Dataset Information

Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.

ABSTRACT: Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker-free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium-mediated method, with efficiency of up to 37.7%, as confirmed by the use of Quickstix strips, histochemical staining, PCR analysis and Southern blotting. Of particular interest, marker-free transgenic wheat plants from various commercial Chinese varieties and their F₁ hybrids were successfully obtained for the first time, with a frequency of 4.3%, using a plasmid harbouring two independent T-DNA regions. The average co-integration frequency of the gus and the bar genes located on the two independent T-DNA regions was 49.0% in T₀ plants. We further found that the efficiency of generating marker-free plants was related to the number of bar gene copies integrated in the genome. Marker-free transgenic wheat plants were identified in the progeny of three transgenic lines that had only one or two bar gene copies. Moreover, silencing of the bar gene was detected in 30.7% of T₁ positive plants, but the gus gene was never found to be silenced in T₁ plants. Bisulphite genomic sequencing suggested that DNA methylation in the 35S promoter of the bar gene regulatory region might be the main reason for bar gene silencing in the transgenic plants.

SUBMITTER: Wang K

PROVIDER: S-EPMC5399001 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.

Wang Ke K Liu Huiyun H Du Lipu L Ye Xingguo X

Plant biotechnology journal 20161220 5

Genotype specificity is a big problem lagging the development of efficient hexaploid wheat transformation system. Increasingly, the biosecurity of genetically modified organisms is garnering public attention, so the generation of marker-free transgenic plants is very important to the eventual potential commercial release of transgenic wheat. In this study, 15 commercial Chinese hexaploid wheat varieties were successfully transformed via an Agrobacterium-mediated method, with efficiency of up to ...[more]

PMID: 27862820

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Project description:BackgroundAgrobacterium rhizogenes-mediated (ARM) transformation is a highly efficient technique for generating composite plants composed of transgenic roots and wild-type shoot, providing a powerful tool for studying root biology. The ARM transformation has been established in many plant species, including soybean. However, traditional transformation of soybean, transformation efficiency is low. Additionally, the hairy roots were induced in a medium, and then the generated composite plants were transplanted into another medium for growth. This two-step operation is not only time-consuming, but aggravates contamination risk in the study of plant-microbe interactions.ResultsHere, we report a one-step ARM transformation method with higher transformation efficiency for generating composite soybean plants. Both the induction of hairy roots and continuous growth of the composite plants were conducted in a single growth medium. The primary root of a 7-day-old seedling was decapitated with a slanted cut, the residual hypocotyl (maintained 0.7-1 cm apical portion) was inoculated with A. rhizogenes harboring the gene construct of interest. Subsequently, the infected seedling was planted into a pot with wet sterile vermiculite. Almost 100% of the infected seedlings could produce transgenic positive roots 16 days post-inoculation in 7 tested genotypes. Importantly, the transgenic hairy roots in each composite plant are about three times more than those of the traditional ARM transformation, indicating that the one-step method is simpler in operation and higher efficiency in transformation. The reliability of the one-step method was verified by CRISPR/Cas9 system to knockout the soybean Rfg1, which restricts nodulation in Williams 82 (Nod-) by Sinorhizobium fredii USDA193. Furthermore, we applied this method to analyze the function of Arabidopsis YAO promoter in soybean. The activity of YAO promoter was detected in whole roots and stronger in the root tips. We also extended the protocol to tomato.ConclusionsWe established a one-step ARM transformation method, which is more convenient in operation and higher efficiency (almost 100%) in transformation for generating composite soybean plants. This method has been validated in promoter functional analysis and rhizobia-legume interactions. We anticipate a broad application of this method to analyze root-related events in tomato and other plant species besides soybean.

