The physiology of Dionysa aretiodes during micropropagation

Abstract

Dionysias are high alpine plants which are restricted in the wild and scarce in cultivation. Fourteen species have been cultured in vitro, but weaning has proved difficult, due to water status difficulties with the plant material. Physiological aspects of this problem have been investigated. Acetate peels from adaxial leaf surfaces showed that control of stomatal density was lost during in vitro culture. Studies of the juxtaposition of glands and stomata showed that, apart from stomata of pot grown (de novo) material, clustering occurred for both structures. Glands appeared to progressively inhibit adjacent stomatal development, from de novo material where no inhibition occurred, to cultures subject to water stress where inhibition was pronounced. Experiments to determine the affect of stomatal density on weaning showed that only plants with very low stomatal densities survived to root. Stomatal apertures, determined by floating leaves in water or ABA solutions, fixing and removing epidermal strips, were found to be similar, independent of treatment. Epidermal strips were removed and stained with sodium cobaltinitrite and ammonium polysulphide solutions to determine potassium accumulation in the guard cells. Low levels of potassium were found for all treatments. Higher accumulations were found in flaccid material. Cation accumulations in leaf material were analysed by atomic emission and atomic absorption for sample solutions prepared by dry combustion. The K:Na ratio was found to drop in vitro cultures,, but could be ameliorated by increasing the pH and/or calcium concentration of the media. No amelioration of stomatal operation was found. Samples of farina from eight species, including in vitro and de novo material, were collected by washing leaves in acetone. Differences in farinal composition, revealed by TLC, were found between species, suggesting the possibility of a chemotaxonomic classification. Some quantitative and qualitative differences were found between in vitro and de novo material. It would appear that some biochemical changes occur during in vitro culture. It is postulated that the morphological adaptions shown by Dionysia aretiodes regulate water loss and account for the apparent lack of stomatal functionality. Physiological differences occur when plant material is cultured in vitro, but no effective treatments to improve weaning were found. The survival and rooting of propagules from culture appears linked to water loss as a result of stomatal density and developmental epidermal patterns.