In vitro propagation and genetic stability analysis of Evolvulus spp. Biotechnological tools for the exploration of native germplasm with ornamental potential

The goal of the present work is to establish a protocol that allows the in vitro propagation of Evolvulus glomeratus and Evolvulus arizonicus. Nodal segments of both species were disinfected by the standard method (ethanol/NaClO/Tween 80) with and without the addition of an antibiotics–antifungal mixture to the Murashige and Skoog (MS) complete medium, growth regulator free. E. arizonicus did not survive to the in vitro culture starting procedure. To determine the nutritional requirements for E. glomeratus , the nodal segments were cultured on different dilutions of the MS macronutrients, being the complete MS the more adequate basal medium. Two types of explants were isolated from E. glomeratus for their in vitro propagation: nodal segments and leaves. The first ones were cultured on complete MS medium supplemented with increasing benzylaminopurine (BA) concentration from 0 to 4.4 µM, and the second ones onto the same basal medium but supplemented with the following naphthalene acetic acid and BA concentration (micromolars): 0.0, 1.3, 2.6, and 5.3 for naphthalene acetic acid and 0.0, 1.1, 2.2, and 4.4 for benzylaminopurine in all possible combinations. Under the conditions applied from the leaves, no shoot regenerations were detected. On the other hand, it was possible to recover an average close to four shoots per explant when the nodal segments were cultured on a medium supplemented with 2.2 µM benzylaminopurine. For the rooting and rustication step, in vitro and ex vitro (with Growing Mix #2 and Perlite as substrate) strategies were tested. The best result was 91.6% efficiency of acclimated plants obtained with the ex vitro procedure using Perlite. The use of intersimple sequence repeat showed no differences among the tested regenerated plants under the applied experiment conditions. The protocol developed here is the starting point for the application of biotechnological techniques for both the massive propagation and the improvement of E. glomeratus and other related species.