Ma T. Piñol

Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants

Abstract Transformation of Nicotiana tabacum cv. Xanthi leaf sections with the pPCV002-ABC (rol genes A, B and C together under the control of their own promoter) or pPCV002-CaMVC (rol gene C alone under the control of the CaMV 35S promoter) construction present in trans-acting Agrobacterium tumefaciens vectors yielded several transgenic root lines. The two types (rolABC and rolC) of transgenic root lines were examined for their nicotine productivity in relation to growth rate and the amount of rolC gene product measured with specific antibodies. In all cases, the changes in the amount of this polypeptide were positively correlated with the capacity of the transgenic roots to grow and produce nicotine. Both capacities were greatly increased when the rolA, rolB and rolC genes were present together, which demonstrates that the activity of the three rol-gene-encoded functions is synergistic. Consistent observations were also made in the corresponding regenerated plants.

MANIPULATION BY CULTURE MIXING AND ELICITATION OF PACLITAXEL AND BACCATIN III PRODUCTION IN TAXUS BACCATA SUSPENSION CULTURES

SummaryExperiments were carried out with Taxus baccata cell lines showing different paclitaxel-producing capacities (between 1.74 and 19.91 mgl−1) when growing in a selected product-formation medium that specifically stimulated the production of taxane to the detriment of cell growth. Through mixing low-, medial- and high-producing lines, it could be observed that paclitaxel productivity in the resulting mixed lines was clearly higher than the mean productivity of the individual lines before mixing. This suggests that culture components generated by high-producing individual lines within the population might induce paclitaxel production. Although the accumulation of paclitaxel and baccatin III was higher when 100 μM methyl jasmonate was added to the subcultures of the mixed lines, the results indicate that exogenously applied methyl jasmonate was not the first factor to stimulate taxane production. The possible effects of methyl jasmonate elicitation and paclitaxel accumulation on cell viability are also considered.

Effect of Benzyladenine and Indolebutyric Acid on Ultrastructure, Glands Formation, and Essential Oil Accumulation in Lavandula Dentata Plantlets

Lavandin (Lavandula dentata) axillary buds were grown in Linsmaier-Skoog (LS) medium solidified with 10 % bactoagar (control) and supplemented with 0.1 mg dm−3 benzyladenine (BA), 0.1 mg dm−3 indolebutyric acid (IBA) or both plant growth regulators. In the studied conditions the axillary buds developed into plantlets. The addition of BA inhibited the formation of glands by 44 % as compared with the control plantlets and also inhibited their development: these plantlets had the highest number of unbroken glands (in pre-secretory state) when compared with plantlets grown in the other conditions. The presence of BA stimulated chloroplast formation, and increased the content of essential oils by 150 % with respect to the control plantlets. It also increased their secretion, and the number of lipid droplets in the chloroplasts, cytosol and plasmalemma. On the contrary, the presence of IBA decreased the essential oil concentration in plantlets by 31 % when compared with the control ones and inhibited their secretion capacity.

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D2 ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.

Immobilization of Galphimia glauca Plant Cell Suspensions for the Production of Enhanced Amounts of Galphimine-B

We tested the capacity of Galphimia glauca cells to produce galphimine-B (G-B) when under the effects of a two-stage culture system: cell immobilization in Ca2+-alginate beads and culture scale-up from shake-flask to two different types of bioreactor (stirred and airlift). In the shake-flask culture, using optimum media for cell growth (first stage) and G-B production (second stage), the G-B yield was similar in both immobilised and free cells. However, while the free cells accumulated G-B within cytoplasmatic compartments, where it could not be recovered without cell disruption, immobilized cells excreted up to 100 % of the G-B produced. Immobilized cells grown in bioreactors running for 14 days with growth medium and an additional 26 days with production medium in batch mode showed a high G-B yield. The stirred bioreactor was the most efficient with a G-B content in the culture medium of 1381 microg.L (-1) at day 24 of culture.

In vitro micropropagation of Ruscus aculeatus

We have developed three protocols for the rapid micropropagation of Ruscus aculeatus. The primary explants utilised were immature embryos, aerial buds excised from rhizomes and shoot buds regenerated from organogenic calli. In order to increase the plant regeneration from the primary explants, we used organogenic calli from cladode, stem and rhizome segments. We tested more than 20 culture media for callus induction and shoot regeneration and the best results were obtained when rhizome segments were cultured on Murashige and Skoog medium supplemented with 0.5 mg dm−3 2,4-dichlorophenoxyacetic acid and 1 mg dm−3 kinetin.

Effect of the univalent cations on the protein and nicotine content in Nicotiana rustica plants

Abstract Nicotiana rustica L. plants treated with Li, Na or Rb in replacement of half the K in the nutrient medium grew less than the control plants. The ratio of nicotine in plant to its dry weight or protein content in the roots (capacity per unit of dry weight or protein to produce nicotine) was similar for the plants treated with Li or Na, but much higher for those treated with Rb. The nutritional stress produced mainly by the Rb drastically reduced its growth and primary metabolism, and stimulated its secondary metabolism.