Vol.:(0123456789) 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-020-01828-z ORIGINAL ARTICLE Acetylsalicylic acid improved antioxidative status and cold storage of encapsulated nodal segments of neem (Azadirachta indica A. Juss.) I. M. G. Padilla 1  · G. Barba‑Espín 2  · J. A. Hernández 2  · A. Piqueras 2  · L. Burgos 2 Received: 18 October 2019 / Accepted: 8 April 2020 © Springer Nature B.V. 2020 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 CaCl 2 ·2H 2 O. 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 incu- bating 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. Key message 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. Keywords Chilling injury · Enzymatic activity · Growth retardant · Medium-term storage · Slow growth · Sodium alginate Introduction The neem tree, Azadirachta indica A. Juss., is an important multipurpose tree, the products of which are globally used in agriculture, medicine, cosmetics and animal health care (Kumar et al. 2013). The neem tree is mainly propagated by seeds. However, as many other recalcitrant tropical seeds, neem seeds are chilling-sensitive and rapidly lose viability even if stored at high relative humidity (Kumar and Mishra 2014; Sacandé et al. 2000). This limits progress in the con- servation and evaluation of germplasm, ex-situ collection, material exchange and, overall, tree improvement. Tissue culture techniques have been described to overcome these limitations. Micropropagation from juvenile (Joshi and Thengane 1996; Shahin-Uz-Zaman et al. 2009) and adult material (Arora et al. 2010; Quraishi et al. 2004) has been described. In that context, organogenesis (Chaturvedi et al. 2004; Srivastava et al. 2009) and somatic embryogenesis (Gairi and Rashid 2005; Rout 2005) protocols have been reported from fower and seed derived material. Thus, it is important to develop a methodology to preserve in vitro cul- ture material and collections of this species for laboratory exchange at medium and long term. Encapsulation of veg- etative propagules has become a potentially cost-efective Communicated by Maurizio Lambardi. * I. M. G. Padilla isabelm.gonzalez.padilla@juntadeandalucia.es 1 Área de Genómica y Biotecnología, IFAPA Málaga, Cortijo de la Cruz, 29140 Málaga, Spain 2 Grupo de Biotecnologia de Frutales. Departamento de Mejora Vegetal, CEBAS-CSIC, Campus Universitario de Espinardo, Edifcio Nº 25, 30100 Murcia, Spain