Catherine Pannetier

Plant regeneration from embryogenic suspension cultures of oil palm (Elaeis guineensis Jacq.)

Suspension cultures of oil palm (Elaeis guineensis Jacq.) were established in a medium with 80 or 100 mg.l−1 2,4-dichlorophenoxyacetic acid and 1 g.l−1 activated charcoal, from calli producing embryogenic cells and protruding proembryos. The suspension was composed of meristematic clumps, breaking away and giving rise to new smaller aggregates. Under the best conditions, the initial weight increased about 4 fold in one month. Embryo differentiation was achieved when plating the clumps either directly, or after a phase in hormone-free liquid medium. In the second case, early maturation occurred in liquid medium. Secondary embryogenesis was reduced, and up to 18.1 % embryos formed shoots. Rooted plants were sucessfully transferred to soil.

Introduction of new traits into cotton through genetic engineering: insect resistance as example

The main goal of gene transfer into cotton is the development of insect-resistant varieties. The stakes are important since cotton protection against insects uses almost 24% of the worlds chemical insecticides market, which is not without consequences on the environment. The first approach was to introduce and express in the cotton genome, genes from the bacterium Bacillus thuringiensis (B.t.) which produces entomopathogenic toxins. The development of an efficient Agrobacterium tumefaciens mediated transformation system was the first step. The expression of B.t. genes was studied and synthetic genes more adapted to a plant genome have been constructed. Studies on their expression in cotton is underway. The second focus was to develop strategies that would minimize the risks of inducing insect resistance. The main approach is to associate several genes coding for entomopathogenic proteins with different modes of action. Genes encoding protease inhibitors were chosen. One possibility is to associate a B.t. gene and a gene encoding a protease inhibitor. Several protease inhibitors were tested in artificial diets on major pests of cotton. The corresponding genes have been introduced into the cotton genome. These various orientations of the research program will be presented.

Wuschel overexpression promotes somatic embryogenesis and induces organogenesis in cotton (Gossypium hirsutum L.) tissues cultured in vitro

Key messageThis work shows that overexpression of theWUSgene fromArabidopsisenhanced the expression of embryogenic competence and triggered organogenesis from some cells of the regenerated embryo-like structures.AbstractAgrobacterium-mediated genetic transformation of cotton was described in the late 1980s, but is still time consuming and largely genotype dependant due to poor regeneration. To help solve this bottleneck, we over-expressed the WUSCHEL (WUS) gene, a homeobox transcription factor cloned in Arabidopsis thaliana, known to stimulate organogenesis and/or somatic embryogenesis in Arabidopsis tissues cultured in vitro. The AtWUS gene alone, and AtWUS gene fused to the GFP marker were compared to the GFP gene alone and to an empty construct used as a control. Somatic embryogenesis was improved in WUS expressed calli, as the percentage of explants giving rise to embryogenic tissues was significantly higher (×3) when WUS gene was over-expressed than in the control. An interesting result was that WUS embryogenic lines evolved in green embryo-like structures giving rise to ectopic organogenesis never observed in any of our previous transformation experiments. Using our standard in vitro culture protocol, the overexpression of AtWUS in tissues of a recalcitrant variety did not result in the production of regenerated plants. This achievement will still require the optimization of other non-genetic factors, such as the balance of exogenous phytohormones. However, our results suggest that targeted expression of the WUS gene is a promising strategy to improve gene transfer in recalcitrant cotton cultivars.

The expression of Bacillus thuringiensis toxin genes in plant cells

Abstract Plants expressing genes encoding δ-endotoxins from Bacillus thuringiensis (Bt) have triggered interest for the control of insect pests. Numerous plant species have been transformed with genes encoding various toxins. The first transformation experiments conducted with bacterial genes showed that their level of expression in plants is too low to confer adequate protection. To circumvent these problems, Bt toxin genes have been modified or resynthesized, dramatically improving their level of expression and the protection afforded. Despite these improvements, problems remain: the control of less susceptible insects and the durable deployment of transgenic plants have yet to be fully addressed.

Assessment of gene flow between gossypium hirsutum and G. herbaceum : evidence of unreduced gametes in the diploid progenitor

In the framework of a gene flow assessment, we investigated the natural hybridization rate between Gossypium hirsutum (AADD genome) and G. herbaceum (AA genome). The latter species, a diploid progenitor of G. hirsutum, is spontaneously present in South Africa. Reciprocal crosses were performed without emasculation between G. herbaceum and G. hirsutum. Neither examination of the morphological characteristics nor flow cytometry analysis of the 335 plants resulting from the G. hirsutum × G. herbaceum cross showed any hybrid features. Of the 148 plants produced from the G. herbaceum × G. hirsutum cross, three showed a hybrid phenotype, and their hybrid status was confirmed by SSR markers. Analysis of DNA content by flow cytometry and morphological traits clearly showed that two of these plants were triploid (AAD). The third plant had a flow cytometry DNA content slightly higher than G. hirsutum. In addition, its morphological characteristics (plant architecture, presence and size of petal spots, leaf shape) led us to conclude that this plant was AAAD thus resulting from fertilization with an unreduced AA gamete of the female G. herbaceum parent. Fluorescent In Situ Hybridization (FISH) and meiotic behavior confirmed this hypothesis. To the best of our knowledge, this is the first description of such gametes in G. herbaceum, and it opens new avenues in breeding programs. Furthermore, this plant material could provide a useful tool for studying the expression of genes duplicated in the A and D cotton genome.

Brachypodium distachyon as a model system for studying genes involved in cell wall synthesis

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in citrus. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x×4x hybridisations and in vitro culture of isolated nucellus. The origin of the plants obtained as a consequence of these two processes is still unclear. In this work, we systematically analyzed the genetic structure (ploidy and allelic constitution at SSR locus) of plants obtained from polyembryonic seeds arising from 2x×4x sexual hybridisations or regenerated from nucellus culture in vitro of different non-apomictic citrus genotypes. Histological studies were also conduced to try to identify the initiation process of polyembryony in nonapomictic genotypes. We demonstrate that all plants obtained from the same undeveloped seed in 2x×4x hybridisations resulted from fission of the original zygotic embryo. Also, the plants obtained from in vitro culture of nucellus were recovered by somatic embryogenesis from cells having the same genotype as the zygotic embryos of the same seed. It appears that in non-apomictic citrus, proembryos or embryogenic cells are formed by fission of the original zygotic embryo and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as result of two different mechanisms that prevent the dominance of the initial zygotic embryo. (Texte integral)