Organ Cultures

Hairy root cultures of anise (Pimpinella anisum L.) were grown in different nutrient media in darkness and under periodic light conditions (58,59). The composition of the essential oils obtained from hairy root cultures differed significantly from that of the fruits. Whereas the major components of the essential oils from the hairy root cultures were the anethole precursor (E)-epoxypseudoisoeugenyl 2-methylbutanoate, zingiberene, /3-bisabolene, geijerene, and pregeijerene, the terpene spectrum of the fruits was dominated by (E)-anethole (Fig. 3). The highest essential oil yield obtained from hairy root cultures was 0.1%, which was comparable to that obtained from the roots of the parent plant and, when considering hairy roots on a dry weight basis, also that of the fruits. With regard to potential biotechnological exploitation, the morphological stability of the "rooty" phenotype is of great importance. In one of four growth media tested, the hairy root cultures revealed high morphological stability with no dedifferentiation or greening even after more than 2 years of culture (60). Encouraged by these results, h3co.

h3co.

(£)-Epoxypseudoisoeugenyl 2-methylbutanoate

Zingiberene

Zingiberene och3

Pregeijerene

Pregeijerene

/^Bisabolene

Geijerene

Geijerene

Figure 3 Flavors from hairy root cultures and fruits of anise (Pimpinella anisum L.).

Matsuda et al. (61) produced mutant hairy roots of musk melon (Cucumis melo L.) with an altered metabolism of essential oils by means of T-DNA insertion mutagenesis. From more than 6500 clones, five fragrant hairy root clones were obtained and the clone emitting the strongest fruity flavor of ripe fresh melons was selected for further investigations. Extraction of the volatile compounds and identification by means of gas-liquid chromatogra-phy-mass spectrometry (GLC-MS) revealed the presence of (Z)-3-hexenol, (2? )-2-hexenal, 1 -nonanol, and (Z)-6-nonenol, which also shape the flavor of melon fruits. Aroma emanation was successfully maintained in the hairy roots when they were subcultured repeatedly for more than 3 years. Preliminary scale-up experiments using a 4-L jar fermenter showed an overall profile of the extracted essential oils that was very similar to that of hairy roots cultured on a routine laboratory scale. When compared with that of the fresh ripe melon fruit, the yield of aroma compounds in this scale-up approach was approximately 6.5-fold higher, indicating possible biotech-nological exploitation. Plant sources of flavor components from cell cultures are summarized in Table 2.

C. Biotransformation by Plant Cell Cultures

As with microbial conversions, cultured plant cells can be employed as "cell factories" to conduct bioconversions of exogenous substrates (Fig. 4). Looking at flavor effectiveness and availability, monoterpenes are of outstanding interest. A comprehensive overview of biotransformations of monoterpenoid alcohols, aldehydes, ketones, and oxides by plant and microbial cell cultures was given by Shin (62). The conversion of monoterpenes, steroids, and indole alkaloids using numerous cell cultures was summarized by Hamada and Furuya (63). Special attention in these reviews was dedicated to the regiospecificity and stereospecificity of the biochemical reactions as well as to immobilization techniques for cells or enzymes.

Immobilized and free cells of kangaroo apple {Solatium aviculare) and of a yam species (Dioscorea deltoidea) were utilized for the oxidation of ( —)-limonene to (Z)- and (£)-carveol and to carvone. Depending on the immobilization medium, either carvone or (Z)- and (E)-carveol were formed predominantly (64).

Suspended cells of chilli pepper (Capsicum frutescens) accumulated vanilla flavor metabolites such as vanillin, vanillic acid, and ferulic acid when fed with isoeugenol. Increased biotransformation rates of isoeugenol could be achieved by immobilizing cells with sodium alginate and applying fungal elicitors. Product yields up to 23 pg ml. were reached by the simultaneous addition of j3-cyclodextrin and isoeugenol (2.5 mM) (65). Further attempts to optimize the biotechnological production of vanilla flavor compounds have been reviewed (66).

Table 2 Possible Plant Sources of Flavor Components from Cell Cultures

Common name

Botanical name

Principal metabolites

Ref.

Paprika

Capsicum frutescens

Capsaicin

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