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Enhancing the production of scopoletin and quercetin 3-O-β-d-glucoside from cell suspension cultures of Tilia americana var. mexicana by modulating the copper and nitrate concentrations

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Abstract

The aerial tissues of Tilia americana var. mexicana produce compounds with anxiolytic activity, such as quercetin 3-O-β-d-glucoside and tiliroside, in addition to ones with anti-inflammatory properties, such as scopoletin. These three compounds were initially identified in callus cultures of apical buds. In the present study, suspension cultures from leaf explant callus were established; the accumulation of scopoletin and quercetin 3-O-β-d-glucoside in these cultures were found to be cell-growth-associated using cell growth and active compound-production kinetics assays. The effects of varying the nitrate and copper concentrations in Murashige and Skoog (MS, 27.4 mM total nitrates and 0.01 µM copper) medium on the growth of a suspension culture of T. americana cells and on the production of active compounds were tested by means of central composite design (CCD) generally used in the response surface methodology (RSM). Cell growth, measured as maximal biomass, improved when the total nitrate concentration decreased in the MS medium to 13.7 mM (p < 0.01) regardless of the copper concentration. As a phytoalexin, scopoletin accumulated rapidly in plants after pathogen infection, in the suspension cultures scopoletin yield was stimulated by increased copper concentration to 1.2 μM (p < 0.01). According to the C:N hypothesis, the carbon excess generated by nitrates reduced to 8.03 mM (p < 0.01) stimulated the production of quercetin 3-O-β-d-glucoside. Cell suspension of T. americana represents a potential biotechnological alternative for industrial exploitation in a stirred-tank bioreactor using a two-phase process: (1) the first step will be to grow the cell suspension, (2) the second stage will consist in handle the suspension culture towards the production of anxiolytic compounds or towards the production of anti-inflammatory compounds. As well as to evaluate another elicitors to stimulate tiliroside production in the T. americana suspension cultures.

Key message

Tilia americana cells grown in a two-phases suspension culture system produce more scopoletin and quercetin 3-O-β-d-glucoside when exposed to increased concentrations of copper and decreased concentrations of total nitrates.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

CCD:

Central composite design

Dt:

Duplication time

DW:

Dried weight

FD:

Factorial design

GI:

Growth index

IBA:

Indol-3-butyric acid

Kin:

Kinetin

µ:

Maximal growth rate

MS:

Murashige and Skoog

rf:

Reference front

RSM:

Response surface methodology

SD:

Star design

TDZ:

Thidiazuron

TPA:

12-O-Tetradecanoylphorbol-13-acetate

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Acknowledgements

This work was supported by Basic Grant 302000 from the Consejo Nacional de Ciencia y Tecnología, México (CONACyT-México) for the doctoral studies of Daniel Cisneros-Torres at the Biotechnology Doctoral Program of UAM-Iztapalapa and by Complementary Grant 99187269 from the Instituto Mexicano del Seguro Social (IMSS).

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Authors and Affiliations

  1. Centro de Investigación Biomédica del Sur (CIBIS), Instituto Mexicano del Seguro Social (IMSS), Argentina #1, 62790, Xochitepec, Morelos, Mexico

    Daniel Cisneros-Torres, Manasés González-Cortazar & Pilar Nicasio-Torres

  2. Doctorado en Biotecnología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de México, Mexico

    Daniel Cisneros-Torres

  3. Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de México, Mexico

    Francisco Cruz-Sosa

  4. División de Ingeniería Química y Bioquímica, Tecnológico de Estudios Superiores de Ecatepec, Ecatepec De Morelos, Estado de México, Mexico

    Aurora Martínez-Trujillo

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  1. Daniel Cisneros-Torres
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  2. Francisco Cruz-Sosa
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  3. Manasés González-Cortazar
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  4. Aurora Martínez-Trujillo
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  5. Pilar Nicasio-Torres
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Contributions

As a Ph.D. student, Daniel Cisneros-Torres participated in all of the experimental work under the advice of the co-authors, in the collection, analysis, interpretation of the data, and the writing of the manuscript. Francisco Cruz-Sosa supervised the establishment of the factorial design experiments, provided scopoletin and quercetin 3-O-β-d-glucoside standards, and he was Daniel Cisneros-Torres’ thesis co-advisor. Manasés González-Cortazar participated in the extraction and in the establishment of analytical methods for compound quantification. Aurora Martínez-Trujillo participated in the concept and design of the factorial design and supported Daniel Cisneros-Torres in the statistical analyses. Pilar Nicasio-Torres supervised the establishment of the cell-suspension cultures and growth and production kinetics, extractions of biomasses; she was also Daniel Cisneros-Torres’s thesis co-advisor, in addition to participating in the writing of the manuscript and approving the final version of the manuscript.

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Correspondence to Pilar Nicasio-Torres.

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Cisneros-Torres, D., Cruz-Sosa, F., González-Cortazar, M. et al. Enhancing the production of scopoletin and quercetin 3-O-β-d-glucoside from cell suspension cultures of Tilia americana var. mexicana by modulating the copper and nitrate concentrations. Plant Cell Tiss Organ Cult 139, 305–316 (2019). https://doi.org/10.1007/s11240-019-01683-7

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