Agrobacterium-mediated Approach Research Articles

Targeting Disease Susceptibility Genes in Wheat Through wide Hybridization with Maize Expressing Cas9 and Guide RNA.

Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight and tan spot, Septoria nodorum blotch, and spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15 to 33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool, not only for targeting susceptibility genes to improve disease resistance without regulatory issues, but also for understanding gene function in wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization and functional analysis of microRNA399 in Cunninghamia lanceolata

The miR399 is a conserved microRNA (miRNA) family, and it has been characterized as an essential regulator of phosphorus transport in plants. However, the biological function of miR399 in Cunninghamia lanceolata is still largely unclear. In this study, the comparison of mature miR399 sequence revealed a high similarity between Arabidopsis thaliana and C. lanceolate, and the pre-miR399 was capable of forming a typical stem-loop hairpin structure. A gene PHOSPHATE 2 (PHO2) was identified as a target of cln-miR399 using 5' rapid amplification of cDNA ends. Furthermore, the relationship between cln-miR399 and PHO2 was further confirmed through a transient co-expression of both genes in Nicotiana benthamiana. To examine the function of miR399 in Arabidopsis, miR399-overexpressing transgenic Arabidopsis thaliana was acquired using Agrobacterium-mediated approach. Real-time PCR showed that the amount of cln-MIR399 transcripts was higher in miR399-overexpressing plants than in wild-type plants, which was accompanied with down-regulation of expression of its target gene AtPHO2. The P content was 1.40 to 1.56-fold higher in the leaves of three transgenic lines than in wild type plants. However, the P content in the roots of the three transgenic lines was 24.5 - 37.2 % less than that in wild type plants. Moreover, the transcriptions of three phosphate transporter genes (PHT1, PHT2, and PHT3) were up-regulated in roots of miR399-overexpressing Arabidopsis plants. Interestingly, the transgenic lines exhibited retarded growth under normal P conditions compared with the wild type. Our findings demonstrate that cln-miR399 may play crucial roles in P transport and plant growth via regulation of its target gene PHO2.

Apple Columnar Gene MdDMR6 Increases the Salt Stress Tolerance in Transgenic Tobacco Seedling and Apple Calli

Recent studies have shown that the MdDMR6 is a key gene for the control of columnar growing habit. However, there has been no research report on the function of the MdDMR6 gene. In the present study, the columnar apple shoot cultures exhibited better tolerance than that of the standard apple shoot cultures under the salt stress. The expression of MdDMR6 was remarkably induced by the salt stress. Subsequently, the MdDMR6 was transferred into tobacco and apple calli via the Agrobacterium-mediated approach. Functional analysis showed that the overexpressing transgenic tobacco and apple calli displayed salt tolerance and ABA-insensitive phenotype through physiological and biochemical analysis. The MdDMR6 overexpression activated the transcript levels of several genes involved in the ABA-dependent stress genes under the salt condition. These results suggest that the MdDMR6 would increase the plant tolerance to the salt stress via activating or interacting other protein to indirectly activate ABA-dependent pathways.

Molecular and phenotypic characterization of transgenic wheat and sorghum events expressing the barley alanine aminotransferase.

The expression of a barley alanine aminotransferase gene impacts agronomic outcomes in a C3 crop, wheat. The use of nitrogen-based fertilizers has become one of the major agronomic inputs in crop production systems. Strategies to enhance nitrogen assimilation and flux in planta are being pursued through the introduction of novel genetic alleles. Here an Agrobacterium-mediated approach was employed to introduce the alanine aminotransferase from barley (Hordeum vulgare), HvAlaAT, into wheat (Triticum aestivum) and sorghum (Sorghum bicolor), regulated by either constitutive or root preferred promoter elements. Plants harboring the transgenic HvAlaAT alleles displayed increased alanine aminotransferase (alt) activity. The enhanced alt activity impacted height, tillering and significantly boosted vegetative biomass relative to controls in wheat evaluated under hydroponic conditions, where the phenotypic outcome across these parameters varied relative to time of year study was conducted. Constitutive expression of HvAlaAT translated to elevation in wheat grain yield under field conditions. In sorghum, expression of HvAlaAT enhanced enzymatic activity, but no changes in phenotypic outcomes were observed. Taken together these results suggest that positive agronomic outcomes can be achieved through enhanced alt activity in a C3 crop, wheat. However, the variability observed across experiments under greenhouse conditions implies the phenotypic outcomes imparted by the HvAlaAT allele in wheat may be impacted by environment.

