Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 26

Jian and Sun (1989) used the controlled rate cooling approach by immersing surface-sterilized kiwifruit stem segments into a precooled (0°C) cryoprotectant solution and lowering the temperature to −10°C at a rate of 1°C/min followed by transfer to liquid nitrogen. After 120 days of storage, the tissue was thawed to 40°C, washed in MS salts and plants were regenerated. Encapsulation–dehydration has been the most frequently employed vitrification-based technique for Actinidia (Susuki et al., 1997; Wu et al., 2000; Bachiri et al., 2001; Zhai et al., 2003). Susuki et al. (1997) used 2-mm-long shoot tips pretreated with 37.8 μM abscisic acid (ABA) for 10 days, or with 100 μM proline for 1 day, and then cultured the explants on media with increasing sucrose concentrations prior to encapsulation in calcium–alginate beads with 0.5 M sucrose. Shoot tips were then dehydrated on medium with 1 M sucrose for 16 h, desiccated over silica gel for 8 h, and immersed in liquid nitrogen. The survival rate was 30% with the ABA pretreatment and 23% with the proline one.

Bachiri et al. (2001) improved the procedure by using shoot tips of different Actinidia species and cultivars. They precultured the alginate beads containing the shoot tips in media with daily increasing sucrose concentrations (0.3, 0.5 and 0.75 M), dehydrated them with silica gel to 20% moisture content and plunged them directly into liquid nitrogen. The range of survival was between 85% and 95%.

Wu et al. (2000) used encapsulated shoot tips of A. deliciosa ‘Tomuri M’ and ‘Tomuri F’ and of A. chinensis precultured in daily increasing sucrose concentrations (0.5, 0.7 and 1.0 M) and dehydrated to 26% moisture content before plunging into liquid nitrogen. Desiccated shoot tips were cryopreserved either by two-step freezing (prefreezing at 0.2°C/min to between −20°C and −40°C followed by immersion in liquid nitrogen) or by direct immersion in liquid nitrogen. The two-step freezing procedure provided better results, with regrowth ranging between 22% and 56%. This protocol was also used by Zhai et al. (2003) who achieved c.45% regrowth using A. deliciosa ‘Tomuri F’ shoot tips. These authors also verified genetic stability using RAPD markers.

Only two protocols related to the application of the vitrification method in Actinidia have been reported (Hakozaki et al., 1996; Xu et al., 2006). Calluses from A. deliciosa ‘Hayward’ seedlings were cultured on medium with 24% or 41% sucrose for 2 days, dehydrated twice (20 min each) in 60% and 100% PVS2 solution (30% glycerol, 15% DMSO, 15% PEG and 13.7% sucrose) and immersed in liquid nitrogen. Frozen tissue was thawed at 37°C and washed in liquid medium with high sucrose content. Surviving calluses were obtained after 15 days (Hakozaki et al., 1996).

Xu et al. (2006) precultured shoot tips (2 mm long) of A. chinensis ‘Ganmi’ in MS basal medium supplemented with 5% DMSO and 5% sucrose for 4 days. The explants were then dehydrated in a solution of PVS (30% glycerol, 15% ethylene glycol, 15% DMSO and 0.4 M sucrose) for 40 min at 0°C and frozen in liquid nitrogen. The regeneration rate achieved after thawing the tissue at 40°C was 51.6%.