Frédéric Jullien

Analysis of gene expression in rose petals using expressed sequence tags

Single‐pass sequences were obtained from the 5′‐ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent.

Biosynthesis of the major scent components 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose O-methyltransferases

In Chinese rose species and in many modern varieties, two methylated phenolic derivatives, 3,5‐dimethoxytoluene and 1,3,5‐trimethoxybenzene, are major scent components. We show that cell‐free extracts of rose petals catalyse the synthesis of 3,5‐dimethoxytoluene and 1,3,5‐trimethoxybenzene by methylation of precursor molecules. An expressed sequence tag approach was used to identify four highly similar O‐methyltransferase sequences expressed specifically in petals and anthers. Thin layer chromatography analysis showed that the activities of these enzymes with different substrates and the proportions of reaction products produced closely mimicked those observed using cell‐free petal extracts, indicating that orcinol O‐methyltransferases are responsible for the biosynthesis of 3,5‐dimethoxytoluene and 1,3,5‐trimethoxybenzene from un‐methylated precursors in this organ.

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.

Direct regenerationin vitro and transient GUS expression inMentha xpiperita.

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient β-glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l−1) and 6-benzylaminopurine (2 mg l−1), followed by selection of regenerated shoots with 200 mg 1−1 kanamycin.

Mannitol and thidiazuron improve in vitro shoot regeneration from spearmint and peppermint leaf disks

In vitro shoot organogenesis of peppermint and spearmint was obtained from leaf disks. Regeneration occurred within six weeks of placement in culture. Best results were obtained when explants were cultured for two weeks onto Murashige and Skoog medium supplemented with 300 mM mannitol, 2.0 μM 6-benzyladenine and 2.0 μM indole-3-butyric acid, and then transferred on a medium without mannitol and containing 0.5 μM α-naphthaleneacetic acid, 9.0 μM 6-benzyladenine and 0.5 μM thidiazuron. Using these culture conditions, percentages of regeneration were 78% for peppermint and 49% for spearmint. Because of its efficiency, this leaf disk regeneration method could be a suitable tool for genetic transformation with Agrobacterium tumefaciens.

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.

Free flavonoid aglycones as markers of parentage in Mentha aquatica, M. citrata, M. spicata and M. x piperita

Abstract External lipophilic methylated flavonoids have been extracted from dried leaves of Mentha aquatica, M. spicata, M. x piperita and M. citrata. After separation and purification, twenty flavonoids have been identified by means of spectrometric methods (UV, EIMS, 1H NMR). The flavonoid patterns of these species and hybrid support the view that M. x piperita may be a hybrid of M. aquatica and M. spicata while a linalool-producing sample of M. citrata may be considered a variety of M. aquatica. Cytological data agree with the observed biochemical results.

Highly oxygenated flavones from Mentha piperita

Abstract Six highly oxygenated flavones have been isolated from the leaves of Mentha piperita . Five known compounds, 5-hydroxy-6,7,8,4′-tetramethoxyflavone, 5,4′-dihydroxy-6,7,8-trimethoxyflavone, 5,3′-dihydroxy-6,7,8,4′-tetramethoxyflavone, 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone and 5,3′,4′-trihydroxy-6,7,8-trimethoxyflavone, are reported for the first time in the genus Mentha . The sixth compound has been identified as 5,6-dihydroxy-7,8,3′,4′-tetramethoxyflavone by UV, NMR and mass spectra.

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).