Overexpression of an MYB-Related Gene FvMYB1 from Fraxinus velutina Increases Tolerance to Salt Stress in Transgenic Tobacco

MYB transcription factors (TFs) in plants play important roles in abiotic stress tolerance. Here, we report that an MYB-related TF gene, named FvMYB1, was isolated from the salt-tolerant tree species Fraxinus velutina Torr. The real-time PCR analysis showed that the FvMYB1 transcript increased remarkably in response to salt treatment. Subsequently, the FvMYB1 gene was transferred into the tobacco genome via the Agrobacterium-mediated approach. PCR and Southern blot verified that the FvMYB1 gene had been expressed in transgenic tobacco. The overexpressing transgenic seedlings displayed better salt tolerance in root length, shoot fresh weight, and shoot/root ratio compared to the wild type. Moreover, along with the superoxide dismutase and peroxidase activities and proline content that were significantly increased in the transgenic plants, the transcriptions of a series of stress-responsive genes were up-regulated under salt treatment. Further, an analysis of the upstream sequence of the FvMYB1 gene revealed that the promoter could enhance GUS gene expression in transgenic tobacco with NaCl treatment. These results indicate that FvMYB1 functions as a transcriptional activator that plays a regulatory role in salt stress tolerance of F. velutina.

Production of oleanane-type sapogenin in transgenic rice via expression of β-amyrin synthase gene from Panax japonicus C. A. Mey.

BackgroundPanax japonicus C. A. Mey. is a rare traditional Chinese herbal medicine that uses ginsenosides as its main active ingredient. Rice does not produce ginsenosides because it lacks a key rate-limiting enzyme (β-amyrin synthase, βAS); however, it produces a secondary metabolite, 2,3-oxidosqualene, which is a precursor for ginsenoside biosynthesis.ResultsIn the present study, the P. japonicus βAS gene was transformed into the rice cultivar ‘Taijing 9’ using an Agrobacterium-mediated approach, resulting in 68 rice transgenic plants of the T0 generation. Transfer-DNA (T-DNA) insertion sites in homozygous lines of the T2 generation were determined by using high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) and were found to vary among the tested lines. Approximately 1–2 copies of the βAS gene were detected in transgenic rice plants. Real-time PCR and Western blotting analyses showed that the transformed βAS gene could be overexpressed and β-amyrin synthase could be expressed in rice. HPLC analysis showed that the concentration of oleanane-type sapogenin oleanolic acid in transgenic rice was 8.3–11.5 mg/100 g dw.ConclusionsThe current study is the first report on the transformation of P. japonicus βAS gene into rice. We have successfully produced a new rice germplasm, “ginseng rice”, which produces oleanane-type sapogenin.

English

In our approach to control reactive oxygen species produced as a result of oxidative stress experienced by cassava roots during harvesting, a phenomena which causes postharvest physiological deterioration in the roots, we transformed cassava variety TMS 60444 with the AtAOX1a gene, driven by the 35S promoter, using agrobacterium-mediated approach. Extracted genomic DNAs of putative transgenic lines were screened using polymerase chain reaction technique (PCR). Messenger RNA was extracted from selected PCR-positive lines for reverse transcription-PCR analysis for gene expression. To screen positive lines for gene function, leaf lobes from two transgenic lines with a line carrying an empty vector and the wild type were subjected to somatic embryogenesis (SE), a known oxidative stress process. The results show that the wild type, at 16 days after initiation (DAI) of the leaf lobes on callus initiation medium, had the highest (100%) number of leaf lobes that produced at least one observed organised embryogenic structure (OES). This was followed by PEV-3, the empty vector plant with 50% OES production, while PB-3 had the least percent (20%) of leaf lobes with OES. PB-3 line also had no OES at all in five out of the seven periods of data collection. During the period, the wild type recorded the highest attainable OES quality score of 2.0 (on a scale of 1-5 where 1=bad and 5=excellent) at the first initiation cycle. Both the transgenic lines and the empty-vector plantlet recorded quality score of 1.0. It seems AtAOX1a only hinders OES development, but exerts little effect on the quality, if OES does not degenerate after development. Key words: Genomic DNAs, reverse transcription-PCR, somatic embryogenesis (SE).

Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco

Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST + Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance. Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST + Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST + Ta6-SFT > Ta1-SST + Ta6-SFT + Ta1-FFT > Ta1-SST + Ta1-FFT > Ta1-SFT + Ta1-FFT > single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants. Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests. An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3-4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass. This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.

Transformation of HBsAg (hepatitis B surface antigen) gene into tomato mediated by Agrobacterium tumefaciens

The plant expression vector pBRSAg was constructed as suitable for transformation via Agrobacterium-mediated approach. It contains all elements for plant expression, such as CaMV 35S promoter, both left and right border sequence for transferred DNA (T-DNA) in Agrobacterium, plant reporter gene gus, and plant selection marker gene hpt. The recombinant binary vector pBRSAg was transformed into Agrobacterium tumefaciens strains by using the freeze-thaw method. Tomato cotyledon explants were transformed by A. tumefaciens and plants were regenerated on selection medium. GUS staining, PCR and PCR-Southern analysis of the plants were positive. It was for the first time shown that the HBsAg (hepatitis B surface antigen) gene was introduced into tomato plants. The expression of transgene is under investigation.

Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with β-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1 mg l−1 indole-3-butyric acid and 15 mg l−1 hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for β-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of 3.9 ± 0.39 transgenic plantlets per explant was achieved in the present transformation system. It took only 2–3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

Populus nigra×Populus deltoids “108” Transformed the Chimeric Genes of Spider Insecticidal and Bt

Using the agrobacterium-mediated approach,the anti-insect gene(the chimeric genes of spider insecticidal and Bt) was successfully transformed into the acceptor of Populus nigra×Populus deltoids "108" as explant.Through PCR analysis,the agarose gel electrophoresis result displayed that six of eight Kanamycin fastness seedlings had target genes;Through PCR-Southern blots analysis,the result showed that the positive rate was 50%,and further indicated that the target genes had been inserted into the genomes of Populus nigra×Populus deltoids "108" plants successfully.

Expression analysis of the calcineurin B-like gene family in rice ( Oryza sativa L.) under environmental stresses

Calcium plays a crucial role as a second messenger in mediating various defense responses under environmental stresses. Calcineurin B-like (CBL) proteins have been implicated as important Ca 2+ sensors in plant-specific calcium signaling. Based on the similarity of sequence, ten CBL genes were identified by searching the rice japonica genome database, which were randomly distribute on chromosomes 1, 2, 3, 5, 10 and 12. By semi-quantitative RT-PCR approach the expression pattern of each gene was detected in various organs at the adult stage and seedlings treated with NaCl, PEG and cold stresses or exogenous ABA. The results showed that the induction of each rice CBL gene was not only responsive to different stress conditions, but also organ specific. In vivo targeting experiment revealed that OsCBL8 localized to the plasma membrane, which was consistent with OsCBL4 and SOS3 previously reported. To elucidate the putative function of OsCBL8 gene, transgenic rice plants overexpressing OsCBL8 gene were generated by the Agrobacterium-mediated approach. The OsCBL8 transgenic rice seedlings showed more tolerance to salt stress than non-transgenic seedlings.

Agrobacterium-Mediated Transformation of Common Bermudagrass (Cynodon dactylon)

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. We have been able to transform the species using Agrobacterium-mediated approach. In seven experiments reported here, a total of 67 plates of calluses and suspensions were infected with Agrobacterium tumefaciens strains, and nine hygromycin B resistant calluses were obtained after selection. Among them two green independent transgenic plants were recovered. The plants growing in pots looked relatively compact at the beginning, but the ploidy level of the plants, as determined by nuclear DNA content, was not altered.