Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of Encyclia mariae (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21-4.4 mu M 6-benzylaminopurine (BA) in combination with 2.69-10.74 mu M naphthalene acetic (NAA), 2.07-8.29 mu M indole-3-butyric (IBA), or 2.85-11.42 mu M indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 mu M BA and 10.74 mu M NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 mu M IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 mu M BA and 5.37 mu M NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 mu M IAA or 4.14 mu M IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.