Abstract
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.
Highlights
Podophyllotoxin (PTOX) is a natural 2,7’-cyclolignan used to obtain the semisynthetic derivatives Etoposide1, Teniposide1 and Etopophos1, which are medicines widely used in cancer chemotherapy
Regarding the height of plantlets regenerated in full strength MS and those regenerated on three-quarters strength (TQS) MS medium, significant differences (p
Our results showed that there was no significant difference in biomass production, but there was a clear difference in PTOX accumulation
Summary
Podophyllotoxin (PTOX) is a natural 2,7’-cyclolignan used to obtain the semisynthetic derivatives Etoposide, Teniposide and Etopophos, which are medicines widely used in cancer chemotherapy. The main source of PTOX is the species Podophyllum hexandrum [1], a wild plant native to the Himalayas, which has been overexploited and is currently endangered [2]. Hairy roots of Hyptis suaveolens improving production of podophyllotoxin. Annual PTOX production is 50–80 tons per year; demand exceeds 100 tons per year [3]. The search for new natural sources that produce PTOX is mandatory, as is research for biotechnological alternatives, to achieve stable and controllable production of this compound [4]. The most important PTOX producer species are Podophyllum hexandrum, Juniperus bermudiana, and Podophyllum peltatum, which produce 43, 22.6, and 4.7 mg g-1 dry weight (DW) of PTOX, respectively [1, 5, 6]
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