A Simple Bioassay for Abscisic Acid and Other Antitranspirants

Abstract

Several diverse bioassays are available for experimental studies of abscisic acid (ABA). They include acceleration of abscission in petiole explants (Addicott et al ., 1949); inhibition of a-amylase synthesis (Goldschmidt and Monelise, 1968); inhibition of growth in wheat coleoptiles (Wright, 1969), wheat embryos (Milborrow, 1967), Lemna (Tillberg, 1975) and Spirodela (van Staden and Bornman, 1970); decrease in the amount of reducing substances formed in barley endosperm (Sivori, Sonvico and Fernandez, 1971); and stomatal closure in Commelina epidermal strips (Tucker and Mansfield, 1971; Ogunkami et al., 1973). While the last of these bioassays refers to stomatal closure, possible injury during preparation of isolated epidermal strips, rhythmic variation in stomatal aperture and the need for accurate measurement of pore size can present some difficulties. A possible solution to these problems associated with stomatal closure was revealed during an examination of the effect of a growth regulator from Eucalyptus (G) in the mung bean rooting bioassay (Dhawan, Paton and Willing, 1979). In this bioassay, G markedly reduced water uptake. An examination of this ABA-like effect of G suggested that water uptake by excised mung bean hypocotyls can provide a direct, quantitative bioassay for the 'antitranspirant activity' of ABA. Seeds of mung bean ( Vigna radiata L.) Wilczek, previously Phaseolus aureus Roxb. cv. Berken) were purchased locally. These were soaked for 3 h in tap water and then sown 2 cm deep in pots containing perlite (Australian Gypsum, Sydney). Seedlings were raised under glass house conditions with day temperature between 27 and 32 °C and night temperature between 18 and 25 °C. A plastic cover was used during the first three days to maintain high humidity. Darkness was imposed during the next 3 days to enhance hypocotyl elongation, but normal light was used during the last 4 days. Under these conditions, 10-day-old seedlings had developed fully expanded primary leaves at the first node. Seedlings selected for uniformity in height (^ 15 cm) and leaf size, and were excised 5 cm below the cotyledons. The excision was done under water to avoid entry of air. Subsequent operations were carried out in a growth cabinet at 25 °C and 12 h fluorescent light (17-5 W m-2) having a gentle air current. The seedling cuttings were cultured in 3 ml narrow mouth glass vials (4-0 cm long, 0*8 cm diam.) filled with water and were protected from wilting under a plastic cover for 1 day. At the end of this adjustment period, cuttings were selected for uniformity of water uptake before transfer to a new set of glass vials which were completely filled with test solutions. Care was taken to ensure that the upper meniscus of the solution was at the top edge of the vials. The uptake of water from each vial was recorded after 24 h by refilling the vial back to the top edge using a finely graduated hypodermic syringe. As the f. wt of cuttings was not markedly affected by treatment and the surface evapora-