Acetylsalicylic acid improved antioxidative status and cold storage of encapsulated nodal segments of neem (Azadirachta indica A. Juss.)

Abstract

The neem tree, Azadirachta indica A. Juss., is an important multipurpose tree globally used in agriculture with recalcitrant and chilling-sensitive seeds. Here, we studied the encapsulation in sodium alginate of in vitro-derived nodal segments of neem as a method to preserve in vitro culture material of this species for laboratory exchange and for the medium-term storage. A good gel complexation was achieved using 3% sodium alginate and 70 mM CaCl2·2H2O. An average bead conversion of 75%, 0.5 cm of shoot length and 2 leaves were recorded after 4 weeks of culture, irrespective of the polymerisation duration (5 to 20 min). The growth of nodal segments was completely inhibited when they were kept at 4 °C or at 8 °C for 4 weeks, while those encapsulated and stored at 12 °C for 4 weeks, sprung up only 20% under optimal conditions, 23 °C. However, bud sprouting of encapsulated nodal segments stored at 12 °C for 4 weeks increased to 75% under optimal conditions by incubating the nodal segments with 25 µM acetylsalicylic acid (ASA) for 4 weeks prior to encapsulation. We observed that this ASA pre-treatment stimulated the antioxidative defenses of the explant, especially ascorbate peroxidase, catalase, peroxidase, dehydroascorbate reductase and glutathione reductase enzymes that could have improved the viability of the buds stored at 12 °C. Additionally, ASA seemed to protect the cell membranes, as observed by the lipid peroxidation results. All these data together led us to suggest the use of ASA in short- and medium-term storage of in vitro-derived nodal segments of neem. Neem nodal segments, encapsulated in alginate beads and stored at 12 °C recovered their viability when were pre-treated with acetylsalicylic acid, which improved buds’ antioxidant defences.