted in a lowered water content of the embryonic axes. Upon flash-drying of axes that had been cryoprotected (singly and in combination), water contents were consistently lower than that of non-cryoprotected axes dried for
Sucrose + Glycerol + DMSO
similar periods (Figure 3.8). Although the general increase in initial water contents after immersion in cryoprotectant solutions compared to non-cryoprotected axes is surprising, reduction in water contents following sucrose cryoprotection of embryonic axes have been reported for other species including Trichilia dregeana (Dumet and Berjak, 1996;
Kioko, 2003), Trichilia emetica (Kioko, 2003) and fifteen species of Amaryllidaceae (Sershen et al., 2007). Tissue dehydration has been achieved through the use of sucrose and other non-penetrating cryoprotectants which are suggested to promote vitrification during freezing (Vanoss et al., 1991; Storey and Storey, 1996; Benson, 2004; 2008;
Fuller, 2004; Volk and Walters, 2006; Day et al., 2008). However, penetrating cryoprotectants such as glycerol do not function by tissue dehydration (Thierry et al., 1997; Benson, 2008). Therefore, the lower water contents recorded for axes cryoprotected with glycerol and flash-dried, when compared to non-cryoprotected axes flash-dried for the same time, were unexpected. Although the reason for this is unknown, Sershen et al. (2007) proposed a possible explanation in that glycerol may change the physical characteristics of the axial tissue which permits more rapid transfer of water from the interior of the tissue to the surface. This water is then lost to the air stream provided by the flash-drier. Furthermore, adding glycerol is like adding dry matter to the explant, which will decrease measured water content. Whatever the direct effects of the cryoprotectants on axis cells of L. kirkii were, the net result was that their application was lethal to all axes if followed by flash-drying and therefore their use was excluded from all subsequent cooling trials.