Determination of a protocol for the in vitro germination of Tillandsia usneoides

The excessive collection of Tillandsia ustenoides (heno, paxtle or Spanish moss) from the wild and the loss of its natural habitat are putting wild populations at risk. Hence, we tried to establish some techniques for the legal production or conservation of viable germplasm. Therefor in vitro seed germination was evaluated. In a prelaminar test contamination was observed in 100% of the explants, so different protocols to overcome contamination were evaluated. In a first bioassay five treatments: 0.5 and 4 mL L^‑1 of Plant Preservative Mixture^® (PPM); 250 mg L¹ and 4 g L^‑1 of chitosan and a control treatment were tested, all of them in combination with 15 g L^‑1 of sugar, MS (Murashige and Skoog) at 25%, coal 2 g L¹ and agar 5.5 g L^‑1. Based on the results of this experiment, a second bioassay was setup in a completely randomized factorial design with factor 1: 250 mg L^‑1 chitosan and 2.5 mL L^‑1 of PPM, and factor 2: four disinfection techniques. The best treatment, without any contamination, was the medium in which 250 mg L^‑1 chitosan was added, in combination with washing steps consisted of soaking with 70% ethanol (3 min) and 5% commercial chlorine (40 min). With this protocol, the highest amount of Tillandsia seeds germinated. The protocol can be used for future experiments where we will focus on the multiplication and conservation of the species.