Transcriptome analysis in pea allows to distinguish chilling and acclimation mechanisms.

In order to distinguish chilling and freezing tolerance mechanisms in pea, responses to cold exposure were compared between the freezing tolerant line Champagne and the sensitive line Terese. Global gene expression was considered in the two lines and associated with morphological, histological and biochemical approaches. The chilling tolerance in both lines was related to responses of the CBF, COR and LEA genes belonging to the CBF regulon, with greater earliness of expression in the Champagne genotype. The freezing tolerance, only observed in Champagne, was associated with acclimation processes such as cellular osmotic stabilization, photosynthesis modifications, antioxidants production, modifications in hormone metabolism, cell wall composition and dynamics.

The flowering locus Hr colocalizes with a major QTL affecting winter frost tolerance in Pisum sativum L.

An understanding of the genetic determinism of frost tolerance is a prerequisite for the development of frost tolerant cultivars for cold northern areas. In legumes, it is not known to which extent vernalization requirement or photoperiod responsiveness are necessary for the development of frost tolerance. In pea (Pisum sativum L.) however, the flowering locus Hr is suspected to influence winter frost tolerance by delaying floral initiation until after the main winter freezing periods have passed. The objective of this study was to dissect the genetic determinism of frost tolerance in pea by QTL analysis and to assess the genetic linkage between winter frost tolerance and the Hr locus. A population of 164 recombinant inbred lines (RILs), derived from the cross Champagne x Terese was evaluated both in the greenhouse and in field conditions to characterize the photoperiod response from which the allele at the Hr locus was inferred. In addition, the population was also assessed for winter frost tolerance in 11 field conditions. Six QTL were detected, among which three were consistent among the different experimental conditions, confirming an oligogenic determinism of frost tolerance in pea. The Hr locus was found to be the peak marker for the highest explanatory QTL of this study. This result supports the hypothesis of the prominent part played by the photoperiod responsiveness in the determinism of frost tolerance for this species. The consistency of three QTL makes these positions interesting targets for marker-assisted selection.

InsP(3)-mediated calcium release induced by heterologous expression of total chicory Leaf RNA.

A calcium dependent-chloride current (I(ni)) was recorded in Xenopus oocytes injected with total RNA from chicory leaf tissues, following depolarization from -35 to +60 mV. However, the signal transduction mechanism mediating I(ni) is unknown. The development of this current was mimicked by intracellular injection of the second messenger InsP(3) in control (non-injected) oocytes. Moreover, InsP(3) injection after I(ni) rundown did not reinitiate the current. The same phenomenon was observed following a second injection into control oocytes. Measurement of InsP(3) production in injected oocytes showed a net increase in the InsP(3) level on depolarization. Moreover, extracellular application of caffeine (5 mM) significantly reduced the number of oocytes displaying I(ni). Also, extracellular application of U-73122, a potent PLC inhibitor, clearly reduced the occurrence of I(ni). These data provide the first evidence that the calcium homeostasis mechanism induced by heterologous expression of total RNA from chicory leaves involves the InsP(3) signaling pathway.

A nonsymbiotic hemoglobin gene is expressed during somatic embryogenesis in Cichorium.

After differential screening of a cDNA library corresponding to genes expressed during the early stages of somatic embryogenesis in leaf tissue from the Cichorium hybrid '474' (C. intybus L., var. sativumxC. endivia L., var. latifolia) a nonsymbiotic hemoglobin cDNA was obtained. Studies of the expression of the gene corresponding to this clone by Northern blot analysis suggest that in Cichorium a nonsymbiotic hemoglobin gene is specifically expressed under somatic embryogenesis-inducing conditions, and that its expression is not related to stress caused by wounding or tissue culture conditions.

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization.

Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10(6) mature pollen protoplasts were fused with 7·10(6) mesophyll protoplasts using a PEG/Ca(2+) method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l(-1)). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l(-1)). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants.

Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.