Rapid cooling 95

Chapter 3: Results and discussion 59

3.9 Cryopreservation trials 94

3.9.1 Rapid cooling 95

Rapid and ultra-rapid cooling is generally considered the most promising method for cryopreservation of recalcitrant embryonic/zygotic axes (Berjak, pers. comm.³). Leprince and Walters-Vertucci (1995) suggested that the partial dehydration of axes can reduce the heat capacity of tissue as well as encouraging the formation of glasses. This partial dehydration of embryonic axes of L. kirkii was achieved by flash-drying only (see section 3.5) as osmotic dehydration, through the use of non-penetrating chemical cryoprotectants, resulted in the death of all the axes (see section 3.7). Although cryoprotection and partial dehydration of recalcitrant embryonic axes has shown to optimise rapid and ultra-rapid cooling rates of larger explants (e.g. Wesley-Smith et al., 1992; 2004a; Kioko et al., 1998; Sershen et al., 2007), the size of explants is a major limiting factor for rapid cooling and avoiding freezing damage (Ryan and Purse, 1985; Wesley-Smith et al., 1992;

Wesley-Smith, 2002; Walters et al., 2008). Ryan and Purse (1985) and Wesley-Smith et al. (1992) suggested that specimens smaller than 100 µm are most amenable to rapid cooling so as to ensure adequate freezing rates throughout the specimen. However, the size of embryonic axes with attached cotyledonary segments of L. kirkii, greatly exceed this recommended size limit. For the current contribution, although rapid (direct immersion in LN) and ultra-rapid (direct immersion in nitrogen slush) cooling showed high root protrusion post-thawing (70 and 100% respectively), no shoot development was observed for either treatment (Table 3.6). The larger size of explants may have contributed to this post-thaw viability loss. In contrast, many rapid cooling techniques such as direct emersion in LN (e.g. Normah et al., 1986; Janeiro et al., 1996; González- Benito, 1998; Kioko, 2003), direct emersion in nitrogen slush (e.g. Wesley-Smith et al., 1992; 2001b; Kioko et al., 1998; Wesley-Smith, 2002; Sershen et al., 2007) and freezing

3 Berjak, P. University of KwaZulu-Natal, Durban, South Africa

in melting isopentane (e.g. Berjak et al., 1999; Walker, 2000; Wesley-Smith et al., 2001b) have been successfully employed for the cryopreservation of recalcitrant embryonic axes. The success of these protocols can be attributed to the cooling rates (Kioko, 2003) as these cooling rates are suggested to move embryonic tissue through the ice nucleation temperatures faster than ice crystal growth can occur, and the intracellular solution is said to form microcrystalline ice or vitrify (Dubochet et al., 1982; James, 1983; Robards and Sleytr, 1985; Wesley-Smith et al., 1992; 2004b; Engelmann, 1993;

Day et al., 2008). Both of these outcomes are considered to be non-injurious to cellular membranes (Kioko, 2003; Benson et al., 2006; Day et al., 2008).

Table 3.6:

Table 3.6:

Table 3.6:

Table 3.6: The effect of different cooling treatments on root protrusion and shoot development from embryonic axes of Landolphia kirkii excised with attached cotyledon segments and subjected to rapid drying to a water content of 0.28±0.04 g g^-1 (n=30)

Treatment Cooling rate (°C min^-1) to -196°C

Root protrusion (%)

Shoot development (%)

Nitrogen slush^† 12000² 100±0^c 0±0^a

LN^† 6000² 70±5^a 0±0^a

Mr Frosty 1 (to -70°C) 80±8.7^b 70±5^c

Mr Frosty 1 (to -70°C) + 6000² 70±5^a 10±8.7^a

+ LN^† Mr Frosty

+ LN

1 (to -70°C) + 14 75±5^ab 10±8.7^a

Cryovials 14 80±0^b 40±10^b

A Kolmogorov-Smirnov test was carried out which showed normal distribution of data. A one-way ANOVA was performed showing significant difference between treatments (p<0.05). Numbers followed by the same letter in the same columns are not significantly different according to the Duncan post-hoc test

2 Wesley-Smith, J. University of KwaZulu-Natal, Durban, South Africa

† Direct immersion of naked embryonic axes

In addition to the cryogen used, the thermal mass of the explant also plays a major role in the cooling rates attained (Meryman, 1956; Bald, 1987; Walters et al., 2008). To achieve cooling rates of 6000 – 12000°C min^-1, as attempted in the present investigation (see Table 3.6), it has been suggested that the total mass of the explant plus water needs to be in the range of 1 – 2 mg (Wesley-Smith, 2002; Walters et al., 2008). At high water contents, smaller explants are required; this ultimately becomes a limiting factor and at high water contents these high cooling rates are not achievable. As discussed previously, the axes of L. kirkii used for this study were excised with approximately 3 mm of each cotyledon attached. Walters et al. (2008) stated that the tissue size affects the thermal mass as well as the surface area to volume ratios, which in turn will affect the cooling rate of the tissue. After dehydration of L. kirkii embryonic axes to the water content chosen to be the most amenable to cryopreservation (i.e. 0.28±0.04 g g^-1), the mass of the single explants were between 3 and 4.5 mg, more than double the recommended mass.

Therefore, due to the mass, in addition to the size of explants employed in the current study, it is unlikely that the extremely high cooling rates necessary to prevent ice crystallisation and to bring about vitrification of partly-dehydrated axes could be attained (Kioko, 2003). Walters et al. (2008) reiterate this, stating that “as the sample dry mass increases, the allowable range of water contents narrows and it becomes physically impossible to cool hydrated tissues fast enough to prevent lethal freezing injury.” Those authors however, differed from Wesley-Smith (2002) with regard to the range of thermal mass required to achieve cooling rates higher than 6000°C min^-1. Wesley-Smith (2002) suggested that if recalcitrant explants have a dry mass of ≤6 mg, a broad range of water contents can be cooled at rates between 6000 and 30000°C min^-1 and, if tissue has a dry mass of <1 mg, cooling rates faster than 60000°C min^-1 are attainable for tissue of a suitable range of water contents.

The lack of survival of embryonic axes of L. kirkii after exposure to rapid and ultra-rapid cooling methods may also be a function of the intracellular water within the tissue.

Although the water content at which these axes were exposed to cryogenic temperatures is quoted as 0.28±0.04 g g^-1, after correction for the high lipid content of the tissue, the water content is closer to 0.39±0.06 g g^-1(see section 3.5). This figure is at the higher

limits of the range of 0.2 – 0.4 g g^-1 suggested by Vertucci and Leopold (1987) to be the amount of non-freezable water within recalcitrant seeds. Therefore, it is very possible that some of the water remaining within the embryonic axes of L .kirkii after flash-drying to 0.39±0.06 g g^-1 (after correction for lipid content) is constituted by the freezable component. Kioko (2003) suggested that even a small proportion of freezable water could provide the basis for lethal ice crystallisation during exposure of axes to rapid cooling protocols, which is dictated by the relatively large thermal mass of the axis/cotyledonary explants.