Although cottonseed is an important source of oil and fiber, the development of cotton embryos has not been investigated as well as development of cotton fiber. The development of cotton embryos in late heart-stage and early cotyledonary stage is less well investigated than the first 10-14 days after anthesis, or the late stages of embryo development during seed-fill and desiccation. This analysis focused on cotton embryos in the late heart-stage and early cotyledonary stage of development (1.5-4.0 mm or about 13-18 DPA). In vitro analyses are important tools for studying embryos in isolation from the endosperm and fiber and when it is necessary to monitor the developing embryo continuously. The original goal of this work was to develop an in vitro culture method that would support continued development of excised zygotic embryos from the early cotyledonary stage into complete plants with true shoots, i.e. true leaves or visible buds and then to use this method to study aspects of developmental regulation during cotyledonary stage and the transition to later stages. Not all embryos were competent to develop true shoots (an apical bud or a leaf plus a bud) in culture. A number of cultural variables were tested and eliminated. Embryo maturity at the time embryos were excised and the presence or absence of light during the first 14 days of culture affected the competence of immature embryos to developed true shoots. The effect of light was verified in several large replicated experiments. Morphological changes occurring during in vivo development were examined microscopically. The transition from heart-stage to early cotyledonary stage and the development of the first leaf from initials to a large structure were identified. Embryonic shoot apices continued to grow in cultured 1-3 mm embryos. The size and shape of light-treated and dark-treated embryonic apices was compared. A germination test of mature seeds identified seedlings with a similar phenotype occurring at similar rates in seedlings and light-cultured embryos and possible causes were discussed.

Chez le pin maritime l’embryogenèse somatique, méthode de multiplication végétative performante, n’est pas optimisée la limite étant le contrôle du développement des embryons somatiques (ES). Nos objectifs ont été 1) d’étudier les mécanismes physiologiques, cellulaires et moléculaires précoces contrôlant la différenciation des ES en réponse à une disponibilité en eau réduite 2) d’évaluer pour l'ES cotylédonaire de 12 semaines son état de maturité, son protéome afin de les comparer à l'embryon zygotique (EZ). 1) Pour le 1er objectif, le rapprochement de données de transcriptomique et de protéomique a été entrepris. Nous avons observé une réponse physiologique et moléculaire ABA-dépendante entrainant une transition précoce de la prolifération vers la différenciation des ES (surexpression de protéines impliquées dans la division cellulaire, l'embryogenèse et la synthèse de l'amidon). Une protéine de type germine et une ubiquitine ligase apparaissent comme marqueurs potentiels de l’embryogenèse somatique précoce du pin maritime, alors que la protéine phosphatase 2C marque la réponse adaptative à l’environnement de culture. 2) La maturité de l’ES cotylédonaire a été étudiée aux niveaux physiologiques (masse sèche, teneur en eau) et biochimiques (teneur en protéines totales, en sucres solubles). Des ES de 10, 12 ou 14 semaines se révèlent semblables. Une méthode de classification hiérarchique ascendante basée sur 9 variables explicatives, montre que l’ES est similaire à l’EZ cotylédonaire frais (protéomes présentant 94% d’homologie). Parmi les protéines communes, 3 familles ont été identifiées (protéines de réserve, protéines de réponse au stress HSP, protéines LEA) ainsi que l’aldose réductase et l’adénosine kinase. Nous les proposons comme marqueurs génériques du stade cotylédonaire de l'embryogenèse tardive du pin maritime. L’ensemble des résultats contribue à une meilleure compréhension de l’embryogenèse somatique du pin maritime. / In maritime pine somatic embryogenesis, a powerful method of vegetative propagation, is not optimized the limitation being the control of the somatic embryo (SE) development. Our objectives were 1) To study the early physiological, cellular and molecular mechanisms controlling SE differentiation in response to a reduced water availability 2) to estimate for cotyledonary SE 12 weeks old, its maturity, its proteome to compare them to the zygotique embryo (ZE). 1) For the 1st objective, transcriptomic and proteomic data were combined. We observed a physiological and molecular answer ABA-dependent, inducing an early transition from proliferation towards SE differentiation (surexpression of proteins involved in the cellular division, the embryogenesis and in starch synthesis). A protein of germin type and an ubiquitine ligase appear as potential markers of the early somatic embryogenesis of maritime pine, while the phosphatase protein 2C stands out the adaptive answer to the environment of culture. 2) Cotyledonary SE maturity was studied at the physiological (dry weight, water content) and biochemical levels (total protein content, soluble sugars). SE of 10, 12 or 14 weeks old appeared very similar. A hierarchical ascendant cluster analysis based on 9 explanatory variables, shows that SE is similar to the fresh cotyledonary EZ; proteome profiling further confirmed high similarity (94.5%) between them. Protein profiling revealed biomarkers belonging to 3 large families of proteins (HSP, LEA and storage proteins) or the aldose reductase and the adenosine kinase. We propose them as generic markers of the cotyledonary stage of the late embryogenesis of the maritime pine. All the results contribute to a better understanding of the somatic embryogenesis of maritime pine.