The effect of charcoal on tissue morphogenesis in vitro.

Abstract

The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving . Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5.0% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1.0% activated charcoal, added before autoclaving . In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolysed The adsorption of 2, 4-dichlorophenoxyacetic acid (2,4-D) by activated charcoal from methanol and aqueous solutions was determinated using HPLC. The amount of the added 2,4-D decreased in both methanol and aqueous solutions in the presence of activated charcoal, compared with those in the absence of activated charcoal. In methanol and aqueous solutions, activated charcoal used at the level of 0.1% significantly reduced 2,4-D. About 68.4% and 60.9% respectively of the added 2,4-D was adsorbed by activated charcoal (1.0%) from these solutions. The changes of inorganic elements in MS-salt solutions, in the presence of activated charcoal, were analysed by SEM-EDX. The concentrations of magnesium (Mg), calcium (Ca), iron (Fe) and zinc (Zn) deceased in the presence of activated charcoal, while the concentrations of potassium (K), copper (Cu), manganese (Mn), phosphorus (P), and sulphur (S) increased in the MS salt solution in the presence of activated charcoal compared with no activated charcoal in the medium. This suggests that activated charcoal adsorbed calcium, magnesium, iron and zinc and released copper, manganese, phosphorus and sulphur. Rooting occurred when 7-day-old seedling hypocotyls of Daucus carota L. Cape Market were placed on MS medium supplemented with 2,4-D, and IAN/NAA in the presence of activated charcoal. Hypocotyls did not produce roots on the 2,4-D containing media in the absence of activated charcoal. The roots were produced polarly on the NAA/IAA-containing media in the presence of activated charcoal. No-polarity of root formation was observed on media supplemented with NAA/IAA without activated charcoal. Different responses of hypocotyls to a series of 2,4-D concentrations (0.5, 1.0, 3.05.0, 8.0, and 10.0 mg l ¯¹) were observed on media supplemented with 0.02, 0.1 and 0.5% activated charcoal. In the NAA/IAA containing media in the presence of activated charcoal, root number per hypocotyl decreased. Root number perhypocotyl, on the media supplemented with NAA and IAA, increased when hypocotyls were pre-cultured on MS medium supplemented with 2,4-D (1.0 mg l ¯¹) for 2-3 days. When hypocotyls were pre-cultured on a 2,4-D containing MS medium for 5 days, embryos emerged from the hypocotyls directly on the medium supplemented with 2,4-D in the presence of activated charcoal. Addition of activated charcoal to MS medium supplemented with 2,4-D resulted in somatic embryogenesis of Daucus carota. Somatic embryos were not formed on the medium in the absence of activated charcoal. In suspension culture, the incorporation of 0.01 to 1.0% concentrations of activated charcoal to the MS medium, irrespective of 2,4-D, increased the number of somatic embryos produced. The maximum number of somatic embryos were produced with 1.0% activated charcoal. Further development of embryos of Daucus carota occurred on the media in the presence of activated charcoal, and the embryos subsequently regenerated normal plantlets. Abnormal somatic embryos followed the addition of 3.0% activated charcoal to the medium.