Bar Gene Research Articles

Agrobacterium tumefaciens-mediated transformation of big bluestem (Andropogon gerardii Vitman)

Big bluestem (Andropogon gerardii Vitman) is native to the Great Plains of North America and is an important warm-season forage and ornamental grass. Recently it has been proposed as one of the more ideal biomass producers for cellulosic ethanol production due to its high biomass yield and low input needs. A robust genetic transformation protocol is needed for germplasm improvement or functional genomics studies in big bluestem. In the present study we report, for the first time the establishment of a reproducible Agrobacterium-mediated transformation protocol for big bluestem. Mature seeds of a big bluestem cultivar Bison were cultured for producing embryogenic calli, which were then used for infection by the Agrobacterium tumefaciens strain AGL1 harboring either the binary vector pTF101.1, which carries the glufosinate resistance (bar) gene or a modified binary vector pMDC32 which carries the hygromycin resistance gene (hpt). Glufosinate ammonium at 6 mg L−1 or hygromycin B at 250 mg L−1 was used effectively to select for resistant callus followed by lowered concentrations of both during regeneration and rooting. Based on PCR test alone or in combination with herbicide leaf painting, the overall transformation frequency was 2.8–3.6 % by using the pTF101.1 vector and 2.4–3.3 % by using the modified pMDC32 vector, both of which are comparable to transformation frequencies reported for other grasses. PCR test showed that all Basta®-resistant plants following the leaf painting contain the bar gene and subsequent Southern blot analysis confirmed that the all tested transgenic plants resulted from independent transgenic events.

Transformation of upland rice with the bar gene and selection for resistance to the herbicide Basta

In this study, Chinese upland rice cultivars including Handao297 and Handao502 were transformed with the anti-herbicide bar gene in order to achieve efficient weed control. PCR amplification, Southern blot analysis and field Basta resistance identification in early T0, T1, and late generation T6 and T11–T13 illustrated that the exogenous bar gene had been integrated into the genome of the upland rice varieties with stable expression in successive generations. Different transformants had a different gene copy numbers, which was not related to Basta resistance. Some of the transgenic lines showed gene silencing, whereas multiple copies of the bar gene were observed in other late generation transgenic lines. Basta resistance can thus be inherited stably among generations, and an optimal Basta concentration and selective pressure are suitable for resistance segregation and breeding of homozygous progenies with high resistance. After three generations of successive Basta resistance selection in 207 T0 plants, we obtained 555 T3 lines. Comprehensive identification and selection for Basta resistance, drought resistance, yield, and other agronomic traits in nine generations yielded two promising transgenic lines, HD297T-31 and HD297T-523. Although both varieties were resistant to 0.1 % Basta, HD297T-31 possessed high (100 %) resistance to Basta while HD297T-523 showed moderate (95 %) resistance. Moreover, during the reproductive stage, the stems, sheaths, leaves, and panicles of HD297T-31 were immune-highly resistant to highly-moderately resistant to 0.5 % Basta, whereas, they were highly resistant–moderately resistant to 1 % Basta. HD297T-523 was immune-highly resistant and immune-highly resistant to 0.5 and 1 % Basta respectively. The plant and stem percentage sensitive to 0.5 % Basta was lower than 1 and 3 % respectively. Since HD297T-31 showed isozygosity, it could be used as a new variety in production.

In vitro propagation and preliminary results of Agrobacterium-mediated genetic transformation of Cordyline fruticosa

