Influence of agar on in vitro cultures: I. Physicochemical properties of agar and agar gelled media

Summary The success of in vitro culture is related to several factors. Beside factors associated with the plant material or the medium composition, the physicochemical characteristics of gelled media can play an important role. In this paper, the latter aspect has been considered and the nature of agar powders has been investigated. Moreover, the process of gel formation for three different media and the availability of water and minerals for the corresponding gels have been studied. Analysis of agar powders showed that they can contain different amounts of impurities and the dialysis of these powders suggested that the impurities might be available to the tissues. Thermal analysis on the hygroscopic properties of the agar brands suggest the importance of these data to obtain comparable and reproducible gelled media. The study on the process of formation of gelled media indicates that there is a critical temperature T_ss which can be used to control the gel processing. In fact, at this temperature, agar powders in water transform into a sol status through a rapid shift of electrical conductivity. Water potential of the medium, water loss from gels over the culture period, and the ease of releasing liquid from gels under pressure were shown to be different for different agar brands. A different availability of water and minerals in Murashige and Skoog medium was deduced from the gels prepared with three agar brands (Oxoid, Merck, and Roth).     

Influence of agar on in vitro cultures: II. Biological performance of ranunculus on media solidified with three different agar brands

Summary The type of gelling agent can influence to a large extent clonal propagation of Ranunculus asiaticus L. through axillary bud stimulation. In preliminary experiments we identified three agar brands (Oxoid=OX, Merck=MK, and Roth=RT) which affect the availability of water and minerals to tissues in different ways. In the present study we investigate the influence of these agars on the in vitro performance of Ranunculus. On OX and MK gels, growth was satisfactory, although the former had a more promotive effect on fresh and dry weight production and on multiplication rate. Growth and development of shoots were poor on RT; shoot clumps showed symptoms of hyperhydricity, with shoots having large dark-green malformed leaves and very elongated petioles. Epidermal strips of leaves from shoots grown on the different gels and collected at the end of the culture period revealed differences according to the agar brand on which the plantlets were cultured. Severe structural deformations of stomata could be detected on RT-grown shoots. The analyses of the sugar content of the gel at the end of the culture period demonstrated that the explants grown on RT gels are strictly dependent on the carbohydrates in the medium. On OX and MK gels the heterotrophic metabolism was lowered compared to RT-grown explants. The agar brand on which plantlets were grown also influenced water retention capacity and water content of the shoots. Experiments with tritiated water were undertaken to better understand the water fluxes inside the vessel and to investigate the difference in “pump function” exerted by shoots cultured on the three gels. Shoots grown on OX media showed the best “pump function,” which would account for the better results obtained on this gel. On the basis of the relationship between gel properties and the growth of Ranunculus shoots, we conclude that the different physiological responses on the three gels are a reflection of different water and nutrient availability in the different media.     

An evaluation of straw-extract agar media for the growth and sporulation of Madurella mycetomatis

Four new media, namely Wheat straw extract agar, Bajra straw extract agar, Jowar straw extract agar and Paddy straw extract agar, were evaluated for their potential to stimulate the growth and sporulation of Madurella mycetomatis in comparison with the conventional Sabouraud dextrose agar and Soil extract agar. Vegetative growth of M. mycetomatis on the four types of Straw extract agars was superior to that obtained on Sabouraud dextrose agar. Isolates of M. mycetomatis sporulated better and faster on the Straw extract agars than on the Sabouraud dextrose agar and Soil extract agar. Straw extract agar is recommended as a sporulation medium for M. mycetomatis. It may prove useful especially for studies of the conidium ontogeny of the fungus for elucidating its taxonomic status.     

An improved (lysine enriched) synthetic medium for the growth of Blastocladiella emersonii on agar media

Numerous combinations of amino acids supported growth ofBlastocladiella emersonii populations on defined media solidified with agar. Lysine was a particularly influential ingredient in these media.     

Suppression of the parasitemia in rodent filariasis (Litomosoides carinii) by immunization with BCG and microfilaria. II. Intravenous BCG application

By intravenous (i.v.) inoculation of living tuberculosis bacteria (BCG) non-specific resistance to microfilariae of Litomosoides carinii (Filarioidea) is induced in cotton rats. This is only possible using the preparation "Immune-BCG Pasteur F" (suspended germs), but not with "Vaccin-BCG pour scarifications" (lyophilized tuberculosis bacteria). After inoculation of Immune-BCG, followed by a challenge infection by 60 infective larvae 6 weeks later, a patent infection develops. However, the level of microfilaraemia is constantly lower than in the control. After challenge infection 12 weeks later, this effect has disappeared. Immune-BCG has no influence on the worm load or the output of microfilariae by the adult worms. If i.v. inoculation of Immune-BCG is combined with a subcutaneous injection of specific antigen--living embryos from the uteri of adult worms--the BCG-activated immune system undergoes specific sensitization. Upon challenge infection 6 weeks later, the microfilaraemia is completely suppressed, but the worm load and production of microfilariae by the adult female worms are normal. If Immune-BCG is injected i.v. 3 days before intraperitoneal injection of freeze-killed microfilariae, there is still constantly reduced microfilaraemia when challenge infection follows 12 weeks later. Obviously, the effect of this relatively weak antigen may be increased by BCG stimulation.