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BackgroundVanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.ResultsFor embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.ConclusionThe subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate...

The spread of Sugarcane yellow leaf virus (SCYLV) in sugarcane plants was studied on Réunion using virus-infected cuttings from four cultivars (R570, R575, R577 and R579). One month after the germination of cuttings in an insect-proof glasshouse, SCYLV was detected by reverse-transcription polymerase chain reaction (RT-PCR) and tissue-blot immunoassay (TBIA) in the leaves, shoots and roots of all cultivars. The distribution of SCYLV in the whole plant did not vary over a 10-to 11-month period of growth. In addition, the spread of SCYLV in sugarcane fields on Réunion was investigated during a survey conducted from 1998 to 2001. Samples were taken in three sugarcane-growing areas, and TBIA was used to detect SCYLV in the three major cultivars (R570, R575 and R579). The percentage of infected stalks varied according to cultivar and growing area, but remained relatively stable for a given cultivar in a given growing area over the 30-month survey period. Cultivar R575 was the most infected cultivar in all three growing areas (mean of 98% infected stalks). The percentage of infected stalks ranged from 16 to 94% in cv. R570 and from 21 to 92% in cv. R579. These results suggested that on Réunion: (i) infected sugarcane stools do not recover from the disease after harvesting; and (ii) the virus is mainly propagated by planting infected cuttings. SCYLV was detected by RT-PCR in the aphid Melanaphis sacchari , a potential vector of this virus. Two months after planting virus-free plants of susceptible cv. R575 in a field surrounded by sugarcane infected with SCYLV, 14% of plants were found infected with the virus. Four months later, 25% of plants were found infected with SCYLV, but no new infections were detected between 6 and 12 months after planting. In the first ratoon crop, 42% of plants were infected with SCYLV after 6 months of growth. Spatial distribution of infected plants suggested that, on Réunion, a small window of time (between 0 and 2 months after planting cuttings) exists during which primary infection can occur. Based on the results obtained in this study, the use of clean planting material for some cultivars and the use of tolerant cultivars should be an efficient means of controlling sugarcane yellow leaf on Réunion.

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The metabolomic analysis of developing Vanilla planifolia green pods (between 3 and 8 months after pollination) was carried out by nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Multivariate data analysis of the (1)H NMR spectra, such as principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA), showed a trend of separation of those samples based on the metabolites present in the methanol/water (1:1) extract. Older pods had a higher content of glucovanillin, vanillin, p-hydroxybenzaldehyde glucoside, p-hydroxybenzaldehyde, and sucrose, while younger pods had more bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A), bis[4-(beta-D-glucopyranosyloxy)-benzyl]-2-(2-butyl)tartrate (glucoside B), glucose, malic acid, and homocitric acid. A liquid chromatography-mass spectrometry (LC-MS) analysis targeted at phenolic compound content was also performed on the developing pods and confirmed the NMR results. Ratios of aglycones/glucosides were estimated and thus allowed for detection of more minor metabolites in the green vanilla pods. Quantification of compounds based on both LC-MS and NMR analyses showed that free vanillin can reach 24% of the total vanillin content after 8 months of development in the vanilla green pods.