A method for the medium-term Storage of plant tissue samples at room temperature and successive cycles of DNA extraction

International audienceBased on the protocol originally described by Stein et al. (2001), we have developed a method that allows for medium-term conservation at room temperature of wheat tissue samples to use for DNA extraction. DNA quality was suitable for analysis by PCR and Southern hybridization, even after 2 months of storage at room temperature. This method allows successive DNA re-extractions from a previously extracted sample and maximization of the DNA yield that can be recovered from precious samples. This method has applications for conservation of leaf samples and management of DNA extraction. Our method can help improve data recovery in many plant molecular genetics research projects

Introduction of a 50 Kb intact DNA fragment into wheat genome by biolistic technology

International audienceGenetic transformation is a powerful tool for studying gene function and regulation of physiological and developmental processes in plants. Although wheat was the last of the major cereal to be transformed, the introduction and expression of transgene in this plant is now quite feasible.Nowadays another challenge in plant transformation technology is the introduction and stable integration of large intact DNA fragments in plant genome. It would make possible the study in one transformation step of multigenic traits or to study the expression of plant gene in their native genomic context. Recently, it has been shown that expression and regulation of a gene frequently require regulatory elements that could be very distantIn the present study we developed a technology, based on biolistic with dephosphorylated cassettes allowing introduction of a 50 kb intact insert DNA in wheat genome. We have chosen Arabidopsis thaliana DNA as large insert fragments to make easier insert detection, and we have flanked the insert by two reporter genes in order to facilitate screening of plants containing the entire cassette.We have first demonstrated that it is possible to obtain transformed plants with a single and intact 50Kb insert, and then we have demonstrated that this insert can be transmitted intact to the T1 progeny.In our knowledge, this is the first time wheat transformation with such a fragment was demonstrated.Developing such technologies could be very useful to improve genetic engineering of wheat which is one of the major food crops in the world

Biolistic transformation of wheat: increased production of plants with simple insertions and heritable transgene expression

International audienceThe feasibility of map-based cloning in wheat has been demonstrated recently, opening new perspectives for a better understanding of wheat plant biology and for accelerating wheat improvement in the coming decades. To validate the function of candidate genes, an efficient transformation system is needed. Here, we have performed two methods for wheat transformation using particle bombardment that ensures the production of transgenic plants with simple integration patterns for research purposes and stable transgene expression for accurate and rapid validation of gene function. To establish this method, we used the bar and pmi selectable genes either as part of whole plasmids, gene cassettes (obtained by PCR or purified on agarose gels), or as dephosphorylated cassettes. The analysis of about 300 transgenic plants showed that the use of gene cassettes or dephosphorylated gene cassettes leads to a majority (50-60 %) of simple integration events. This is significantly higher than the number of simple events obtained with whole plasmids (9-25 %). Moreover, the decrease of the quantity of DNA from 500 to 5 ng/mu l for PCR-amplified cassettes used for transformation increased the number of single integration events. The transformation efficiency remained stable at 2.5 %, and a higher number of plants expressing the transgenes were obtained with the dephosphorylated cassette. No correlation was observed between the complexity of the events and stability of expression of the transgene, suggesting that plasmid sequences could be involved on transgene silencing. The inheritability of the transgene was demonstrated in T1 and T2 generations. These results show that biolistic transformation of dephosphorylated gene cassettes provides an easy and efficient route to produce backbone vector-free transgenic wheat carrying and expressing intact and single transgenes

Molecular and FISH analyses of a 53-kbp intact DNA fragment inserted by biolistics in wheat (Triticum aestivum L.) genome

International audienceA large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing

The<em> Brachypodium distachyon</em> UGT Bradi5gUGT03300 confers type II fusarium head blight resistance in wheat

International audienceFusarium head blight (FHB), caused by fungi belonging to the Fusarium genus, is a widespread disease of wheat (Triticum aestivum) and other small-grain cereal crops. The main causal agent of FHB, Fusarium graminearum, produces mycotoxins mainly belonging to type B trichothecenes, such as deoxynivalenol (DON), that can negatively affect humans, animals and plants. DON detoxification, mainly through glucosylation into DON-3-O-glucose, has been correlated with resistance to FHB. A UDP-glucosyltransferase from the model cereal species Brachypodium distachyon has been shown to confer resistance both to initial infection and to spike colonization (type I and type II resistances, respectively). Here, the functional characterization of transgenic wheat lines expressing the Bradi5g03300 UGT gene are described. The results show that, following inoculation with the fungal pathogen, these lines exhibit a high level of type II resistance and a strong reduction of mycotoxin content. In contrast, type I resistance was only weakly observed, although previously seen in B. distachyon, suggesting the involvement of additional host-specific characteristics in type I resistance. This study contributes to the understanding of the functional relationship between DON glucosylation and FHB resistance in wheat

Impacts de la surexpression du gène codant le facteur de transcription GAMYB sur la synthèse des protéines de réserve chez le blé tendre (Triticum aestivum)

Impacts de la surexpression du gène codant le facteur de transcription GAMYB sur la synthèse des protéines de réserve chez le blé tendre (Triticum aestivum). 5. Colloque du Réseau Français de Biologie des Graine

Identification of determining factors for meiotic recombination targeting in bread wheat

Identification of determining factors for meiotic recombination targeting in bread wheat. 12. French 3R Meetin

Relationships between puroindoline A-prolamin interactions and wheat grain hardness

International audienceGrain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α-and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr) 8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindolineprolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines