This report presents a simple, rapid and accessible validated method for quantification of eight major plant growth regulators (PGR): cytokinins (6-(γ,γ-dimethylallylamino)purine (2-iP), benzylaminopurine (BA) and zeatin), auxin (indole-3-acetic acid; IAA), jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) and abscisic acid (ABA) by liquid chromatography mass spectrometry. This method was tested in eight species including agricultural, ornamental and medicinal species: St. John’s wort, African violet, banana, American elm, tobacco, potato, sweet wormwood, and fennel. The method has good reproducibility and good sensitivity with %RSD (percent relative standard deviation) between 1 and 10% for all matrices and recovery values of 89 to 118% for all analytes. Method detection limits were 50.65 ng/g, 203.4 ng/g, 50.65, ng/g, 50.65 ng/g, 203.4 ng/g, 12.7 ng/g, 193 pg/g and 3.08 ng/g, for SA, IAA, zeatin, JA, GA3, ABA, 2-iP, and BA, respectively. Our results with a range of plant species show that this method represents a simple, low-cost method for analysis of PGRs, and may also serve as an useful starting point for the analysis of other related PGRs, as demonstrated by inclusion of the SA derivative, acetylsalicylic acid, and the JA derivatives: 12-oxo-phytodienoic acid and JA-isoleucine. The efficiency of this method will enable its incorporation into the plant tissue culture work flow and through characterization of endogenous PGR levels, will allow for improved method development for recalcitrant species facilitating fundamental and applied studies in plant morphogenesis, propagation and conservation.
This is a preview of subscription content, log in to check access.
The authors gratefully acknowledge the financial support of this work by the National Sciences and Engineering Research Council (NSERC) of Canada [Grant Number 46741] and the Gosling Research Institute for Plant Preservation (GRIPP) [Grant Number 050294].
LAEE participated in conception and design, data acquisition, analysis and interpretation, MRS participated in conception and design, WBG participated in conception and design and data analysis and PKS participated in conception and design and data interpretation. All authors participated in manuscript preparation and gave final approval of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
AOAC International (2013) Appendix K: Guidelines for dietary supplements and botanicals. In: Official methods of analysis. AOAC International, Arlington, Appendix K: 1–32Google Scholar
Arteca RN (1996) Manipulation of growth and photosynthetic processes by plant growth regulators. In: Plant growth substances. Springer, Boston, pp 240–272Google Scholar
Assani A, Chabane D, Foroughi-Wehr B, Wenzel G (2006) An improved protocol for microcallus production and whole plant regeneration from recalcitrant banana protoplasts (Musa spp.). Plant Cell Tissue Organ Cult 85:257–264. doi: 10.1007/s11240-005-9058-yCrossRefGoogle Scholar
Cai B-D, Ye E-C, Yuan B-F, Feng Y-Q (2015) Sequential solvent induced phase transition extraction for profiling of endogenous phytohormones in plants by liquid chromatography-mass spectrometry. J Chromatogr B 1004:23–29. doi: 10.1016/j.jchromb.2015.09.031CrossRefGoogle Scholar
Gaspar T, Kevers C, Penel C, Greppin H (1996) Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Dev Biol Plant 32:272–289CrossRefGoogle Scholar
Hewezi T, Jardinaud F, Alibert G, Kallerhoff J (2003) A new approach for efficient regeneration of a recalcitrant genotype of sunflower (Helianthus annuus) by organogenesis induction on split embryonic axes. Plant Cell Tissue Organ Cult 73:81–86. doi:10.1023/A:1022689229547CrossRefGoogle Scholar
Li H, Murch SJ, Saxena PK (2000) Thidiazuron-induced de novo shoot organogenesis on seedlings, etiolated hypocotyls and stem segments of Huang-qin. Plant Cell Tissue Organ Cult 62:169–173. doi: 10.1023/A:1006491408762CrossRefGoogle Scholar
Li G, Lu S, Wu H et al (2015) Determination of multiple phytohormones in fruits by high-performance liquid chromatography with fluorescence detection using dispersive liquid-liquid microextraction followed by precolumn fluorescent labeling. J Sep Sci 38:187–196. doi: 10.1002/jssc.201401131CrossRefPubMedGoogle Scholar
Mithila J, Hall J, Victor JMR, Saxena P (2003) Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulia ionantha Wendl.). Plant Cell Rep 21:408–414. doi: 10.1007/s00299-002-0544-yCrossRefPubMedGoogle Scholar
Mundhara R, Rashid A (2006) Recalcitrant grain legume Vigna radiata, mung bean made to regenerate on change of hormonal and cultural conditions. Plant Cell Tissue Organ Cult 85:265–270. doi: 10.1007/s11240-005-9061-3CrossRefGoogle Scholar
Nguyen AH, Hodgson LM, Erskine W, Barker SJ (2016) An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes. Plant Cell Tissue Organ Cult 127:623–635. doi: 10.1007/s11240-016-1087-1CrossRefGoogle Scholar
Pliego-Alfaro F, Monsalud MJR, Litz RE, Gray DJ, Moon PA (1996) Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos. Plant Cell Tissue Organ Cult 44:63–70. doi: 10.1007/BF00045914CrossRefGoogle Scholar
Sanago MH, Murch SJ, Slimmon TY, Krishnaraj S, Saxena PK (1995) Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium × hortorum Bailey). Plant Cell Rep 15:205–211. doi: 10.1007/BF00193721CrossRefPubMedGoogle Scholar
Shukla MR, Jones AMP, Sullivan JA et al (2012) In vitro conservation of American elm (Ulmus americana): potential role of auxin metabolism in sustained plant proliferation. Can J For Res 42:686–697. doi: 10.1139/x2012-022CrossRefGoogle Scholar
Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–130PubMedGoogle Scholar