Tinkering with the C-Function: A Molecular Frame for the Selection of Double Flowers in Cultivated Roses

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La morphogenèse florale chez le rosier (identification et caractérisation de gènes impliqués dans le phénomène fleur double et optimisation d'un protocole de transformation génétique stable)

Le phénomène fleur double (ou duplicature), particulièrement important chez les rosiers modernes cultivés (Rosa x hybrida), consiste en une augmentation significative du nombre de pétales par rapport au type sauvage. Nos modèles d étude sont R. chinensis cv Old Blush , rosier à fleur double et R. chinensis Mutabilis , rosier à fleur simple. L objectif de la thèse était d étudier les phénomènes moléculaires impliqués dans la duplicature. Peu de données étaient disponibles sur le développement floral du rosier. Trois approches complémentaires ont été abordées: (i) la caractérisation de gènes candidats potentiellement impliqués dans le contrôle du nombre de pétales par fleur, (ii) le développement d un outil pour l analyse du transcriptome: une puce à ADN rosier et (iii) l optimisation d un protocole de transformation génétique stable chez le rosier qui permet, par des expériences de génétique fonctionnelle, de valider les fonctions potentielles des gènes étudiés.Petal traits heavily influence flower quality. My thesis work focused on understanding the molecular events controlling petal morphogenesis and number in rose, by using different approaches. Although flower development has been well characterized in model species (i.e. Arabidopsis), very little is known in ornamental plants. We established a detailed description of the rose flower development from organs initiation until opening of the bud. To identify genes involved in flower development we used three approaches: (i) a candidate gene approach allowed us to identify and functionally analyzed a set of genes potentially associated with this phenomenon; (ii) a microarrays approach (using the available rose ESTs) is being used to compare transcriptome of rose plants presenting contrasted floral phenotypes. (iii) we developed a genetic transformation protocol for 2 rose species (modern and botanic) to validate the function of our candidate genes.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Versatile somatic embryogenesis systems and transformation methods for the diploid rose genotype Rosa chinensis cv Old Blush.

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Somatic embryogenesis and transformation of the diploid Rosa chinensis cv Old Blush

International audienceSomatic embryogenesis was induced from in vitro-derived leaf explants of Rosa chinensis cultivar (cv) Old Blush. Calli producing embryos with expanded cotyledons (RcOBType1 embryos) were obtained. Further refinements of the callus maintenance medium generated a more typical rose embryogenic callus (RcOBType2) displaying high levels of secondary embryogenesis and embryos with limited cotyledon expansion Agrobacterium tumefaciens-mediated transformation assays using β-glucuronidase (GUS) reporter gene showed that both types of embryos were competent for transformation. Under selection conditions, transformed RcOBType1 explants produced non chimaeric transformed embryos, from which shoots could be adventitiously regenerated. In contrast to RcOBType1, transformed RcOBType2 embryos directly yielded transformed shoots when repeatedly cultured in selective regeneration conditions. Transformation efficiency ranged between three to nine percent and shoots suitable for rooting were obtained within 6-8 months. Transgenic plants were transferred into the greenhouse and molecularly confirmed. The availability of transformation methods in a diploid rose, R. chinensis cv. Old Blush, will be useful for gene functional studies

pur4 Mutations Are Lethal to the Male, But Not the Female, Gametophyte and Affect Sporophyte Development in Arabidopsis[C][W]

Purine metabolism is crucial in living cells and involves three complex pathways in plants: the de novo synthesis, the salvage, and the degradation pathways. The relative importance of each pathway in plant development and reproduction, however, is still unclear. We identified two T-DNA insertions in the Arabidopsis (Arabidopsis thaliana) PUR4 gene (At1g74260) that encodes formylglycinamidine ribonucleotide synthase (EC 6.3.5.3), the fourth enzyme in the de novo purine biosynthesis pathway. The mutated alleles were never transmitted through the pollen of heterozygous plants but could be inherited through the female gametophyte, indicating that de novo purine synthesis is specifically necessary for pollen development. Because the pur4 mutations were lethal to the male gametophyte, homozygous pur4 plants could not be obtained. However, the reproductive phenotype of hetererozygous plants carrying the pur4-2 mutated allele was more severe than that carrying the pur4-1 mutated allele, and pur4-2/+ plants showed slightly delayed early development. We showed that the pur4-2 allele produces an antisense transcript and that the amount of PUR4 mRNA is reduced in these plants. Transient expression of a translational fusion with the green fluorescent protein in Arabidopsis plantlets showed that the formylglycinamidine ribonucleotide synthase protein is dually targeted to chloroplast and mitochondria, suggesting that at least some steps of the de novo purine biosynthesis pathway can take place in both organelles in Arabidopsis, a dual location previously thought to be a peculiarity of ureide-forming tropical legumes

Genomic approach to study floral development genes in Rosa sp.

International audienceCultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower