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Preflowering Levels of Phytohormones in Sorghum. I. Analytical Procedure 1
Author(s) -
Dunlap J. R.,
Hanks A. H.,
Morgan P. W.
Publication year - 1981
Publication title -
crop science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.76
H-Index - 147
eISSN - 1435-0653
pISSN - 0011-183X
DOI - 10.2135/cropsci1981.0011183x002100010023x
Subject(s) - ethyl acetate , chromatography , high performance liquid chromatography , abscisic acid , acetic acid , gibberellin , diethyl ether , elution , column chromatography , bioassay , biology , chemistry , botany , biochemistry , genetics , gene
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 R f intervals, eluted with methanol, and analyzed with high performance liquid chromatography (HPLC) operating in the reverse‐phase mode (µ Bondapak C 18 ) 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 GA 3 , GA 4 , and/or GA 7 , by comparison to standards chromatographed on paper and bioassay ed. The putative GA 3 , was the most abundant GA‐like compound present. Additional quantities of putative IAA, ABA, and GA 3 , were obtained by mild acid hydrolysis of the final aqueous residue.