200 W. A. VENDRAME AND A. A. KHODDAMZADEH
Classical techniques are generally operationally complex since they
require the use of sophisticated and expensive programmable freezers.
In some cases, slow freezing can be performed with a domestic or
laboratory freezer (Kartha and Engelmann 1994; Engelmann 1997).
In vitrification-based procedures, cell dehydration is performed prior
to freezing by exposure of samples to concentrated cryoprotective media
and/or air desiccation, followed by rapid cooling. As a result, all fac-
tors that affect intracellular ice formation are avoided. Vitrification-
based procedures offer practical advantages in comparison to classi-
cal freezing techniques, such as ultra-rapid freezing, which is more
appropriate for complex organs (shoot tips, embryos) containing a vari-
ety of cell types, each with unique requirements under conditions of
freeze-induced dehydration. By precluding ice formation in the sys-
tem, vitrification-based procedures are operationally less complex than
classical cryopreservation (e.g., they do not require the use of con-
trolled freezers) and have greater potential for broad applicability,
requiring only minor modifications for different cell types (Engelmann
1997).
New cryopreservation techniques are more appropriate for com-
plex organs, such as embryos and shoot apices and can be used
in any tissue culture basic laboratory (Rao 2004). In addition, these
techniques increased the applicability of cryopreservation to a wide
range of plant materials, especially to non-cold hardy tropical plant
species (Hirai and Sakai 2003). Seven vitrification-based procedures
have been developed: (1) encapsulation–dehydration; (2) vitrifica-
tion; (3) encapsulation–vitrification; (4) dehydration; (5) pregrowth;
(6) pregrowth–dehydration; and (7) droplet freezing (Engelmann 2000;
Panis et al. 2005).
Encapsulation–dehydration is a very popular cryopreservation tech-
nique and is based on the technology developed for producing syn-
thetic seeds, that is, the encapsulated explants in calcium alginate beads
(Redenbaugh 1993). Encapsulation explants are then pre-cultured in
liquid medium with a high sucrose concentration and partially des-
iccated before freezing. Encapsulation allows submission of explants
to very drastic treatments, such as pre-culture with high sucrose con-
centrations and desiccation to low moisture contents. Pre-treatment
conditioning before encapsulation is important for sensitive organs,
such as somatic embryos and PLBs, and can enhance the propag-
ule survival during pre-culture and encapsulation (Engelmann 2011).
Encapsulation–dehydration techniques (Fabre and Dereuddre 1990;
Dereuddre et al. 1991) have been developed for the cryopreservation of
a number of species (Engelmann and Takagi 2000; Gonzalez-Arnao and
Engelmann 2006; Sakai and Engelmann 2007; Engelmann et al. 2008;