Effects of ultrasound on cell growth and secondary metabolite production in plant cell cultures

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Effects of ultrasound on cell growth and secondary metabolite production in plant cell cultures


Author: Lin, Lidong
Title: Effects of ultrasound on cell growth and secondary metabolite production in plant cell cultures
Degree: Ph.D.
Year: 2003
Subject: Hong Kong Polytechnic University -- Dissertations
Plant cell culture
Metabolism, Secondary
Cells -- Growth
Department: Dept. of Applied Biology and Chemical Technology
Pages: xxvii, 224 leaves : ill. (some col.) ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b1686646
URI: http://theses.lib.polyu.edu.hk/handle/200/4299
Abstract: Plant cell culture is an important tool of plant biotechnology. One of its potential applications is the production of phytochemicals or plant secondary metabolites, especially those derived from naturally-rare and slowly-growing species. This project is aimed to study a new measure, the application of low-energy ultrasound (US), for the stimulation of secondary metabolite production in plant cell cultures. Ultrasound, particularly at sublethal doses for living organisms, can produce a number of biological effects which are of potential significance in biotechnology. However, there have been few studies on the bioeffects of US in plant cell cultures. The main objectives of the project are to gain better knowledge and understanding of the bioeffects of low-intensity US on plant cells and to explore the beneficial effects of US for secondary metabolite production in plant cell cultures. Three types of plant cell culture with tremendous commercial potential were used in this project, including Panax ginseng (producing saponins), Lithospermum erythrorhizon (producing shikonins) and Taxus chinensis (producing taxol). Suspension cultures of the plant cells were exposed to US in an ultrasonic bath at various US powers (maximum 82 mW/cm3) and exposure periods (maximum 8 min). A number of novel and interesting effects of US on cell growth, viability and metabolism were found and the most remarkable effect was the promotion of secondary metabolite production. Under suitable exposure conditions, US increased the yields of secondary metabolites significantly, e.g., up to 60-70% increase in the saponin yield of P. ginseng cells and the shikonin yield of L. erythrorhizon, and up to 3-fold increase in the taxol yield of T. chinensis. The growth and viability of cells were usually depressed immediately after the exposure to US, but recovered gradually to levels similar to those of a normal culture in a few days, with virtually no net loss of biomass yield at the end of the culture period. The effects of US on the yield of secondary metabolites and cell viability showed a significant correlation with the total US energy applied. Other effects of US on plant cells found in our work include the increase in the intracellular product release and membrane permeability, the decrease of cell volume and water content and the enzymatic browning reactions. Further analysis of the sonicated plant cells showed that the US exposure stimulated the activities of some key enzymes related to plant secondary metabolism such as phenylalanine ammonia-lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO). The enhancement of plant cell secondary metabolism by US appears to be a result of US-induced defense response of the plant cells. The oxidative burst, rapid production of active oxygen species, H2O2, and the increase in cross-membrane ion fluxes, Ca2+ influx and K+ efflux/H+ influx, occurred immediately after US treatment. As those are the characteristic events of plant defense responses to pathogens and elicitors, the US may be acting as an abiotic elicitor of plant defense metabolisms to stimulate the secondary metabolite production of plant cells. The mechanical stress arising from the hydrodynamic events in the sonicated media, particularly acoustic cavitation and microstreaming, is the most possible cause of the US-induced effects in plant cell cultures. To maximize the secondary metabolite yields, US stimulation was combined with other yield-enhancing strategies, e.g., in situ product extraction or the two-phase culture and elicitor treatment. In the aqueous-organic two-phase culture systems, sonication further increased the shikonin yield of L. erythrorhizon cell culture by about 30% and taxol yield of T. chinensis by 100%. In addition, the shikonin and taxol excreted from the cells was increased to 60-70% from about 20% in the single-phase culture, due to the increase of the cell membrane permeability by sonication and the in situ solvent extraction. On the other hand, with the combined use of US stimulation and methyl jasmonate elicitor in the Taxus culture, the taxol yield was increased by nearly 16-fold. In conclusion, US was found to be a simple and effective stimulus for secondary metabolite production in plant cell cultures. The effects of US on plant cell growth and metabolism are mostly attributed to the mechanical stress created by the vibrating US in the culture media. The enhancement of secondary metabolite synthesis may be a result of US-induced plant cell defense metabolism. The results and findings from this project make new contributions to the development of plant cell culture technology for the production of valuable plant secondary metabolites and to fundamental understanding of the bioeffects of US.

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