Plants are valuable sources of a wide range of secondary metabolites used as additives, fragrances, flavours pharmaceuticals and agrochemicals such as biopesticides. Many of them are produced in nature as defence mechanisms against attack by environmental conditions or pathogens. Despite the advances in synthetic organic chemistry, mankind still depends upon biological sources for most of the secondary metabolites, which are structurally complex, so difficult to synthesize. For centuries they have been obtained directly from wild plants or crops. Now biotechnology offers the opportunity to exploit cells, tissues, organs an entire organisms by growing them in in vitro systems. This technology has the potential of manipulating the cultures in order to obtain the desired compounds.

Although plant cells are biosynthetically totipotent, which means that each cell in culture retains complete genetic information and hence is able to produce the range of chemicals found in parent plants (Ramachandra and Ravishankar, 2002), many times, in in vitro conditions, they do not biosynthesize certain secondary metabolites. In these cases they may be challenged by certain biotic or abiotic stress factors called elicitors.

Grindelia species are widespread in South American semiarid regions. Most of these genuses are used in folk medicine as antispasmodic and diuretic, among other purposes. Grindelic acid and minoritary hydroxylated metabolites were isolated from G. pulchella aerial parts (Guerreiro et al. 1981). These metabolites showed bioactive properties towards Tenebrio molitor larvae species and phytopatogen fungi and bacteria. Furthermore, the resins of several species of the genus Grindelia such as G. chiloensis, G. camporum and G. glutinosa, particularly rich in diterpene acid derivatives, have been extensively studied due to their possible industrial applications like pine resin and raw material for the naval stores industry (Hoffmann and McLaughlin, 1986; Timmermann et al. 1987; Ravetta and Soriano, 1998; Zavala and Ravetta, 2001; Zavala and Ravetta, 2002; Wassner and Ravetta, 2005).

Searching for a biotechnological approach as an alternative for stable production of grindelic acid seems to be quite promising since a wide range of derivatives could be obtained by both chemical and biological transformations.

The objective of this work was to establish in vitro cultures of G. pulchella in order to study the possibility of producing stable amounts of grindelic acid.

G. pulchella in vitro cultures in solid media were established from seedling leaves. The latter were obtained from seeds which were surface disinfected and germinated in aseptic conditions. Basal culture media was suplemented with several plant growth regulators and the best conditions were chosen. Cell suspensions in liquid cultures were initiated by transferring fast growing and friable calli into basal liquid media added with the most suitable phytoregulator supplementation. Cell submerged cultures with detectable levels of grindelic acid were selected. Furthermore, different stress factors and combinations of them were used to elicit cell suspensions. These experiments demonstrated that the combination of heavy metals salts (CuSO₄) and the solvent dimethylsulfoxide,  which is traditionaly used to increase cell permeability (Kurina et al. 2000), increased grindelic acid production 3.6 times compared with the control assays. In this condition cell growth was strongly inhibited. In contrast, the addition of the elicitos jasmonic acid, a proved mediator in plant-wounding and plant-microbe interactions, neither affected cell growth nor grindelic acid accumulation.

This work results in a valuable contribution to establish in vitro culture conditions for the wild specie G. pulchella in order to produce the diterpene grindelic acid. Further elicitation experiments such as the use of biotic agents and combinations of biotic and abiotic elicitors would allow improving grindelic acid production. According to these results, other mediators different of jasmonic acid should be involved in the transduction of the elicitor signals in the regulation of the expression of this type of metabolites in G. pulchella.

References

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HOFFMANN, J.J. and McLAUGHLIN, S.P. Grindelia camporum: potential cash crop for the arid southwest. Economic Botany, 1986, vol. 40, p. 162-169.

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ZAVALA, J.A. and RAVETTA, D.A. The effect of irrigation regime on biomass and resin production of Grindelia chiloensis. Field Crop Research, 2001, vol. 69, no. 3, p. 227-236.

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