Philippe Vain

Brachypodium as a model for the grasses: Today and the future

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-IΔD86) for nematode resistance in transgenic rice plants

Abstractu2002We have used a genotype-independent transformation system involving particle gun bombardment of immature embryos to genetically engineer rice as part of a programme to develop resistance to nematodes. Efficient tissue culture, regeneration, DNA delivery and selection methodologies have been established for elite African varieties (‘ITA212’, ‘IDSA6’, ‘LAC23’, ‘WAB56-104’). Twenty-five transformed clones containing genes coding for an engineered cysteine proteinase inhibitor (oryzacystatin-IΔD86, OC-IΔD86), hygromycin resistance (aphIV) and β-glucuronidase (gusA) were recovered from the four varieties. Transformed plants were regenerated from all clones and analysed by PCR, Southern and western blot. Detectable levels of OC-IΔD86 (up to 0.2% total soluble protein) in plant roots were measured in 12 out of 25 transformed rice lines. This level of expression resulted in a significant 55% reduction in egg production by Meloidogyne incognita.

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

Abstractu2002An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T0) and their progeny (T1). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed.

Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup

AbstractnTransgene integration, expression level and stability have been studied, across two generations, in a population of rice plants transformed using a new dual binary vector system: pGreen/pSoup. pGreen is a small Ti binary vector unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. We engineered both pGreen and pSoup to contain each a different T-DNA. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units (no transgene in pSoup) or with a pSoup vector containing an aphIV and gfp expression units (no transgene in pGreen). High plant transformation frequencies (up to 40%) were obtained using herbicide resistance (bar) or antibiotic resistance (aphIV) genes. Around 80% of the independently transformed plants expressed unselected reporter genes (gusA or gfp) present in the vectors. Backbone sequences transfer was frequent (45% of lines) and occurred often in multicopy lines. Around 15–20% of the rice plant lines contained a single T-DNA integration without backbone. Integration of additional transgene copies did not improve expression levels in either T0 plants or T1 progenies. Nearly all multicopy lines contained transgenes integrated at several loci in the plant genome, showing that T-DNAs from either pGreen or pSoup frequently integrated at unlinked loci. Precise determination of loci number required the analysis of transgene presence in progeny. Segregation of transgene phenotype was generally misleading and tended to underestimate the real number of transgenic loci. The contribution of this new dual-binary vector system to the development of high-throughput rice transformation systems and to the production of marker-free transgenic rice plants is discussed.

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment

Abstract.The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene (aphIV) and the unselected reporter gene (gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T0), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T0 plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T0) and their progeny (T1): (1) absence of transgene expression in T0 plants (41% of lines), (2) sterility of T0 plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.

The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants

Abstractu2008u2008Segregating T1, T2 and T3 transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected β-glucuronidase (gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing the likelihood of an additive effect on transgene expression level is discussed.

A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants

Brachypodium distachyon is emerging as a new model system for bridging research into temperate cereal crops, such as wheat and barley, and for promoting research in novel biomass grasses. Here, we provide an adapter ligation PCR protocol that allows the large-scale characterization of T-DNA insertions into the genome of Brachypodium. The procedure enables the retrieval and mapping of the regions flanking the right and left borders (RB and LB) of the T-DNA inserts and consists of five steps: extraction and restriction digest of genomic DNA; ligation of an adapter to the genomic DNA; PCR amplification of the regions flanking the T-DNA insert(s) using primers specific to the adapter and the T-DNA; sequencing of the PCR products; and identification of the flanking sequence tags (FSTs) characterizing the T-DNA inserts. Analyzing the regions flanking both the LB and RB of the T-DNA inserts significantly improves FST retrieval and the frequency of mutant lines for which at least one FST can be identified. It takes approximately 16 or 10 d for a single person to analyze 96 T-DNA lines using individual or batch procedures, respectively.

Agrobacterium -Mediated Transformation of Brachypodium distachyon

Brachypodium distachyon is an attractive genomics and biological model system for grass research. Recently, the complete annotated genome sequence of the diploid line Bd21 has been released. Genetic transformation technologies are critical for the discovery and validation of gene function in Brachypodium. Here, we describe an efficient procedure enabling the Agrobacterium-mediated transformation of a range of diploid and polyploid genotypes of Brachypodium. The procedure relies on the transformation of compact embryogenic calli derived from immature embryos using either chemical selection alone or a combination of chemical and visual screening of transformed tissues and plants. Transformation efficiencies of around 20% can routinely be achieved using this protocol. In the context of the BrachyTAG programme (BrachyTAG.org), this procedure made possible the mass production of Bd21T-DNA mutant plant lines.

Gene insertion patterns and sites.

During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is also an essential step in the safety assessment of genetically modified crops. This chapter describes the standard experimental procedures used to analyze transgene insertion patterns and loci in cereals and grasses transformed using Agrobacterium tumefaciens or direct transfer of DNA. Methods and protocols enabling the determination of the number and configuration of transgenic loci via a combination of inheritance studies, polymerase chain reaction, and Southern analyses are presented. The complete characterization of transgenic inserts in plants is, however, a holistic process relying on a wide variety of experimental approaches. In this chapter, these additional approaches are not detailed but references to relevant bibliographic records are provided.