Open AccessImproving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets
Author(s) -
Crescencio Bazaldúa,
Alexandre Cardoso-Taketa,
Gabriela TrejoTapia,
Brenda Hildeliza Camacho-Díaz,
Jesús Arellano,
Elsa Ventura-Zapata,
Marı́a Luisa Villarreal
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0222464
Subject(s) - podophyllotoxin , sulforhodamine b , hairy root culture , agrobacterium , explant culture , botany , biology , high performance liquid chromatography , chromatography , chemistry , biochemistry , cytotoxicity , transformation (genetics) , in vitro , stereochemistry , gene
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 .