Tetraclinis articulata (Vahl) Masters is an endangered tree growing in coastal and arid environments that is widely exploited by the timber and resin industry, among other applications. In this context, the use of in vitro techniques is highly encouraged for its propagation. We present a protocol for micropropagation using twigs from adult trees as a source of explants. The Schenk and Hildebrandt basal medium (SH) supplemented with 30 g L−1 sucrose, 6.5 g L−1 plant agar, 4.0 mg L−1 6-benzyladenine (BA), and 0.05 mg L−1 1-naphthaleneacetic acid (NAA) provided the optimum multiplication rate (90.48 ± 9.52 explants with basal shoots and 2.58 ± 0.29 basal shoots per explant). Application of activated charcoal (AC) or ½ Knop solution in a liquid overlay produced significantly longer shoots. Supplementation of solid media with indole-3-butyric acid (IBA) or NAA gave low rooting percentages (<17%). Addition of 0.9 g L−1 AC improved rooting (40%) but rooting performance was optimal (66.7%) after a pulse treatment consisting of 4 h immersion in liquid SH medium without growth regulators, followed by 8 weeks of cultivation. Rooted microplants were successfully acclimatized (93.33%) in a peat moss and vermiculite mixture (1:1 v/v ratio). The genetic stability of the in vitro regenerated plantlets was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Explant survival and growth remained higher than 90% after 28 weeks of cold storage at both 4 °C and 10 °C. The protocol presented here allows for largescale T. articulata production and could be applied for both ex situ conservation strategies and industrial purposes.
The effect of different doses of Cd (0.05, 0.1 and 0.2 mM) and subsequent period in a Cd-free medium on growth, the antioxidant status and the polyamine (PA) pattern was studied using in vitro cultured nodal segments of carnation. The Cd within the tissues increased in parallel with its concentration in the culture medium, inhibited growth, altered the concentration of some minerals and decreased the levels of pigments and the total antioxidants. However, the concentration of ascorbate (Asc) + dehydroascorbate (DHA) and the Asc redox status remained unaffected, and malondialdehyde (MDA) increased only with 0.2 mM Cd. Cd also affected PA metabolism, decreasing the total PA concentration and disturbing the relative predominance of each PA fraction. Cd exposure increased the total putrescine (Put)/(spermidine (Spd) + spermine (Spm)) ratio, and an opposite pattern was recorded during the phase in Cd-free medium. Regarding individual amines, Cd induced significant changes mainly in the free Put levels. Our results suggest that Cd produces oxidative stress and that PA (especially free Put and the total Put/(Spd+Spm) ratio), are good indicators of the stress caused by Cd.
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