**2.4 Droplet-vitrification**

The droplet-vitrification technique is a modification of the basic vitrification protocol that involves placing the sample within a droplet of 1-10 µl of cryoprotective solution on a piece of aluminium foil before immersion in LN (as opposed to 1-2 mL of cryoprotective solution in the original protocol), as shown in Fig. 1.

Fig. 1. Cryopreservation procedure for the droplet vitrification method.

This approach achieves higher cooling and re-warming rates, as the small volume of liquid allows a higher rate of heat transfer to and from the sample (Sakai & Engelmann, 2007). Cooling rates are increased to >130°C/sec (Panis & Lambardi, 2005), therefore facilitating the direct transition of intracellular water from a liquid state to a glassy state far more rapidly, thus minimising water crystallisation. The droplet-vitrification protocol has been successfully applied in the cryopreservation of garlic and chrysanthemum (Kim et al., 2009), yams (Leunufna & Keller, 2005), lily (Chen et al., 2011), potato (Yoon et al., 2006) and other plants (Sakai & Engelmann, 2007).

#### **2.5 Encapsulation-vitrification**

Another modification of the vitrification approach termed encapsulation/vitrification combines elements of the encapsulation/dehydration method with the vitrification method. As with the standard encapsulation method, shoot tips or calluses are first encapsulated in alginate beads and then the encapsulated material is incubated in a vitrification solution to promote sufficient dehydration and vitrification rather than dehydration under a constant airflow, which is very time consuming and relatively imprecise (Hirai & Sakai, 1999). Successful protocols have been established for potato (Hirai & Sakai, 1999), gentian (Tanaka et al., 2004), strawberry (Hirai et al., 1998) and pineapple (Gamez-Pastrana et al., 2004).

structure (such as shoot tips and embryos) that have been successfully cryopreserved with this

The droplet-vitrification technique is a modification of the basic vitrification protocol that involves placing the sample within a droplet of 1-10 µl of cryoprotective solution on a piece of aluminium foil before immersion in LN (as opposed to 1-2 mL of cryoprotective solution

procedure (Takagi et al., 1997; Touchell et al., 1992; Vidal et al., 2005).

Fig. 1. Cryopreservation procedure for the droplet vitrification method.

This approach achieves higher cooling and re-warming rates, as the small volume of liquid allows a higher rate of heat transfer to and from the sample (Sakai & Engelmann, 2007). Cooling rates are increased to >130°C/sec (Panis & Lambardi, 2005), therefore facilitating the direct transition of intracellular water from a liquid state to a glassy state far more rapidly, thus minimising water crystallisation. The droplet-vitrification protocol has been successfully applied in the cryopreservation of garlic and chrysanthemum (Kim et al., 2009), yams (Leunufna & Keller, 2005), lily (Chen et al., 2011), potato (Yoon et al., 2006) and other

Another modification of the vitrification approach termed encapsulation/vitrification combines elements of the encapsulation/dehydration method with the vitrification method. As with the standard encapsulation method, shoot tips or calluses are first encapsulated in alginate beads and then the encapsulated material is incubated in a vitrification solution to promote sufficient dehydration and vitrification rather than dehydration under a constant airflow, which is very time consuming and relatively imprecise (Hirai & Sakai, 1999). Successful protocols have been established for potato (Hirai & Sakai, 1999), gentian (Tanaka et al., 2004), strawberry (Hirai et al., 1998) and pineapple (Gamez-Pastrana et al., 2004).

**2.4 Droplet-vitrification** 

in the original protocol), as shown in Fig. 1.

plants (Sakai & Engelmann, 2007).

**2.5 Encapsulation-vitrification** 
