RESUMEN
A protocol for extracting polysaccharides from cell walls has been modified and used to analyze histochemically two fruits with opposite characteristics. Grapes are nonclimacteric fruits and are harvested at full maturity. In contrast, kiwi fruits are climacteric and are harvested and consumed before they are physiologically mature. The two fruits were analyzed histochemically using two protocols. One method is defined as chemical, and is based on subsequential extractions of pectins by chemical agents. The other is defined as enzymatic because it removes pectins using pectinase followed by hot ammonium oxalate. In both protocols, two types of hemicellulosic polymers are removed by 1 M and 4 M/KOH leaving a cellulosic residue on the slide. Both protocols remove the same amount of pectins, thus confirming their precision. The sum of hemicellulose and the cellulosic insoluble residue are equivalent using the two methods, but the relative amounts of the cellulose and hemicellulosic polymers were dependent upon the method of extraction. When the enzyme was used to extract the pectins, there was less cellulose and more hemicellulose. The removal of polysaccharides by ammonium oxalate and by guanidinethiocyanate in the enzymatic and the chemical protocols, respectively, yielded approximately the same amount of removed material. Similar results were obtained from both fruits. Grape, being softer than kiwi fruit, was relatively richer in pectic substances and less rich in hemicellulose and cellulose polymers. No difference in cell wall material could be ascribed to the different ripening habits.
Asunto(s)
Pared Celular/química , Frutas/química , Polisacáridos/aislamiento & purificación , HistocitoquímicaRESUMEN
Heating plant sections at 90 C with 0.5% aqueous ammonium oxalate is required to remove pectins. When applied to tissues rich in starch such as potato, this step produces heavy dextrinization of the starch which hinders subsequent evaluation of the extinction values of the cell walls. To overcome this a method has been devised to brush away the starch granules from the sections with a thin paint brush, just after paraffin removal by xylene. The slide is then processed as usual: pectins are removed by heat treatment, cell walls are stained with PAS and the stain intensity can be evaluated by photometry.