Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets

English version

Autoři: Crescencio Bazaldúa ^aff001; Alexandre Cardoso-Taketa ^aff002; Gabriela Trejo-Tapia ^aff001; Brenda Camacho-Diaz ^aff001; Jesús Arellano ^aff003; Elsa Ventura-Zapata ^aff001; María Luisa Villarreal ^aff002
Působiště autorů: Departamento de Biotecnología, Centro de Desarrollo de Productos Bióticos, Instituto Politécnico Nacional, Yautepec, Morelos, México ^aff001; Laboratorio de Plantas Medicinales, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Cuernavaca, México ^aff002; Laboratorio de Botánica Estructural, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Cuernavaca, México ^aff003
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0222464

Souhrn

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L^-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (μ) was 0.11 d^-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g^-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens.

Klíčová slova:

Biology and life sciences – Anatomy – Integumentary system – Hair – Organisms – Bacteria – Agrobacteria – Microbiology – Plant microbiology – Plant science – Medicine and health sciences – Physical sciences – Chemistry – Chemical compounds – Organic compounds – Vitamins – B vitamins – Thiamine – Carbohydrates – Organic chemistry – Analytical chemistry – Mass spectrometry – Liquid chromatography-mass spectrometry – Research and analysis methods – Chromatographic techniques – Liquid chromatography – High performance liquid chromatography – Biological cultures – Suspension cultures – Spectrum analysis techniques – Specimen preparation and treatment – Mechanical treatment of specimens – Sonication

Zdroje

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Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets

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