A method for in vitro propagation through axillary shoot proliferation and induction of adventitious shoots as well as Agrobacterium-mediated transformation of Cordyline fruticosa is presented. In vitro Cordyline shoots were cultured on MS medium supplemented 0–4mgl−1 thidiazuron (TDZ) for their multiplication. MS medium supplemented with 0.1mgl−1 TDZ proved to be the most effective in which 14 shoots per explants were obtained. Leaf segments were excised from in vitro axillary shoots and cultured on MS medium supplemented with TDZ (0–5mgl−1) for callus induction. Following induction, the non-embryogenic calluses were proliferated on MS medium without plant growth regulators. A rapid and efficient method for producing transgenic plants has been developed for Cordyline via Agrobacterium-mediated transformation. Leaf segments from in vitro shoots were co-cultivated with Agrobacterium tumefaciens EHA105, which harbored the binary vector carrying the bar and β-glucuronidase (GUS) genes in the T-DNA region. The results showed that the number of plants expressing GUS gene first increased with inoculation time and bacterial density (OD600), and then dramatically decreased with the increment of both factors. The highest percentage of transformation efficiency (85%) was obtained when the leaf segment explants were inoculated with Agrobacterium for up to 40min at OD600 of 1.0 followed by 80 and 75% at OD600 of 0.8 and 1.2, respectively. The transgenes were confirmed in transgenic plants using GUS primers and gave the expected band size of 750bp. The results suggested that infection time and Agrobacterium density could have some effects on the transformation efficiency. Southern blot analysis was used for definitive confirmation of tDNA integration in Cordyline and confirmed the results of histochemical GUS assay and PCR amplifications. Regenerated plantlets were acclimatized in greenhouse.

Genetic transformation of cultivated jute (Corchorus capsularis L.) by particle bombardment using apical meristem tissue and development of stable transgenic plant

An in vitro plant regeneration and genetic transformation protocol was established in jute (Corchorus capsularis L. var JRC321). One-day-old apical, meristematic tissues of germinating seedlings were used as explants. Multiple shoots were regenerated from each explant using Murashige and Skoog basal medium containing 1.78 µM benzylamino purine and 4.92 µM indole-3-butyric acid. Transformation was carried out in three independent sets (each set comprising of three independent experiments each comprising three replications with 35 explants per replication) using the bialaphos resistance gene (bar), synthetically designed for high level plant expression. The positive transformants containing the bar gene were selected in growth medium containing 2.5 mg/l bialaphos. Polymerase chain reaction (PCR), Southern and northern blots, real-time quantitative PCR, western blot and enzymatic assay of five putative transformants from three independent sets provided evidence for full-length gene integration into the genomic DNA of transformed jute, as well as high level expression of the transgene. Analysis of the T1 plants revealed a stable inheritance of the transgene through the progenies. The data presented in this report showed considerable advancement in jute transformation and should improve future genetic engineering strategies to be employed for improvement of this very important fibre crop.

A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed.

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.

Electroporation and morphogenic potential of Gentiana kurroo (Royle) embryogenic cell suspension protoplasts

This article presents our further in vitro studies into the morphogenic potential of gentian cells, organs, and tissues after modification of their genome. The objective was to study the effect of electroporation and the introduction of foreign genes on the morphogenic potential of Gentiana kurroo embryogenic cell suspension protoplasts. Protoplasts were electroporated with DNA plasmids carrying nptII and bar genes. The stability of cell membranes, the contents of electroporation buffer, the length of electric pulse, the number of pulses and the strength of the electric field were studied. We determined the highest electroporation efficiency by evaluating the highest protoplast survival rate under specific physical conditions. The best results were achieved in the presence of EB1 electroporation buffer where the viability of protoplasts was 70.1%. Protoplast survival at this higher level required culture temperatures near 0EC, and a 20 μs electric pulse with an electric field of 1.0 kV/cm. After seven days of agarose embedded protoplast culture, a selective agent – kanamycin – was introduced to the medium. The cell transformation effect was improved by a long term culture of callus, regenerated somatic embryos and transformants in the presence of 50 mg/l kanamycin.

Pollen‐mediated Gene Flow in Camelina sativa (L.) Crantz

ABSTRACTCamelina sativa (L.) Crantz is a biofuel crop with application on the Great Western Plains of North America that is being developed using genetic engineering. Before release of genetically engineered cultivars, the potential for pollen‐mediated gene flow (PMGF) needs to be assessed to determine if they can coexist with conventional cultivars without causing market harm. Medium‐scale field experiments (40‐m diameter) were conducted in 2011 and 2012 to quantify PMGF using a seed‐expressed florescent marker and bar gene that confers resistance to glufosinate [2‐amino‐4‐(hydroxymethylphosphinyl) butanoic acid] in Alberta, Canada. More than 17 million seeds were screened to quantify outcrossing. Pollen‐mediated gene flow best fit an exponential decay model in which the highest average PMGF (0.78%) occurred adjacent to the donor crop and rapidly declined to 0.09% by 20 m. Pollen‐mediated gene flow was leptokurtic, with a 50% reduction in gene flow 1.5 m from the pollen source. No directional trends were detected, suggesting wind dispersal was not influencing experimental results. The risk of PMGF at longer distances is minimal, but not zero, in this primarily self‐pollinating species.

