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CHAPTER 6
CONCLUSION AND FUTURE PERSPECTIVES
This work aimed at developing artificial cultivation systems of sweet basil for the extraction of RA in consideration of its commercial value as ingredient of medicinal, herbal and plant protection products. A number of experiments were conducted with sweet basil seedlings of different cultivars (green-leaved “Genovese” and “Superbo”, and purple-leaved “Dark Opal”) grown in both in vitro and in vivo culture. The latter system was based on greenhouse hydroponic system, in particular on floating system. In all experiments, several CADs of interest (caffeic acid, caftaric acid, chlorogenic acid, cicoric acid, cynarin, ferulic acid, t-cinnamic acid, p-coumaric acid, rosmarinic acid) were quantified using HPLC and HPLC-MS. Apart from rosmarinic acid (RA) and a methylated form of this substance, the levels of other selected CADs were close or below the detection limit (0.05 mg g-1 DW) in all the analyzed samples, irrespective of genotype, growing conditions or plant tissue. Work is in progress to elucidate the nature and the origin (genuine in vivo biosynthesis or artefact during the methanol extraction) of the methylated form of RA detected in our samples.
All the determinations were conducted on fresh samples as in preliminary experiments it was found that desiccation at 70°C reduced markedly (up to 40%) RA content in basil tissues. Desiccation is generally used for post-harvest preservation of medicinal plant materials; dried materials is easy to handle and process and less prone to microbial degradation. If fresh material has to be used for the extraction of the metabolites of interest, greenhouse cultivations must be located very close to the processing facilities and cold rooms may be necessary for short-term storage, following a production-delivery scheme similar to the one used by fresh-cuts products industry. Of course, detailed economical analysis is necessary to compare the overall costs of RA production from fresh or dry sweet basil material.
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In general, the RA concentrations found in sweet basil grown either in vitro or in
vivo were very high in comparison with those reported in the literature for this
species. The highest RA content were detected in young leaves of hydroponically-grown plants fertilized with 0.5 mol m3 NO3- (up to 10% DW) and in
micro-propagated plantlets in RITA™ vessels (up to more than 15% DW).
Among the three cultivars of sweet basil under investigation, the purple-leaved cultivar contained less RA than green-leaved genotypes. The former cultivar is very rich in anthocyanins pigments and a competition between RA and anthocyanins synthesis, which have a common precursor (4-coumaroyl-CoA), can be hypothesized.
In general, floating raft system provided a suitable growing method of sweet basil for agro-industrial production of RA, as a large amount of biomass with high concentration of this compound can be produced in few weeks. The highest RA content was detected in root and leaf tissues at full bloom; RA content was up to 10% DW in the youngest leaves. Even if the root biomass production is limited, in comparison with the shoot (above or less 10% of total dry mass), the utilization of the roots system for the extraction of RA may be hypothesized, as the floating system facilitates the harvesting of those tissues, clean and easy to processed.
Our experiments allowed the optimization of N nutrition of hydroponically-grown plants. It was found that the standard N concentration used in hydroponic culture (10.0 mol m-3 or higher) could be reduced considerably, with important implications from the environmental point of view. The use of a total NO3- concentration of 5.0
mol m-3 resulted in optimal plant growth, while the supply of much lower concentration (0.5 mol m-3 NO3- ) induced a strong increase of RA accumulation (up
to 97 mg g-1 DW in the young leaves), but a simultaneous reduction of biomass production. In contrast, the addition of NH4+ to the nutrient solution was detrimental
to both biomass accumulation and RA synthesis and its use should be avoided in hydroponic culture of sweet basil.
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mild NaCl salinity. These results have some implications from the operational point of view, as they suggest that poor quality irrigation water can be used in water culture of sweet basil and that the aeration of nutrient solution is not a crucial factor for optimal plant growth and RA production of this species.
As regard in vitro culture, high RA accumulation levels were detected in micropropagated plantlets. The content of this metabolite varied during the micropropagation phases and in dependence of BA concentration in the growing medium, which however affected differently purple-leaved and green-leaved plants. In fact, increasing BA concentration from 0.1 to 1 mg L-1 resulted in higher RA accumulation in green-leaved genotypes whereas an opposite effect was observed in the purple-leaved Dark Opal. Plant tissue content of RA was also affected by the type of cultural vessel adopted for explants growth. The highest RA accumulation was found in RITA™-cultured shoots and this was associated with completely-heterotrophic growth and large accumulation of ethylene in the internal atmosphere, as compared to other vessel types.
The levels of RA determined in micropropagated plants exceeded by far the concentrations reported in the literature for in vitro-grown sweet basil tissues or cells. On the other hand, the concentrations were similar to those reported for the plants grown in vivo, whereas the biomass production was dramatically lower. Due to the high cost and low biomass production of in vitro growing systems, greenhouse hydroponic culture seems much more cost-effective and probably easier from the operational point of view. Hydroponic production of sweet basil and other herbs for fresh or minimally-processed vegetables market is a well-developed technology that can be easily applied to the production of biomass to be extracted for active principles. Production efficiency could be improved by the cultivation of selected varieties, as sweet basil RA content may vary markedly in dependence of the genotype.
Moreover, future work may be done at the aim to elucidate the biochemical and molecular mechanisms underlying the shift from primary and secondary metabolism
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and the influence of growing factors on RA synthesis. For that, in vitro culture could represent a suitable model.
Tyrosine aminotransferase (TAT), hydroxycinnamoyl-CoA: hydroxyphenyllactate hydroxycinnamoyl transferase (RAS) and PAL are key-enzymes of RA biosynthesis. Genes sequences encoding for these enzymes were isolated in leaf tissues of sweet basil plants (cv. Genovese) grown both in vivo and in vitro. Real time PCR experiments are in progress to investigate the RNA expression levels of these genes in plants grown under conditions that have been found to affect markedly RA accumulation, for instance in N-deficient or NH4+-fed plants.
