Constitutive Overexpression of a Conifer WOX2 Homolog Affects Somatic Embryo Development in Pinus pinaster and Promotes Somatic Embryogenesis and Organogenesis in Arabidopsis Seedlings

Although full sequence data of several embryogenesis-related genes are available in conifers, their functions are still poorly understood. In this study, we focused on the transcription factor WUSCHEL-related HOMEOBOX 2 (WOX2), which is involved in determination of the apical domain during early embryogenesis, and is required for initiation of the stem cell program in the embryogenic shoot meristem of Arabidopsis. We studied the effects of constitutive overexpression of Pinus pinaster WOX2 (PpWOX2) by Agrobacterium-mediated transformation of P. pinaster somatic embryos and Arabidopsis seedlings. Overexpression of PpWOX2 during proliferation and maturation of somatic embryos of P. pinaster led to alterations in the quantity and quality of cotyledonary embryos. In addition, transgenic somatic seedlings of P. pinaster showed non-embryogenic callus formation in the region of roots and subsequently inhibited root growth. Overexpression of PpWOX2 in Arabidopsis promoted somatic embryogenesis and organogenesis in a part of the transgenic seedlings of the first and second generations. A concomitant increased expression of endogenous embryogenesis-related genes such as AtLEC1 was detected in transgenic plants of the first generation. Various plant phenotypes observed from single overexpressing transgenic lines of the second generation suggest some significant interactions between PpWOX2 and AtWOX2. As an explanation, functional redundancy in the WOX family is suggested for seed plants. Our results demonstrate that the constitutive high expression of PpWOX2 in Arabidopsis and P. pinaster affected embryogenesis-related traits. These findings further support some evolutionary conserved roles of this gene in embryo development of seed plants and have practical implications toward somatic embryogenesis induction in conifers.Peer Reviewe

Identificación y regulación transcripcional de genes arogenato deshidratasa implicados en la biosíntesis de kignina en pino marítimo

La fenilalanina es un aminoácido esencial para la síntesis de proteínas pero también un precursor de una gran variedad de compuestos del metabolismo secundario que son esenciales para el crecimiento, desarrollo y defensa de las plantas. La biosíntesis de la pared celular secundaria que tiene lugar durante la formación de madera en árboles implica la biosíntesis masiva de lignina, un polímero que no contiene nitrógeno pero que deriva metabólicamente de la fenilalanina. Por lo tanto, estas plantas requieren una coordinación metabólica precisa entre la biosíntesis de fenilalanina y la biosíntesis de lignina para asegurar su desarrollo y crecimiento anual. En este estudio, hemos encontrado que la enzima arogenato deshidratasa, que cataliza el último paso en la ruta biosintética de la fenilalanina en las plantas, se regula transcripcionalmente a través de la interacción directa con el factor de transcripción PpMYB8. El análisis transcripcional de plantas de pino marítimo silenciadas para PpMyb8 sugiere que este factor de transcripción está directamente involucrado en la biogénesis de la pared celular secundaria y en los procesos de muerte celular. En conjunto, estos resultados indican que un único factor de transcripción coordina la biosíntesis de fenilalanina y la acumulación de lignina durante la formación de madera en las coníferas.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

El análisis transcriptómico de embriones somáticos y cigóticos de pino revela diferencias de expresión en diferentes rutas metabólicas

La embriogénesis es un proceso complejo en las plantas, pero comprenderlo en las coníferas es especialmente crítico no sólo para poder hacer estudios comparativos con angiospermas sino también para producir embriones viables y de calidad con diferentes propósitos. Actualmente falta una visión comparativa global de los genes implicados en la embriogénesis somática y cigótica del pino. En este trabajo presentamos un análisis del transcriptoma en tres etapas del desarrollo de los embriones, identificando procesos biológicos conservados y funciones genéticas activas durante el proceso de embriogénesis somática y cigótica. La mayoria de las diferencias son más significativas a medida que avanza el desarrollo. Cuando comparamos etapas de desasrrollo similares, como es el estadio embrión maduro encontramos 1640 genes sobreexpresados en embriones cigóticos frente a 4814 genes en embriones somáticos.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Caractérisation et variation d'une famille multigénique, l'ADN ribosomique 5S nucléaire, chez quatre espèces forestières des genres larix M. (Pinaceae) et Quercus L. (Fagaceae)

Not availableNon disponibl

Qualité génétique de lots de graines récoltés dans des vergers clonaux de pin maritime

International audienc

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

Douglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programs is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation are essential biotechnology tools in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Litvay et al. 1985). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realise increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France

Flowering intensity and phenology in clonal seed orchards of maritime pine

International audienc

Pollen contamination and mating structure in maritime pine ( Pinus pinaster Ait.) clonal seed orchards revealed by SNP markers

Maritime pine ( Pinus pinaster Ait.) is a major forest tree species in south-western Europe. In France, an advanced breeding program for this conifer species has been underway since the early 1960s. Open-pollinated seed orchards currently supply more than 90% of maritime pine seedlings for plantation forestry. However, pollen contamination and mating structure have been poorly studied in such seed orchards whereas they could impact genetic gains and diversity. We analyzed these features in three maritime pine clonal seed orchards. We addressed biological (tree genotype, age, flowering phenology) and environmental factors (vicinity with external pollen sources, orchard structure, soil type, climatic conditions) that are expected to determine the genetic composition of seed lots. Genetic analyses were based on an optimized set of 60 SNP markers and performed on 2,552 seedlings with Cervus software (likelihood inference methodology). Pollen contamination rates were highly variable between seed lots (from 20 to 96%), with a mean value of 50%. Interpretative factors included the distance between the seed orchard and external pollen sources, rain during the pollination period, seed orchard age, soil conditions and seed parent identity. All parental genotypes from the seed orchards contributed to the offspring as pollen parents, but differences in paternal reproductive success were detected. Finally, the overall self-fertilization rate was estimated at 5.4%, with considerable variability between genotypes (from 0% to 26%). These findings are useful to formulate recommendations for seed orchard management (seed orchard location, soil and climate optimal conditions, minimum age for commercial seed lots harvesting) and for identifying new research perspectives (exploring links between pollen contamination and climatic data, genetic control of flowering traits)