The quantitative determination of antioxidative-protective and some storage substances of seeds and leaves: Modification of methodological approach

Abstract

The modification was done with a purpose to find the integral medium for extraction of some organic and nonorganic substances responsible for seed germination and plant growth: phytine, PO4 ion, as well as, stress protecting substances of a different origin: total soluble phenols and thiols in seeds and leaves. This modification implied the selection of the extraction medium suitable for the quantitative isolation of substances from the common extracts of the initial material (seed, leaf). It was shown that highly diluted 0.15% tri-sodium-citrate can be used as a universal extraction medium for phytine, ortho-phosphate ion, soluble total thiols and total phenols. This medium allowed the efficient extraction of phytine and phosphate from degermed milled maize seeds. Also, it is sensitive enough to make difference between their content in young and mature maize leaves. Two weighted samples of 0.1-0.5 g, from the average sample of 25 seeds or seedlings, five young plants, as well as, two leaves (above and bellow maize cob) from three plants in the tasseling stage, with the addition of 8-15 ml of the above mentioned solution, provided a spectrophotometrical determination of these substances in 2-3 replicates. The procedure was developed to be suitable for the introduction and breeding, including a unique minimum average sample for all observed parameters. The application of precise and rapid spectrophotometrical methods, modified for large sample series, provides the reduction in the number of replicates to two (the statistical minimum), hence there is enough space for the introduction of the other desirable analyses.

U ovom radu izvršena je modifikacija metoda za određivanje organskih i neorganskih supstanci odgovornih za klijanje semena i rast biljaka: fitin i slobodni PO4 jon, kao i antistresnih faktora različitog porekla: ukupnih rastvorljivih tiola i fenola, u semenu i biljci, a koja se sastojala u izboru ekstrakcionog sredstva pogodnog za kvantitativno izolovanje navedenih supstanci iz zajedničkog ekstrakta početnog materijala (seme, list). Pokazalo se da je 0,15% tri-natrijum-cirtat podjednako efikasan za ukupne toile, fitin, slobodni PO4 jon i ukupne fenole. Dalje, dve odvagane supstance od 0,1 do 0,5 g iz prosečnog uzorka: 25 semena, odnosno klijanaca; pet mladih biljaka sa 5-7 listova, kao i dva lista (listovi ispod i iznad klipa) sa po tri biljke u fazi metličanja, sa 8 do 15 ml datog rastvora omogućava spektrofotometrijsko određivanje navedenih supstanci u dva, odnosno tri ponavljanja. Procedura je razrađena za potrebe introdukcije i selekcije, gde se raspolaže sa minimalnim prosečnim uzorkom koji se može upotrebiti za analize. U analitičkom smislu problem predstavlja mala masa uzorka kod sitnih semena i klijanaca, što zahteva minimalnu odvaganu supstancu i broj ponavljanja. Primena preciznih i brzih spektrofotometrijskih metoda, modifikovanih za velike serije, omogućava smanjenje broja ponavljanja na dva (statistički minimum), tako da preostaje dosta prostora (ml ekstrakta, g polaznog uzorka) da se uvedu i druge željene analize.