Efficient protocol for in vitro propagation of Laelia rubescens Lindl. from asymbiotic seed germination

Abstract

Laelia rubescens Lindl. is an orchid species that is currently threatened by fragmentation of its habitat. Compared to other species of the genus Laelia, this species has been less studied, and so far, well-characterized protocols for its successful in vitro establishment and conservation have not been reported. Here we describe a protocol for asymbiotic germination, in vitro development, and acclimatization of L. rubescens. Tretrazolium tests were used to monitor seed viability during 12 weeks after open pollination. A factorial experiment for asymbiotic germination was established using four different capsule ages and four different basal culture media: Knudson C, Phytamax, Vacint and Went, half-strength Murashige and Skoog, and full-strength Murashige and Skoog. Then, the effect of pineapple juice and coconut water, both at 100 ml L−1, on seedling growth was also quantified. Pine bark nuggets, pumice, tezontle, and rubber mulch were evaluated for acclimatization, to determinate their suitability on survival and ex vitro growth of L. rubescens. Temporal-course analyses of seed viability revealed that seeds from fruits nine weeks after hand self-pollination are already suitable for in vitro germination. Phytamax supplemented with peptone (100 g L−1) was the best medium to promote seed asymbiotic germination, which was strongly inhibited by NH4 as inorganic nitrogen source. Pineapple juice (100 ml L−1) had a synergetic effect with peptone to enhance seedling growth. All of the substrates for acclimatization allowed rooting and growth but the higher survival percentages and dry weight were obtained with pumice rock. This protocol can be useful for conservation, establishment of germplasm banks, and for L. rubescens breeding programs .