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

The 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/µl 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.     

A set of modular binary vectors for transformation of cereals

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.     

Analysis of promoters in transgenic barley and wheat

Advances in the genetic transformation of cereals have improved the prospects of using biotechnology for plant improvement, and a toolbox of promoters with defined specificities would be a valuable resource in controlling the expression of transgenes in desired tissues for both plant improvement and molecular farming. A number of promoters have been isolated from the important cereals (wheat, barley, rice and maize), and these promoters have been tested mostly in homologous cereal systems and, to a lesser extent, in heterologous cereal systems. The use of these promoters across the important cereals would add value to the utility of each promoter. In addition, promoters with less sequence homology, but with similar specificities, will be crucial in avoiding homology-based gene silencing when expressing more than one transgene in the same tissue. We have tested wheat and barley promoters in transgenic barley and wheat to determine whether their specificity is shared across these two species. The barley bifunctional α-amylase/subtilisin inhibitor ( Isa ) promoter, specific to the pericarp in barley, failed to show any activity in wheat, whereas the wheat early-maturing ( Em ) promoter showed similar activity in wheat and barley. The wheat high-molecular-weight glutenin ( HMW-Glu ) and barley D-hordein ( D-Hor ) and B-hordein ( B-Hor ) storage protein promoters maintained endosperm-specific expression of green fluorescent protein (GFP) in wheat and barley, respectively. Using gfp , we have demonstrated that the Isa and Em promoters can be used as strong promoters to direct transgenes in specific tissues of barley and wheat grain. Differential promoter activity across cereals expands and adds value to a promoter toolbox for utility in plant biotechnology.     

叶绿体遗传转化：植物导入外源基因的新途径

叶绿体转化系统,独立于传统的核转化,为植物导入外源基因提供了新途径,它有如下优点：超量表达目的的基因;以定点整合方式导入外源基因从而消除了位置效应及基因沉默;具有原核表达方式,能以多 反子的形式表达多个基因;母系遗传方式可防止基因扩散;基因产物区域化并能提供适于某些产物发挥功能的小环境等。随着这项技术在越来越多的领域发挥作用,其优势性逐渐得到认同。本文着重对此技术的特点,发展及应用作一综述。     

叶绿体遗传转化：植物导入外源基因的新途径

叶绿体转化系统,独立于传统的核转化,为植物导入外源基因提供了新途径。它有如下优点：超量表达目的基因;以定点整合方式导入外源基因从而消除了位置效应及基因沉默;具原核表达方式,能以多顺反子的形式表达多个基因;母系遗传方式可防止基因扩散;基因产物区域化并能提供适于某些产物发挥功能的小环境等。随着这项技术在越来越多的领域发挥作用,其优越性逐渐得到认同。本文着重对此技术的特点、发展及应用作一综述。     

Phylogenetic and molecular analysis of the ribulose-1,5-bisphosphate carboxylase small subunit gene family in banana

Despite being the number one fruit crop in the world, very little is known about the phylogeny and molecular biology of banana (Musa spp.). Six banana rbcS gene families encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from six different Musa spp. are presented. For a comprehensive phylogenetic study using Musa rbcS genes, a total of 57 distinct rbcS sequences was isolated from six accessions that contained different combinations of the A and B ancestral/parental genomes. As a result, five of the six members of the rbcS gene family could be affiliated with the A and/or B Musa genomes and at least three of the six gene families most likely existed before Musa A and B genomes separated. By combining sequence data with quantitative real-time PCR it was determined that the different Musa rbcS gene family members are also often multiply represented in each genome, with the highest copy numbers in the B genome. Expression of some of the rbcS genes varied in intensity and in different tissues indicating differences in regulation. To analyse and compare regulatory sequences of Musa rbcS genes, promoter and terminator regions were cloned for three Musa rbcS genes. Transient transformation assays using promoter-reporter-terminator constructs in maize, wheat, and sugarcane demonstrated that the rbcS-Ma1, rbcS-Ma3, and rbcS-Ma5 promoters could be useful for transgene expression in heterologous expression systems. Furthermore, the rbcS-Ma1 terminator resulted in a 2-fold increase of transgene expression when directly compared with the widely used Nos terminator.     

Transgenic plastids in basic research and plant biotechnology

Facile methods of genetic transformation are of outstanding importance for both basic and applied research. For many years, transgenic technologies for plants were restricted to manipulations of the nuclear genome. More recently, a second genome of the plant cell has become amenable to genetic engineering: the prokaryotically organized circular genome of the chloroplast. The possibility to directly manipulate chloroplast genome-encoded information has paved the way to detailed in vivo studies of virtually all aspects of plastid gene expression. Moreover, plastid transformation technologies have been intensely used in functional genomics by performing gene knockouts and site-directed mutageneses of plastid genes. These studies have contributed greatly to our understanding of the physiology and biochemistry of biogenergetic processes inside the plastid compartment. Plastid transformation technologies have also stirred considerable excitement among plant biotechnologists, since transgene expression from the plastid genome offers a number of most attractive advantages, including high-level foreign protein expression and transgene containment due to lack of pollen transmission. This review describes the generation of plants with transgenic plastids, summarizes our current understanding of the transformation process and highlights selected applications of transplastomic technologies in basic and applied research.     

Artificial and engineered chromosomes: developments and prospects for gene therapy

At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals.     

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.     

The distribution of transgene insertion sites in barley determined by physical and genetic mapping

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.     

Hygromycin resistance as an efficient selectable marker for wheat stable transformation

Summary A highly efficient method for stable wheat transformation using hygromycin resistance as a selectable marker is described. Young embryogenic calli growing from immature wheat embryos were transformed using a gunpowder-driven microparticle accelerator. Transgenic wheat plants were determined by PCR amplification of transgene fragments and confirmed by Southern hybridization, activity of the transgene expression and by analysis of the progeny. The hpt gene was as good as or a better selectable marker than the bar gene with an average efficiency (number of transgenic plants relative to the number of bombarded calli) of 5.5% compared with 2.6% for the bar gene.     

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives

Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.     

Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile “transgene maintenance and control” strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.     

The effect of additional virulence genes on transformation efficiency,transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system

We assessed the effect of four different virulence (vir) gene combinations on plant transformation efficiency and transgene behaviour in rice using the pGreen/pSoup dual binary vector system. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units with, or without, the virG542, virGN54D, virGwt or the virG/B/C genes added to the backbone. Additonal vir gene(s) significantly altered plant transformation efficiency and the integration of vector backbone sequences. However, no differences in transgene copy number, percentage of expressing lines and expression levels could be detected. Addition of virGwt was the most beneficial, doubling the overall performance of the pGreen/pSoup vector system based on transformation frequency, absence of backbone sequence integration and expression of unselected transgenes. In 39 of the plant lines, the additional vir genes were integrated into the rice genome. The contribution of super dual binary pGreen/pSoup vectors to the development of efficient rice transformation systems and to the production of plants free of selectable marker genes are discussed.