Jean-Claude Caissard

Localization of production and emission of pollinator attractant on whole leaves of Chamaerops humilis (Arecaceae)

Volatile compounds, which frequently play important roles in plant-insect interaction, can be produced either by flowers to attract pollinators or by leaves to deter herbivores. The specialized structures associated with odor production differ in these two organs. The European dwarf palm Chamaerops humilis represents a unique intermediate between these two. In previous work, its leaves were shown to produce volatile organic compounds (VOCs) that attract pollinators only during flowering. Because the leaf sinuses look like a gland, the sinus was examined histologically and with environmental scanning electron microscopy (ESEM) for evidence that the sinus emits VOCs. Volatile compounds emitted by the different parts of the leaf were extracted by washes and headspace then analyzed by gas chromatograph-mass spectrometer (GC-MS). The sinus does not have the expected gland-like structure; the VOCs are actually produced by the whole leaf, even if the composition of the VOCs emitted by the sinus slightly differs. Thus, attraction of pollinators does not result from specialized secreting cells in leaves of flowering European dwarf palms. The results are discussed in the context of a convergent evolution of leaves toward petals.

High efficiency transformation of peppermint (Mentha × piperita L.) with Agrobacterium tumefaciens

Transgenic peppermint (Mentha × piperita L.) plants were obtained by using Agrobacterium tumefaciens-mediated gene transfer. The effects of the coculture period and of the Agrobacterium strain were tested. 10% transformed plants were regenerated by leaf disk culture after inoculation with strain EHA105MOG harbouring β-glucuronidase and neomycin phosphotransferase II genes, with a coculture period of 5 days. Rooting of regenerated plants was achieved on selective medium with 150 mg/l kanamycin. The presence of transgenes in DNA was shown through PCR and Southern blot hybridization and transgene product activity via histoenzymatic GUS test and leaf callus assay. Transgenic plants were successfully acclimatized in the greenhouse.

A simple and efficient method for in vitro shoot regeneration from leaves of lavandin (Lavandula××intermedia Emeric ex Loiseleur)

Abstract A shoot regeneration system from leaves of lavandin (Lavandula×intermedia Emeric ex Loiseleur) was developed. The best results were obtained using a set of four different media. Callus was obtained on Murashige and Skoog (MS) medium containing 9 μm 6-benzylaminopurine and 4.5 μmα-naphthaleneacetic acid. After 2 weeks of culture, calli were transferred onto MS medium supplemented with 18 μm 6-benzylaminopurine to trigger bud regeneration. Shoot elongation was then stimulated by 1 μm gibberellin A3. Rooting was induced with 1 μm indole-3-butyric acid. All plantlets survived to greenhouse acclimatization. This is the first description of bud regeneration from leaves of lavandin.

Agrobacterium tumefaciens-mediated transformation of Mentha spicata and Mentha arvensis

The stable integration of GUS and NPTII genes in Mentha arvensis and M. spicata has been achieved by Agrobacterium tumefaciens-mediated gene transfer. Transformation assays were performed by cocultivating plant leaf disks with either GV2260/GI or EHA105/MOG Agrobacterium strains. Transgenic plants were selected on medium containing 150 mg l−1 kanamycin. Transgene presence and structure was studied by the use of PCR analysis and Southern blot hybridization. Transgene expression was evaluated by RT-PCR and transgene product activity by a histoenzymatic GUS assay.

Agrobacterium-mediated transformation of lavandin (Lavandula x intermedia Emeric ex Loiseleur)

Lavandin (Lavandula x Emeric ex Loiseleur) is an aromatic plant, the essential oil of which is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. The qualitative or quantitative modification of its terpenes‐containing essential oil by genetic engineering could have important scientific and commercial applications. In this study, we report the first Agrobacterium tumefaciens‐mediated gene transfer into lavandin. The transformation protocol was optimized by lengthening precultivation and cocultivation periods and by testing five different bacterial strains. We obtained transformed callus lines at a frequency of 40–70 with strains AGL1/GI, EHA105/GI and C58/GI. Transgenic shoots were regenerated from these kanamycin resistant calli and rooted on selective medium with 150 mg l-1 kanamycin. The final percentage of transgenic plants obtained varied from 3 to 9, according to the strain used, within 6 months of culture. The presence of the introduced β‐glucuronidase and neomycin phosphotransferase II genes was shown both by PCR and Southern blot analysis. Transgene expression was investigated using histoenzymatic β‐glucuronidase assays, leaf callus assays and RT‐PCR. Results showed that both β‐glucuronidase and neomycin phosphotransferase II genes were expressed at a high level in at least 41 of the transgenic plants regenerated. This efficient transformation strategy could be used to modify some genetic traits of lavandin (flower colour, pathogens resistance) and to study the biosynthesis of the major monoterpene components of its essential oil (linalool, linalyl acetate, camphor and 1,8‐cineole).

Extracellular Localization of the Diterpene Sclareol in Clary Sage (Salvia sclarea L., Lamiaceae)

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.

Production and Emission of Volatile Compounds by Petal Cells

We localized the tissues and cells that contribute to scent biosynthesis in scented and non-scented Rosa x hybrida cultivars as part of a detailed cytological analysis of the rose petal. Adaxial petal epidermal cells have a typical conical, papillate shape whereas abaxial petal epidermal cells are flat. Using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that, in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis, was localized in both epidermal layers. These results are discussed in view of results found in others species such as Antirrhinum majus, where it has been shown that the adaxial epidermis is the preferential site of scent production and emission.