Isabel Arrillaga

Up-Regulation of 1-Deoxy-d-Xylulose-5-Phosphate Synthase Enhances Production of Essential Oils in Transgenic Spike Lavender

Spike lavender (Lavandula latifolia) is an aromatic shrub cultivated worldwide for the production of essential oils. The major constituents of these oils are monoterpenes, which are obtained from isopentenyl diphosphate and dimethylallyl diphosphate precursors through the plastidial methylerythritol phosphate (MEP) pathway and/or the cytosolic mevalonate pathway. 1-Deoxy-d-xylulose-5-P synthase (DXS) catalyzes the first step of the MEP pathway. A cDNA coding for the Arabidopsis (Arabidopsis thaliana) DXS was constitutively expressed in spike lavender. Gas chromatography/mass spectrometry analyses revealed that transgenic plants accumulated significantly more essential oils compared to controls (from 101.5% to 359.0% and from 12.2% to 74.1% yield increase compared to controls in leaves and flowers, respectively). T0 transgenic plants were grown for 2 years, self-pollinated, and the T1 seeds obtained. The inheritance of the DXS transgene was studied in the T1 generation. The increased essential oil phenotype observed in the transgenic T0 plants was maintained in the progeny that inherited the DXS transgene. Total chlorophyll and carotenoid content in DXS progenies that inherited the transgene depended on the analyzed plant, showing either no variation or a significant decrease in respect to their counterparts without the transgene. Transgenic plants had a visual phenotype similar to untransformed plants (controls) in terms of morphology, growth habit, flowering, and seed germination. Our results demonstrate that the MEP pathway contributes to essential oil production in spike lavender. They also demonstrate that the DXS enzyme plays a crucial role in monoterpene precursor biosynthesis and, thus, in essential oil production in spike lavender. In addition, our results provide a strategy to increase the essential oil production in spike lavender by metabolic engineering of the MEP pathway without apparent detrimental effects on plant development and fitness.

Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves

We generated transgenic spike lavender (Lavandula latifolia) plants constitutively expressing the limonene synthase (LS) gene from spearmint (Mentha spicata), encoding the LS enzyme that catalyzes the synthesis of limonene from geranyl diphosphate. Overexpression of the LS transgene did not consistently affect monoterpene profile in pooled leaves or flowers from transgenic T(0) plants. Analyses from cohorts of leaves sampled at different developmental stages showed that essential oil accumulation in transgenic and control plants was higher in developing than in mature leaves. Furthermore, developing leaves of transgenic plants contained increased limonene contents (more than 450% increase compared to controls) that correlated with the highest transcript accumulation of the LS gene. The levels of other monoterpene pathway components were also significantly altered. T(0) transgenic plants were grown for 2 years, self-pollinated, and the T(1) seeds obtained. The increased limonene phenotype was maintained in the progenies that inherited the LS transgene.

Assessments of somaclonal variation in micropropagated shoots of Cedrus: consequences of axillary bud breaking

Reversed-phase HPLC analysis and random amplified polymorphic DNA (RAPD) markers were used to monitor DNA methylation status and genetic stability of C. atlantica and C. libani shoots generated through axillary bud proliferation. Average DNA methylation in C. atlantica or C. libani seedlings and mature 200-year-old trees of C. libani was 19.8, 19.5 and 22.3%, respectively. These global amounts showed no significant variation after the in vitro establishment of seedling-originated shoot stocks. In contrast, in vitro culture caused a significant decrease in the amount of 5-methylcytosine in genomic DNA of the tissue culture (TC) progenies of one of the adult C. libani genotypes. This DNA demethylation event accompanied an enhancement of the regrowth capacity of this genotype. Detected RAPD variation between mother plants and their TC progenies was species-related, with C. libani TC progenies being genetically more stable than those of C. atlantica. Nevertheless, similarity indices ranged from 0.97 to 1 among mother plants and their TC progenies. Furthermore, the analyses of molecular variance (AMOVA) suggest that RAPD variation among the mother plants and their TC progenies might be considered as not significant. The application of various statistical approaches, including cluster-based genetic distance methods and AMOVA, demonstrates that RAPD markers discriminate C. atlantica and C. libani appropriately.

Somatic Embryogenesis and Plant Regeneration from Hypocotyl Cultures of Digitalis obscura L.

Summary Hypocotyl sections from 20-day-old seedlings of Digitalis obscura L. were used to examine the effect of IAA on somatic embryogenesis. Although embryogenesis was obtained with all IAA concentrations (0.1, 0.5 and 1 ppm) and under both illumination conditions tested (16 h photoperiod and darkness), incubation in photoperiod increased yield of embryos and shortened the embryo induction time. Optimum results were obtained with 1 ppm IAA. About 30 % of the embryos obtained developed into complete plantlets when transferred to basal medium without growth regulators.

In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.

Somatic embryogenesis from hypocotyl callus cultures of Digitalis obscura L.

Hypocotyl-derived calli obtained in agar solidified medium with several growth regulator combinations gave rise to proembryonal masses and globular embryos when transferred to liquid media with lower growth regulator and higher NH4HO3 levels. By transferring cultures from liquid media to different solidified media, new embryo formation took place, but further development of these embryos or those previously induced depended on the characteristics of these media. Normal development was only achieved on 8 g/l agar solidified medium without growth regulators. Typical cotyledonary embryos developed into whole plants when transferred to this same medium.

Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves

Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.

Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender

Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.

Plant hormones and Agrobacterium tumefaciens strain 82.139 induce efficient plant regeneration in the cardenolide-producing plant Digitalis minor

Summary Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation and adventitious bud differentiation, by varying the amount and source of plant hormones. Shoot regeneration was also obtained after infection of D. minor with the wild-type Agrobacterium tumefaciens strain 82.139, which induced shooty tumours. These shoots were not transgenic, as revealed by nopaline assays and the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated gusA gene. Plants were easily rooted and transplanted into the greenhouse. Shoot cultures of D. minor accumulated up to 226 μg cardenolides per g dry mass when cultured on agar-solidified medium. Cardenolide content was significantly lower in submerged shoot cultures, the effect being related to a high incidence of hyperhydricity. Greenhouse-grown plants presented the highest amounts of these metabolites. This is the first report on micropropagation of the Balearic endemic medicinal plant species D. minor . The induction of caulogenic galls by A. tumefaciens wild strain 82.139 opens new possibilities for the biotechnological improvement of Digitalis spp.