Production of Secondary Metabolites by in vitro Culture

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The first report on the production of ginsenosides in ginseng callus was published in 1970 by Japanese authors (Furuya et al., 1970). They detected by TLC and column silica gel chromatography analysis a large amount of ginsenoside Rg and a small amount of ginsenoside Rb in ginseng callus derived from petiole of cultivated ginseng grown on MS medium without glycine and supplemented with 1 mg/l 2,4-D. Later Furuya et al. (1973) found that the kind and amount of saponins in the callus of the same origin are about the same as in the ginseng root. By means of TLC and column chromatography analysis they isolated the ginsenosides Rb1 and Rg1, panaxadiol, panaxatriol and oleanolic acid. Simultaneously they obtained a mixture of phytosterols consisting of a large amount of ^-sitosterol and a small amount of campesterol and stigmasterol. The presence of ginsenosides Rb1 and Rg1 was confirmed by NMR.

The effect of auxins on saponin production in ginseng callus was studied by Furuya et al. (1983a) who found that the saponin production depends on the presence of 2,4-D in the medium. Habituated calli growing without plant growth regulators showed significantly lower capacity for saponin production than normal cells. When the media with 2,4-D-requiring and habituated calli were supplemented with IAA, the production of saponins in both calli was not significantly affected. Khodalovskaya et al. (1995) reported on the promoting effect of 4-chlorine phenoxyacetic acid (4-CPA) on the biosynthesis of ginsenosides in ginseng callus. Furthermore, it was found that semicarbazide and particularly thiosemicarbazide inhibited the production of phytosterols and promoted the biosynthesis of saponins in the presence of mevalonic acid (Furuya et al, 1983b, Linsefors et al., 1989). Odnevall and Bjork (1989a) who studied the relationship between the morphological state and ginsenoside formation found that the maximum ginsenoside production occurred in tissue cultures consisting of cell aggregates and differentiated roots on a medium supplemented with 2,4-D and kinetin under light conditions after 2-4 days in the stationary phase. The dynamics of the biosynthesis of ginsenosides during a one growth cycle of callus cell culture of ginseng was studied by Konstantinova et al. (1995) who found that maximum ginsenoside accumulation occurred on the 50th-80th day of subculture. Ginsenoside accumulation in ginseng root cultures depended on the carbon source (Odnevall and Bjrk, 1989b). Maximum ginsenoside content (5.2-5.7 mg/g dry weight) was determined under effect of sucrose and fructose.

Further work was aimed at the study of saponin production in ginseng cell suspension cultures cultured in media with different plant growth regulators (Furuya et al., 1983c). It was shown that the growth in rotary shaking cultures was about 1.8 times greater than in reciprocal cultures, while the saponin production was about the same, and the most effective hormonal condition was the combination of IBA with kinetin. Russian authors (Bulgakov et al., 1996) have isolated a cephazolin-resistant cell line Ic-Ceph which produced 2.3 times greater amounts of ginsenosides during three years than their non-selected counterparts.

Biotransformation ability of ginseng root and callus cultures was studied by Furuya et al. (1989). They found that root cultures are able to convert (RS)-2-phenylpropionic acid into (RS)-2-phenylpropionyl ^-D-glucopyranoside, (2RS)-2-O-(2-phenylpropionyl)-D-glucose, (2S)-2-phenylpropionyl 6-O-^-D-xylopyranosyl-^-D-glucopyranoside and a myo-inositol ester of (R)-2-phenylpropionic acid. The total conversion reached 100 per cent at day three. Compared with the root culture, callus culture showed lower glycosylation ability. As shown by Ushiyama et al. (1989), root cultures of ginseng are also able to convert aromatic carboxylic acid into glucose and/or sophorose substituted conjugates. Ginseng cell cultures are able to transform digitoxigenin into nine compounds including a new compound digitoxigenin ^-D-glucoside malonyl ester (Kawaguchi et al., 1996).

The adoption of plant cell cultures as an industrial process depends greatly on economics. Lipsky (1992) reported on the mathematical model for the functional relationship between the nominal costs of biomass and secondary metabolites and the plant cell growth characteristics in a multicycle growth system for Panax ginseng grown in various types of bioreactors.

The largest market segment for ginseng cell cultures is believed to be the food and beverage industry because such products do not require long study trials and have mass appeal. Furthermore, the content of ginsenoside in such products needs not be verified and such production costs can be reduced. The only notable application to the food and beverage industry is probably the production of ginseng cell mass used in the manufacture of ginseng drinks in

Japan. China and Southeast Asia will be the target market for ginseng-containing drinks and fast foods in the near future.

Abbreviations

B5—Culture medium (Gamborg et al, 1968) BA—6-Benzylaminopurine

CaMV 35S promoter—Cauliflower mosaic virus 35S promoter

2,4-D—2,4-Dichlorophenoxyacetic acid

DIG—Digoxigenin

DMSO—Dimethylsulfoxide

GA3—Gibberellic acid

GUS—^-glucuronidase

HPLC—High pressure liquid chromatography IAA—Indole-3-acetic acid IBA—Indole-3-butyric acid

MS—Culture medium (Murashige & Skoog, 1962) NAA—a-phthaleneacetic acid NOS—Nopaline synthetase PCR—Polymerase chain reaction

Ri—Root-inducing plasmid from Agrobacterium rhizogenes RM—Culture medium (Linsmaier & Skoog, 1965) T-DNA—Transferred DNA of the Ti plasmid Ti—Tumour-inducing plasmid from Agrobacterium tumefaciens TLC—Thin layer chromatography X-gluc—X-glucuronide

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