title: Determination of abscisic acid and its glucosyl ester in embryogenic callus cultures of Vitis vinifera in relation to the maturation of somatic embryos using a new liquid chromatography-ELISA analysis method.
abstract: The levels of abscisic acid (ABA), its conjugate ABA-GE, and IAA were determined in embryogenic calli of Vitis vinifera L. (cv. Mencía) cultured in DM1 differentiation medium, to relate them to the maturation process of somatic embryos. To achieve this goal, we developed an analytical method that included two steps of solid-phase extraction, chromatographic separation by HPLC, ABA-GE hydrolysis, and sensitive ELISA quantification. Because the ABA immunoassay was based on new polyclonal antibodies raised against a C4'-ABA conjugate, the assay was characterized (detection limit, midrange, measure range, and cross-reaction) and validated by a comparison of the ABA data obtained with this ELISA procedure and with a physicochemical method (LC-ESI-MS/MS). Radioactive-labeled internal standards were initially added to callus extracts to correct the losses of plant hormones, and thus assure the accuracy of the measurements. The endogenous concentration of ABA in the embryogenic callus cultured in DM1 medium was doubled at the fifth week of culture, concurring with the maturation process of somatic embryos, as indicated by the accumulation of carbohydrates observed through histological analysis. The ABA-GE content was higher than ABA, decreasing at 21 days of culture in DM1 medium but increasing thereafter. The data suggest the involvement of the synthesis and conjugation of ABA in the final stages of development in grapevine somatic embryos from embryogenic callus. IAA levels were low, suggesting that auxin plays no significant role during the maturation of somatic embryos. In addition, the lower ABA levels in calli cultured in DM differentiation medium with PGRs, a medium presenting high precocious germination and deficiencies in somatic embryo development indicate that an increase in ABA content during the development of somatic embryos in grapevine is necessary for their correct maturation.
authors.affiliation: Departamento de Biología Vegetal y Ciencia del Suelo, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain. Electronic address: mjpradoc@gmail.com.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Prado
authors.affiliation: Departamento de Ingeniería y Ciencias Agrarias, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain. Electronic address: alargs@unileon.es.
authors.forename: Asier
authors.initials: A
authors.lastname: Largo
authors.affiliation: Departamento de Biología Vegetal y Ciencia del Suelo, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain. Electronic address: crisdominguezcastro@gmail.com.
authors.forename: Cristina
authors.initials: C
authors.lastname: Domínguez
authors.affiliation: Departamento de Fisiología Vegetal, Universidad de Santiago de Compostela, Campus Sur, 15872 Santiago de Compostela, Spain. Electronic address: mvictoria.gonzalez@usc.es.
authors.forename: María Victoria
authors.initials: MV
authors.lastname: González
authors.affiliation: Departamento de Biología Vegetal y Ciencia del Suelo, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain. Electronic address: mrey@uvigo.es.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rey
authors.affiliation: Departamento de Ingeniería y Ciencias Agrarias, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain. Electronic address: mlcenm@unileon.es.
authors.forename: María Luz
authors.initials: ML
authors.lastname: Centeno
mesh_terms: D000040:Abscisic Acid / Q000032:analysis* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004952:Esters / Q000032:analysis / Q000378:metabolism; D007210:Indoleacetic Acids / Q000032:analysis* / Q000378:metabolism; D010937:Plant Growth Regulators / Q000032:analysis* / Q000378:metabolism; D058439:Plant Somatic Embryogenesis Techniques; D027843:Vitis / Q000196:embryology / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D004952:Esters; D007210:Indoleacetic Acids; D010937:Plant Growth Regulators; C030737:indoleacetic acid; D000040:Abscisic Acid
keywords: Abscisic acid; Grapevine; Immunoassay; Somatic embryo maturation; Somatic embryogenesis