In vitro propagation of rare and endangered serpentine fern species

Abstract

The carried-out studies concerned two serpentine fern species: Asplenium adulterinum and A. cuneifolium, as well as ferns not connected but also occurring on serpentine rocks, such as: A. septentrionale and Polypodium vulgare. The growth and development of gametophytes of all fern species were affected by Murashige and Skoog (MS) medium composition [Murashige and Skoog (Physiol Plant 15:473-497, 1962)]. Diluted MS medium to one half, and modified MS medium provided the best results in terms of fresh weight increase. The gametophytes growing in these two media (1/2 MS and MS own modification) had the correct shape and formed the most numerous gametophytes in the first and following rows. Measurements of the fresh weight of gametophytes of A. adulterinum and P. vulgare revealed that growth enhanced with sucrose concentrations between 20 and 30 g L-1. Lower sucrose concentration (5-10 g L-1), decreased the fresh weight of gametophytes and diminished their size. Higher sucrose concentration (40 g L-1) reduced gametophyte growth, though they did not show any discoloration or decay. A. septentrionale tolerated all the sucrose concentrations; it was only without sucrose in the medium that the fresh weight of gametophytes was smaller, as was also their number. The influence of several sugars, such as glucose, fructose, mannose, sucrose, maltose, and lactose (in the same concentration of 0.087 M) on growth and development of gametophytes was studied. The results of investigations showed that each of the fern species studied attained the best increase in the fresh weight, and produced the largest gametophytes in a medium supplemented with sucrose. Application of glucose provided similar results; with the exception of A. adulterinum, which preferred maltose in the medium, and A. cuneifolium, which grew equally well on medium with glucose and mannose. The remaining sugars, and particularly fructose, inhibited the increase of protallial fresh weight or caused its decay. Moreover, fructose turned out to be the most toxic: gametophytes of all studied fern species decayed when fructose was used in the medium. The studies show that in vitro cultures are an effective method of creating prothallia gene bank for serpentine fern species. The best medium for long-term storage of prothallia of A. adulterinum and A. cuneifolium was 1/2 MS without phytohormones, at low temperature of +8°C, under cool white fluorescent lamp of 0.15 μmol·m-2·s-1. The number of sporophytes obtained in gametophyte cultures of all the studied species was low. From thousands of gametophytes used, only three to five sporophytes developed. The propagation of sporophytes was tested on media containing kinetin (KIN) and 1-naphtalene acetic acid (NAA). Low doses of these hormones stimulated development of fronds, and higher doses of KIN decreased their size but increased the number of initiated buds. The process of regeneration of new sporophytes was also possible. Rooting of ferns was performed in perlite with a liquid medium, and the influence of diverse soils on the fern survival was evaluated. Plants acclimated well outside in a garden cold frame. The sporophytes reached maturity and produced spores in their second or third year of cultivation.