Project description:BackgroundGeneration of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Co-transformation methods that enable the removal of selectable marker genes have been extensively used because they are simple and clean. Few comparisons are currently available between different strain/plasmid co-transformation systems, and also data are related to variation in co-transformation frequencies caused by other details of the vector design.ResultsIn this study, we constructed three vector systems for the co-transformation of allotetraploid Brassica napus (B. napus) mediated by Agrobacterium tumefaciens and compared these co-transformation methods. We tested a mixed-strain system, in which a single T-DNA is harbored in two plasmids, as well as two "double T-DNA" vector systems, in which two independent T-DNAs are harbored in one plasmid in a tandem orientation or in an inverted orientation. As confirmed by the use of PCR analysis, test strips, and Southern blot, the average co-transformation frequencies from these systems ranged from 24 to 81% in T0 plants, with the highest frequency of 81% for 1:1 treatment of the mixed-strain system. These vector systems are valuable for generating marker-free transgenic B. napus plants, and marker-free plants were successfully obtained in the T1 generation from 50 to 77% of T0 transgenic lines using these systems, with the highest frequency of 77% for "double T-DNA" vector systems of pBID RT Enhanced. We further found that marker-free B. napus plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T0 generation. Two types of herbicide resistant transgenic B. napus plants, Bar + with phosphinothricin resistance and Bar + EPSPS + GOX + with phosphinothricin and glyphosate resistance, were obtained.ConclusionWe were successful in removing selectable marker genes in transgenic B. napus plants using all three co-transformation systems developed in this study. It was proved that if a appropriate mole ratio was designed for the specific length ratio of the twin T-DNAs for the mixed-strain method, high unlinked co-insertion frequency and overall success frequency could be achieved. Our study provides useful information for the construction of efficient co-transformation system for marker-free transgenic crop production and developed transgenic B. napus with various types of herbicide resistance.

Project description:BackgroundMapping and map-based cloning of genes that control agriculturally and economically important traits remain great challenges for plants with complex highly repetitive genomes such as those within the grass tribe, Triticeae. Mapping limitations in the Triticeae are primarily due to low frequencies of polymorphic gene markers and poor genetic recombination in certain genetic regions. Although the abundance of repetitive sequence may pose common problems in genome analysis and sequence assembly of large and complex genomes, they provide repeat junction markers with random and unbiased distribution throughout chromosomes. Hence, development of a high-throughput mapping technology that combine both gene-based and repeat junction-based markers is needed to generate maps that have better coverage of the entire genome.ResultsIn this study, the available genomics resource of the diploid Aegilop tauschii, the D genome donor of bread wheat, were used to develop genome specific markers that can be applied for mapping in modern hexaploid wheat. A NimbleGen array containing both gene-based and repeat junction probe sequences derived from Ae. tauschii was developed and used to map the Chinese Spring nullisomic-tetrasomic lines and deletion bin lines of the D genome chromosomes. Based on these mapping data, we have now anchored 5,171 repeat junction probes and 10,892 gene probes, corresponding to 5,070 gene markers, to the delineated deletion bins of the D genome. The order of the gene-based markers within the deletion bins of the Chinese Spring can be inferred based on their positions on the Ae. tauschii genetic map. Analysis of the probe sequences against the Chinese Spring chromosome sequence assembly database facilitated mapping of the NimbleGen probes to the sequence contigs and allowed assignment or ordering of these sequence contigs within the deletion bins. The accumulated length of anchored sequence contigs is about 155 Mb, representing ~ 3.2 % of the D genome. A specific database was developed to allow user to search or BLAST against the probe sequence information and to directly download PCR primers for mapping specific genetic loci.ConclusionsIn bread wheat, aneuploid stocks have been extensively used to assign markers linked with genes/traits to chromosomes, chromosome arms, and their specific bins. Through this study, we added thousands of markers to the existing wheat chromosome bin map, representing a significant step forward in providing a resource to navigate the wheat genome. The database website ( http://probes.pw.usda.gov/ATRJM/ ) provides easy access and efficient utilization of the data. The resources developed herein can aid map-based cloning of traits of interest and the sequencing of the D genome of hexaploid wheat.

Project description:BackgroundGene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible.ResultsIn this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right- and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2-10%. Interestingly, while large deletions (> 100 bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato.ConclusionsGene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.

Dataset Information

Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.

Publications

Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties.

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