Influence of Ca2+ and 6-benzyladenine on chestnut (Castanea sativa Mill.) in vitro shoot-tip necrosis

Shoot apex necrosis affects in vitro chestnut cultures particularly during the rooting stage. Lack of cytokinins or Ca2+ deficiency in the culture medium have been reported to be responsible for occurrence of tip necrosis. In the present research the effects of three Ca2+ levels (3, 9 and 18 mmol l-1) were tested on C. sativa 'Garrone rosso' and 'Clone 46' shoots treated for rooting. Tissue Ca2+ content of the apical, middle and basal portion of both brown and healthy shoots was determined. In a second trial the tip necrosis related effect of the local (tip) application of CaCl2 (3 mmol l-1), BA (5 μmol l-1) and CaCl2 + BA (3 mmol l-1 and 5 μmol l-1) was tested on Clone 46 shoots, during rooting. With regard to Ca2+ concentration in the rooting medium, no significant difference could be detected from the three tested Ca2+ levels on tip necrosis but the highest one caused a drop in rooting ability. Regardless of Ca2+ concentration, Garrone rosso showed a lower percentage of the disorder and a higher affinity for calcium in terms both of uptake and ion translocation. The data of the second trial showed that the local application of 5 μmol l-1 BA completely eliminated tip necrosis while the application of CaCl2 + BA delayed the appearance of the disorder. Apex calcium content of shoots treated locally revealed that the most the healthy tissue contained higher calcium levels than the necrotic one. BA treated shoots contained the lowest ion levels, independently from their status.