Improving fruit quality in tomato (Lycopersicum esculentum Mill) under heat stress by silencing the vis 1 gene using small interfering RNA technology

Constantly elevated temperatures cause an array of physio-biochemical changes in tomato (Lycopersicum esculentum Mill.) which make the fruit ripen quickly and up to 50 % yield loss. The development of tomato cultivars, using genetic engineering approaches which delay ripening, offers a new way to keep tomatoes healthy under heat stress. Over-expression of small heat shock protein gene, viscosity 1 (vis 1) plays a role in increasing juice viscosity, early ripening and tissue soften which emphasizes the importance of this gene in premature ripening. The aim of this work was therefore to develop a useful system for silencing the vis1 gene using small interfering RNA strategy. Agrobacterium strain GV3101 harbouring the binary vector pICBV19 containing the gus and bar genes was used to adapt the transformation process in this study. The primers were designed to amplified the first exon of the vis 1 gene and the amplified fragment was used for cloning into the pFGC5941 at XhoI/NcoI site at sense orientation then additional fragment was subsequently cloned at BamHI/XbaI to form sense/antisense cloned fragment interrupted by the CHSA-intron Agrobacterium strain LBA4404 with the binary vector pFGC5941 which harbors vis1 gene under the control of the 35S promoter containing bar gene under the control of a mannopine synthase 2′(Mas2′) as selectable marker, was used to reduce the expression of vis1 gene in fruit. Polymerase chain reaction (PCR), RT–PCR and northern blotting analysis were applied to detect putative transgenic plants. Significantly, silencing of vis 1 gene was potently occur and new transgenic tomato cultivars were produced with enhanced ripening qualities for recommendation for growing under heat stress.

Tissue Culture Independent Agrobacterium tumefaciens Mediated In Planta Transformation Method for Tropical Maize (Zea mays.L)

Tropical maize is recalcitrant to tissue culture regeneration because of its poor response to in vitro regeneration after transformation. In this context, the present study has developed the tissue culture independent in planta transformation protocol for tropical maize by transferring plumular meristem cells of germinating seeds of tropical maize genotype through Agrobacteriumtumefaciens infection. The protocol was developed by using Agrobacterium strain EHA105 containing vector pCAMBIA3301 carrying cry1Ab, gus and bar genes. The expression of transgene gus in T0 plants was confirmed by measuring the hydrolysis rate of the fluorescent substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) assay whereas the presence of cry1Ab gene was confirmed by polymerase chain reaction and T0 plants were allowed to grow in glass house into whole plant until maturity and were selfed to produce seeds of T1 generation. The presence of transgene and its segregation was studied in T1 generation through Southern and enzyme-linked immunosorbent assay confirming the presence of transgene and its expression respectively. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 75–90 days.

High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection.

The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

Rice transgene flow: its patterns, model and risk management.

Progress has been made in a 12year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21 pollen recipients, we have elucidated the patterns of transgene flow to different types of rice. The frequency to male sterile lines is 10(1) and 10(3) higher than that to O.rufipogon and rice cultivars. Wind speed and direction are the key meteorological factors affecting rice transgene flow. (ii) A regional applicable rice gene flow model is established and used to predict the maximum threshold distances (MTDs) of gene flow during 30years in 993 major rice producing counties of southern China. The MTD0.1% for rice cultivars is basically ≤5m in the whole region, despite climate differs significantly at diverse locations and years. This figure is particularly valuable for the commercialization and regulation of transgenic rice. (iii) The long-term fate of transgene integrated into common wild rice was investigated. Results demonstrated that the F1 hybrids of transgenic rice/O.rufipogon gradually disappeared within 3-5years, and the Bt or bar gene was not detectable in the mixed population, suggesting the O.rufipogon may possess a strong mechanism of exclusiveness for self-protection. (iv) The flowering time isolation and a 2-m-high cloth-screen protection were proved to be effective in reducing transgene flow. We have proposed to use a principle of classification and threshold management for different types of rice.

Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation.

A plant-derived 0.3 kb constitutive promoter was obtained from AtTCTP expression analysis, and successfully applied to the expression of a selectable marker gene for production of transgenic creeping bentgrass plants. The isolation and use of an efficient promoter is essential to develop a vector system for efficient genetic transformation of plants, and constitutive promoters are particularly useful for the expression of selectable marker genes. In this study, we characterized a small size of the constitutive promoter from the expression analysis of Arabidopsis thaliana translationally controlled tumor protein (AtTCTP) gene. Histochemical and fluorometric GUS analyses revealed that a 303 bp upstream region from the start codon of the AtTCTP gene showed strong GUS expression throughout all plant tissues, which is approximately 55 % GUS activity compared with the cauliflower mosaic virus 35S promoter (35Spro). To examine the possible application of this promoter for the development of genetically engineered crops, we introduced pCAMBIA3301 vector harboring the 0.3 kb promoter of AtTCTP (0.3kbpro) that was fused to the herbicide resistance BAR gene (0.3kb pro ::BAR) into creeping bentgrass. Our transformation results demonstrate that transgenic creeping bentgrass plants with herbicide resistance were successfully produced using 0.3kb pro ::BAR as a selectable marker. Northern blot analysis revealed that the transgenic plants with 0.3kb pro ::BAR showed reduced but comparable expression levels of BAR to those with 35S pro ::BAR. Moreover, the transcription activity of the 0.3 kb promoter could be increased by the fusion of an enhancer sequence. These results indicate that the 0.3 kb AtTCTP promoter can be used as a plant-derived constitutive promoter for the expression of selectable marker genes, which facilitates its use as an alternative to the 35S promoter for developing genetically engineered crops.

Expression of a cystatin transgene can confer resistance to root lesion nematodes in Lilium longiflorum cv. ‘Nellie White’

Lilium longiflorum cv. 'Nellie White' assumes a great economic importance as cut flowers, being one of the most valuable species (annual pot plants value above $20,000,000) in terms of wholesales in the US. The root lesion nematode Pratylenchus penetrans (RLN) constitutes one of the main pests for lily producers due to the significant root damage it causes. Our efforts have focused on the generation of soybean hairy roots (as a transient test model) and stable transgenic lilies overexpressing a modified rice cystatin (Oc-IΔD86) transgene and challenged with root lesion nematodes. Lily transformation was achieved by gene gun co-bombardment using both a pBluescript-based vector containing the cystatin gene and pDM307 that contains a bar gene for phosphinothricin selection. Both soybean hairy roots and lilies overexpressing the OcIΔD86 transgene exhibited enhanced resistance to RLN infection by means of nematode reduction up to 75 ± 5% on the total number of nematodes. In addition, lily plants overexpressing OcIΔD86 displayed an increase of plant mass and better growth performance in comparison to wild-type plants, thereby demonstrating an alternative strategy for increasing the yield and reducing nematode damage to this important floral crop.

Development of some transgenic rice lines containing the herbicide resistance gene BAR and study on inheritance of this BAR gene up to the T2 generation

Development of some transgenic rice lines containing the herbicide resistance gene BAR and study on inheritance of this BAR gene up to the T2 generation

Low frequency paternal transmission of plastid genes in Brassicaceae.

