Preflowering Levels of Phytohormones in Sorghum. I. Analytical Procedure1

Abstract

Procedures are described for the analysis of indole‐3‐acetic acid (IAA), abscisic acid (ABA), and gibberellin (GA)‐like compounds in sorghum, Sorghum bicolor (L.) Moench. Shoots of seedlings were harvested, frozen in liquid N, lyophilized, ground in a Wiley mill, and replicate samples extracted with methanol/water. After vacuum removal of methanol the residual was fractionated by partitioning against petroleum ether and ethyl acetate. Free, acidic forms of the phytohormones were recovered in the final ethyl acetate fraction, and a portion of the conjugated forms were obtained by mild acid hydrolysis of the aqueous residue followed by partitioning into ethyl acetate. Fractions containing IAA‐, ABA‐, or GA‐like compounds were further purified by ascending paper chromatography. Chromatograms of purified extracts were cut into various Rf intervals, eluted with methanol, and analyzed with high performance liquid chromatography (HPLC) operating in the reverse‐phase mode (µ Bondapak C18) for IAA or ABA and with the lettuce hypocotyl bioassay for GA‐like activity. Substantial quantities of IAA and ABA were obtained from sorghum tissue samples; recovery averaged 40 ± 10%. The identity of IAA and ABA was tentatively confirmed by: (a) identical retention times as authentic standards on gas‐liquid chromatography, (b) co‐chromatography with their respective standards on HPLC operated at different flow rates or solvent compositions. Gibberellin‐like compounds were tentatively identified as GA3, GA4, and/or GA7, by comparison to standards chromatographed on paper and bioassay ed. The putative GA3, was the most abundant GA‐like compound present. Additional quantities of putative IAA, ABA, and GA3, were obtained by mild acid hydrolysis of the final aqueous residue.