Rossioglossum grande (Lindl.) Garay & G.C. Kenn is a Mexican native orchid threatened to extinguish according to the NOM-059-Semarnat-2010. In vitro asymbiotic germination is a conservation tool widely used for threatened orchids. Generate basic knowledge about in vitro response of R. grande was the object of the work, comparing seed germination in Knudson C medium (KC) versus Dalla Rosa y Laneri medium (DR), modified adding iron chelate and coconut water. We distinguished three different stages of culture: the sowing culture (CM) and two subcultivated culture (C1 and C2). Experimental design was totally aleatory. Anova, Tukey’s test (p≤ 0.05) y Chi-square were used to analyze germination variables averages. After 60 days from sowing (CM), seedling was more developed, and in general it had more percentage of green and vital protocorms using DR medium. After 12 months without subcultivate, we assist at regeneration of protocorms in DR medium in a significant way. In the subcultivation C1 and C2 we test similar responses. We assume that use of iron chelate and coconut water improves the protocorms development and their regeneration of R. grande in in vitro conditions.