Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 182

Advances in breeding and genetic engineering require that the genetic diversity of pineapples and their close relatives should be conserved and used for pineapple improvement. While in vitro techniques offer the opportunity for rapid propagation, they also offer the convenience of medium- and long-term storage of germplasm and facilitate its safe distribution.

Low temperatures (16–20°C) have been used to extend subculture times for up to 4 years (Sugimoto et al., 1991). Zee and Munekata (1992) observed that reducing the nutrient salts of MS medium to one-quarter was successful for medium-term (12 months), low-input maintenance of micropropagated pineapple cultures.

Cryopreservation of pineapple and other tropical species is enhanced if endogenous cryoprotectants (such as sugars) are increased before freezing because they increase the stability of the membranes, promote intracellular dehydration by increasing the osmotic pressure, thus reducing injuries that may cause cell death after freezing (Gámez-Pastrana et al., 2004; Martinez-Montero et al., 2005).

Excised shoot tips (0.5–1 mm with several primordial leaves) from micropropagated plants were precultured on MS medium with 0.3 M sucrose for 48 h before undergoing vitrification (González-Arnao et al., 1998; Martinez-Montero et al., 2005; Souza et al., 2016). González-Arnao et al. (1998) used a plant vitrification solution described as PVS2 which contained 30% glycerol, 15% ethylene glycol and 15% dimethyl sulfoxide (DMSO) in 0.4 M sucrose at 0°C for 7 h before immersion in liquid nitrogen. Martinez-Montero et al. (2005) later modified the vitrification solution by omitting DMSO and using 50% sucrose and 50% glycerol and achieved reasonable survival (25–45%). Gámez-Pastrana et al. (2004) introduced an encapsulation method in which pineapple apices were embedded in 3% calcium alginate beads before preculture and vitrification as above, and 54% and 84% of apices from ‘MD-2’ and ‘Puerto Rico’ survived, respectively.

Souza et al. (2016), on the other hand, used a droplet-vitrification cryopreservation technique which is showing wide application with many plant species. In this method aluminium foil strips with shoot tips suspended in PVS2 droplets were used, while shortening the interval in the cryoprotectant to 45 min, before immersion in liquid nitrogen. This gave the best survival (44–86%) of the majority of genotypes tested while causing less injury to the tissues.