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24-h photoperiod and 3,000 lux

24-h photoperiod and 3,000 lux version to and stable accumulation of volatiles, especially in oxyfunctionalization reactions. One example of economical relevance could be the regioselective biotransformation of valencene into the sesquiterpene ketone nootka-tone. After six hours of incubation, almost 70% of the substrate was transformed with no other detectable volatile by-products. Natural nootkatone with its grapefruitlike odor and bitter taste is a sought-after flavor compound with limited availability. As was found repeatedly in earlier work with plant cell cultures, flavor formation was favored by slow growth, an indication that flavor syntheses may be a general characteristic of a heterostatic physiology of the cell (26). The preceding findings disagree with early statements that either the cytotoxicity of the once formed volatiles or their instability in the surrounding medium compartment would prevent flavors from being accumulated in elevated concentration.

The addition of heat-inactivated microbial homogenates to growing plant cell cultures was discussed as a means of inducing metabolic cascades involved in chemical defense. As some flavor compounds interfere with bacterial or fungal growth, their synthesis was thought to be triggered by simulating a respective microbial attack. This so-called elicitation was demonstrated by the addition of autoclaved fungal mycelium to Petroselinum cells that responded with the formation of volatile phthalides, coumarins, and phenolics (97). A phototrophic state of the cells was required, because only the plastid differentiated cells tolerated the higher cytokinin concentrations and the fungal homoge-nate. This kind of ecological stress typically leads to the secretion of the newly formed compounds into the surrounding nutrient medium, where they unfold their bio-activity, and from where they can be easily recovered. The use of lipophilic traps (solvent or adsorbent) simulating a natural morpholicical accumulation site can be combined with the elicitor approach.

Cell immobilization is an expanding area of biotechnology. The characteristics of continuous operation, reuse of the biocatalyst, ease of process control, and improved bio-catalyst stability should prove especially beneficial for cultured plant cells, as immobilized cells are similar in many respects to the tissues of intact plants. Aggregated cells face heterogeneous microenvironments, release intracellular products, and divide slower. These and other factors may redirect plant secondary metabolism. The use of immobilized plant cells in column or membrane reactors may offer better perspectives for a future application on an industrial scale. If single enzymes were rate limiting, as particularly in biotransformations, it should be possible to select for higher yields by making use of the somaclonal variation that builds up in plant cell cultures, or by enhanced mutagenesis. Increased levels of essential oils were reported for mutant plants of Pelargonium and other species regenerated from UV or ethyl methane sulfonate treated callus cultures; this type of approach is somewhat restricted by the laborious procedures and assays required (98).

In spite of the achievements it should not be overlooked that de novo syntheses as well as biotransformations using plant cells will always have to compete not only with field plants, but also with microbial and enzyme technologies. Plant cells are not very adaptable to extreme conditions of cultivation, and the logical consequence would rather be to use in vitro plant cells of suitable cultivars as a convenient source of genes and to link these plant genes to microbial operators in future rDNA experiments.

Specialized Plant Cells

The biotechnological ideal of an immobilized plant cell with active flavor metabolism, available in any amount, was created by nature: mature fruits. It has been known for a long time that aged fruit tissues are able to take up exogenous substrates, metabolizing them to flavor compounds. The same holds true for intact fruits and volatile substrates. A procedure was developed to expose fruits during storage to vapors of precursors of volatile flavors. By analogy with controlled atmosphere (CA) storage, this biotechnological concept was termed PA (for precursor atmosphere) storage. One example refers to the drying of banana tissue (99). Although many compounds of banana flavor showed a good retention in the starchy matrix of the tissue, the concentrations of some highly volatile carboxylic esters decreased by 50 to 80% during freeze drying. When bananas were submitted to PA-storage in 3-methylbutanol vapors before dehydration, the fruits rapidly accumulated volatile impact components. By managing endogenous organized enzymes and their substrates, a dried fruit with an amount of 3-methylbutyl esters comparable to that of the genuine fresh fruit was finally obtained (Table 9). As a result, a preceding PA storage can compensate for flavor deficiencies caused by one-sided breeding, by improper transport and storage, or by physical losses during thermal processing operations.

Table 9. Effect of a Preceding Precursor Atmosphere (PA) Storage on Impact Volatiles of Freeze-Dried Banana Slices
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