Plastid-encoded genes are maternally inherited in most plant species. Transgenes located on the plastid genome are thus within a natural confinement system, preventing their distribution via pollen. However, a low-frequency leakage of plastids via pollen seems to be universal in plants. Here we report that a very low-level paternal inheritance in Arabidopsis thaliana occurs under field conditions. As pollen donor an Arabidopsis accession (Ler-Ely) was used, which carried a plastid-localized atrazine resistance due to a point mutation in the psbA gene. The frequency of pollen transmission into F1 plants, based on their ability to express the atrazine resistance was 1.9 × 10(-5). We extended our analysis to another cruciferous species, the world-wide cultivated crop Brassica napus. First, we isolated a fertile and stable plastid transformant (T36) in a commercial cultivar of B. napus (cv Drakkar). In T36 the aadA and the bar genes were integrated in the inverted repeat region of the B. napus plastid DNA following particle bombardment of hypocotyl segments. Southern blot analysis confirmed transgene integration and homoplasmy of plastid DNA. Line T36 expressed Basta resistance from the inserted bar gene and this trait was used to estimate the frequency of pollen transmission into F1 plants. A frequency of <2.6 × 10(-5) was determined in the greenhouse. Taken together, our data show a very low rate of paternal plastid transmission in Brassicacea. Moreover, the establishment of plastid transformation in B. napus facilitates a safe use of this important crop plant for plant biotechnology.

Disease Occurrence in Transgenic Rice Plant Transformed with Silbene Synthase Gene and Evaluation of Possible Horizontal Gene Transfer to Plant Pathogens

Genetic engineering is being used to enhance disease resistance and nutritional value of crops including rice plant. Considering the fast-growing agricultural biotechnology and rapidly increasing global area of transgenic crops, the risk evaluation on environment is necessary. In this study, we surveyed the difference of disease occurrence between transgenic rice variety, Iksan526 transformed with peanut stilbene synthase gene and non-transgenic rice varieties, Dongjin and Nampyeong in the field. Moreover, the possibility of gene transfer from transgenic rice to bacterial and fungal pathogens was investigated. The results of this study indicated that there was no significant difference in the occurrence and severity of the diseases between Iksan526 and Dongjin or Nampyeong. In addition, the results suggested that rice pathogen, such as Xanthomonas oryzae pv. oryzae, Rhizoctonia solani and Magnaporthe grisea did not take up stilbene synthase and bar genes under natural conditions. Moreover the transformed DNA was not transferred to the pathogens even in repetitive contacts.

Blueberry (Vaccinium corymbosum L.).

Vaccinium consists of approximately 450 species, of which highbush blueberry (Vaccinium corymbosum) is one of the three major Vaccinium fruit crops (i.e., blueberry, cranberry, and lingonberry) domesticated in the twentieth century. In blueberry the adventitious shoot regeneration using leaf explants has been the most desirable regeneration system to date; Agrobacterium tumefaciens-mediated transformation is the major gene delivery method and effective selection has been reported using either the neomycin phosphotransferase II gene (nptII) or the bialaphos resistance (bar) gene as selectable markers. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining the optimal conditions for efficient plant regeneration, reliable gene delivery, and effective selection. The protocol has led to successful regeneration of transgenic plants from leaf explants of four commercially important highbush blueberry cultivars for multiple purposes, providing a powerful approach to supplement conventional breeding methods for blueberry by introducing genes of interest.

Downregulation of Glucan Synthase-Like (TaGSL) genes in wheat leads to inhibition of transgenic plant regeneration

RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants.

Application of RNA interference methodology to investigate and develop SCMV resistance in maize.

Specific fragments of the sugarcane mosaic virus (SCMV) coat protein gene (cp) were amplified by reverse transcriptionpolymerase chain reaction and used to construct a marker free small interfering RNA complex expression vector against SCMV. In planta transformation was performed on maize (Zea mays) inbred line 8112 mediated by Agrobacterium tumefaciens. PCR and Southern blot analyses demonstrated successful integration of the cp segment into the 8112 genome. The in planta transformation frequency was 0.1%, and the cotransformed frequency with the cp and bar genes was 0.034%. Real-time quantitative PCR of samples from different transgenic plant organs showed that the expression of the cp gene fragment in transgenic plants was variable and that the highest expression level occurred in the tassels and leaves and the lowest expression occurred in the roots. Real-time quantitative PCR was also used to measure how gene expression in transgenic T2 generation plants inoculated with SCMV changes over time. The results showed that the hairpin RNA structure transcribed from the cp gene interfered with SCMV infection and transgenic maize lines were not equally effective in preventing SCMV infection. Our findings provide a valuable tool for controlling plant viruses using RNA interference and the posttranslational gene silencing approach.

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.