Castanea spp. hybrid clones in vitro conservation: synthetic seeds vs. slow growth storage

Chestnut fruit production has declined due to ink disease (Phytophthora cinnamomi). Hybrid clones produced from controlled crosses between Castanea sativa x C. crenata and C. sativa x C. mollissima were micropropagated to test the susceptibility to P. cinnamomi. Since in vitro clonal conservation is relevant for germplasm preservation, in this work, we compared two different techniques: slow growth storage (SGSt) and synseeds. Sucrose and mannitol concentrations (0.16 and 0.22 M) were tested during SGSt over different storage periods (1, 3 and 6 months). Capsules with nodal segments (5-7 mm) were transferred at three different conservations (empty tubes vs tubes filled with sterile distilled water or 30% glycerol solution). When SGSt was tested, the lowest survival after storage was observed with mannitol. After 6 months of conservation at 4 degrees C and darkness, a high survival percentage (93 +/- 1.5%; P<1%) was achieved with 0.22 M sucrose. The best multiplication rate (P<5%) was achieved with 0.16 M sucrose (2.4 +/- 0.15) after 1 month and with 0.22 M sucrose (1.83 +/- 0.14), after 6 months of cold storage. Synseeds conservation was not affected by neither the Na-alginate and CaCl2 concentration used during encapsulation or conservation period. The lowest survival percentage was observed when 30% glycerol was used and the best result (100%; P<1%) was obtained with sterile distilled water. The germination time ranged from 16-24 days (after 1-6 months). After 6 months the best result of germination rate (85.3%; P<5%) was achieved with sterile distilled water; in the second multiplication cycle, the best result was scored with 100 mM CaCl2/sterile distilled water (1.80 +/- 0.25; P<5%). Overall, the best storage treatments for these cultures were achieved with synseeds stored with sterile distilled water and with SGSt with 0.22 M (suc.).