An In Vitro Protocol for Propagating Castanea sativa Italian Cultivars

Castanea sativa cv. 'Garrone Rosso' and 'Marrone di Castel del Rio' are two of the most prized varieties in Italy due to their valuable and healthy nuts used for fresh consumption and in the confectionery industry. Despite the growing demand for chestnuts, there are constraints regarding plant propagation that hamper the renewal and new planting of orchards in different areas. Castanea sativa is susceptible to diseases that have caused a reduction in its area of production. For this reason, in vitro culture represents a valuable technique for germplasm preservation and plant multiplication enabling production of a high number of plants for use in breeding programs. Here we present an in vitro micropropagation protocol for Italian Castanea sativa cv. 'Marrone di Castel del Rio' and cv. 'Garrone Rosso' to contribute to the preservation and enhancement of the Italian germplasm. Nodal explants were used as the starting material for in vitro establishment. The cv. 'Marrone di Castel del Rio' showed a high percentage of survival explants (92%) when subjected to long bleach exposure (25 min), in contrast to what was observed for the 'Garrone Rosso' cultivar. Ascorbic acid was found to be the best compound to counteract phenol exudation. The MS3B and DKW media supplied with 0.5 mg/L BAP were effective for in vitro establishment, while the DKW medium (0.1 mg/L BAP and 0.05 mg/L IBA) was preferable for the proliferation phase. A double-layer rooting methodology was used and 35% rooting was observed with 25 mg/L IBA rooting treatment.