T-DNA integration patterns in transgenic plants mediated by &lt;I&gt;Agrobacterium tumefaciens&lt;/I&gt;

Yang, Lin; Fu, Feng; Li, Wan-Chen

doi:10.3724/sp.j.1005.2011.01327

Cited by 5 publications

(3 citation statements)

References 51 publications

(41 reference statements)

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“…This method does not require laborious preparations and the use of radioactive probes in DNA hybridizations. Although the method was only recently developed (Liu and Chen, 2007;Yang et al, 2011), primer design appears to be crucial for eliminating nonspecific PCR bands. For example, we obtained the same flanking sequence (alpha tubulin 5) from a transformant line using 3 different AD primers.…”

Section: Discussionmentioning

confidence: 99%

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos

Uçarlı¹,

Tufan²,

Gürel³

2015

Genet. Mol. Res.

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ABSTRACT. Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of cocultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these Genomic integration of transgenes in mature barley embryos expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T 0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

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Section: Discussionmentioning

confidence: 99%

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos

Uçarlı¹,

Tufan²,

Gürel³

2015

Genet. Mol. Res.

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“…These procedures are associated with stresses such as wounding and osmotic stress, and GM plants obtained through tissue culture are more variable than those obtained using the floral‐dip technique (Montero et al, 2011). Agrobacterium and other pathogen infections may also cause mutations (Yang et al, 2011). In total, many factors can induce unintended effects, including random mutations, somatic mutations, gene sequence insertions, positional effects, inductive effects, mutations in the tissue culture process, and pleiotropy, which may emerge at any stage of the GE plant development process (Wang et al, 2019) (Liu et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…Two RNAi‐based GE rice lines (Ubi5 and Ubi6) were transformed with the same transformation vector, which included promoters derived from ubiquitin genes and IR sequences; however, no hpt marker genes were present. Yang et al (2011) found that the insertions of the Ubi5 and Ubi6 constructs were located in an intergenic site at 26.82 and 7.69 Mb on chromosome 6, respectively (unpublished), and each as a single copy. The researchers further verified this insertion site via PCR using three specific PCR primers (China patent: CN201911243983.0).…”

Section: Methodsmentioning

confidence: 99%

RNAi‐based genetically engineered rice resistant to black‐streaked dwarf virus does not show adverse genetic effects: A multi‐omics analysis

Huang,

Wang,

Zhu

et al. 2024

Plants People Planet

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Societal Impact StatementAdvancements in science and technology enable us to address the challenges of the times: hunger, malnutrition, climate change, and dwindling natural resources. One of the most significant innovations in food and agriculture is the advent of genetic engineering, which may help us address the aforementioned challenges. Millions of people could benefit from the application of genetically engineered crops in agriculture and food. However, it is vital that biosafety assessments of unintended effects are carried out to identify any potential risks to humans, the environment, or society.Summary The study compared the extent of siRNA, transcriptomic and metabolomic changes in the leaves of four RNAi‐based genetically engineered rice lines to evaluate whether RNAi‐based genetically engineered rice that is resistant to black‐streaked dwarf virus experiences changes not seen in the non‐genetically engineered counterpart. siRNA, transcriptome, and metabolome profiling were conducted. siRNAs with different lengths and abundance were highly enriched in the transcript of rice; however, relative expression analysis of eight potential off‐target genes revealed that there was no decrease in gene expression in the RNAi‐based genetically engineered lines, meaning that no off‐target phenomena were caused as a result of siRNA derived from invert repeat sequences of rice black‐streaked dwarf virus in this study. This study identified some differentially expressed genes and differentially accumulated metabolites in the RNAi‐based genetically engineered lines; however, they were not enriched in pathways detrimental for humans, animals and the environment. Only 30 differentially expressed genes were significantly enriched in the plant hormone signal transduction pathway via a combined analysis using two‐way orthogonal partial least squares model. These genes belonging to salicylic acid, jasmonic acid, cytokinin, abscisic acid, and brassinosteroid pathway involved in pathways related to virus resistance or infection response were significantly changed. Taken together, there was no off‐target in RNAi‐based genetically engineered rice lines and may have a positive effect on virus resistance overall in this study.

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A novel double T-DNA system for producing stack and marker-free transgenic plants

Wang

Dong

et al. 2016

Biol Plant

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This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7, containing six target genes, was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of intact two T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least six target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.

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T-DNA integration patterns in transgenic plants mediated by <I>Agrobacterium tumefaciens</I>

Cited by 5 publications

References 51 publications

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos

RNAi‐based genetically engineered rice resistant to black‐streaked dwarf virus does not show adverse genetic effects: A multi‐omics analysis

A novel double T-DNA system for producing stack and marker-free transgenic plants

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