Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

AbstractA nativerepABCreplication origin, ori pRi, was previously reported as a single copy plasmid inAgrobacterium tumefaciensand can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors forAgrobacterium-mediatedtransformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::RirepABCoperon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed thatAgrobacteriumcells with the RepBY299Hmutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299Hmutation on transformation and quality plant production, the RepBY299Hmutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299Hmutation inAgrobacteriumcells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

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Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens

Transgenic Research ◽

10.1007/s11248-010-9458-6 ◽

2010 ◽

Vol 20 (4) ◽

pp. 773-786 ◽

Cited By ~ 15

Author(s):

Xudong Ye ◽

Edward J. Williams ◽

Junjiang Shen ◽

Susan Johnson ◽

Brenda Lowe ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Transgenic Plants ◽

Replication Origin ◽

Single Copy ◽

Crop Species ◽

Binary Vectors ◽

Enhanced Production

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Receiving of genetic-modified wheat plants with heterolous ornitin-δ-aminotransferase gene

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v22.952 ◽

2018 ◽

Vol 22 ◽

pp. 222-227

Author(s):

O. M. Honcharuk ◽

O. V. Dubrovna

Keyword(s):

Triticum Aestivum ◽

Transgenic Plants ◽

Bread Wheat ◽

Binary Vector ◽

Triticum Aestivum L ◽

Callus Cultures ◽

Pcr Analysis ◽

Genetic Construct ◽

Agrobacterium Mediated Transformation

Aim. Receiving of genetically modified plants of bread wheat with heterologous ornithine‑δ‑aminotransferase gene. Methods. Agrobacterium-mediated transformation of callus cultures in vitro, PCR-analysis. Results. By Agrobacterium-mediated transformation of the morphogenic calluses of bread wheat (Triticum aestivum L.) using the AGLO strain containing the binary vector pBi-OAT with the target ornithine-δ-aminotransferase (oat) and selective neomycinphosphotransferase II (nptII), transgenic plants-regenerators have been obtained. Conclusions. As a result of the genetic transformation of Zimoyarka variety, 12 wheat regenerants were obtained in the genome which revealed a complete integration of the genetic construct containing the oat and nptII transgenes. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine‑δ‑aminotransferase gene, PCR-analysis.

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Effect of nuclear matrix attachment regions on transgene expression in tobacco plants.

Acta Biochimica Polonica ◽

10.18388/abp.2001_3898 ◽

2001 ◽

Vol 48 (3) ◽

pp. 637-646 ◽

Cited By ~ 16

Author(s):

W Nowak ◽

M Gawłowska ◽

A Jarmołowski ◽

J Augustyniak

Keyword(s):

Transgenic Plants ◽

Reporter Gene ◽

Transgene Expression ◽

Binary Vector ◽

Special Property ◽

Matrix Attachment Regions ◽

Plant Populations ◽

Tobacco Plants ◽

Gus Reporter ◽

Loop Domains

Matrix attachment regions (MARs) are thought to participate in the organization and segregation of independent chromosomal loop domains. Although there are several reports on the action of natural MARs in the context of heterologous genes in transgenic plants, in our study we tested a synthetic MAR (sMAR) with the special property of unpairing when under superhelical strain, for its effect on reporter gene expression in tobacco plants. The synthetic MAR was a multimer of a short sequence from the MAR 3' end of the immunoglobulin heavy chain (IgH) enhancer. This sMAR sequence was used to flank the beta-glucuronidase (GUS) reporter gene within the T-DNA of the binary vector pBI121. Vectors with or without the sMARs were then used to transform tobacco plants by Agrobacterium tumefaciens. Transgenic plants containing the sMAR sequences flanking the GUS gene exhibited higher levels of transgene expression compared with transgenic plants which lacked the sMARs. This effect was observed independently of the position of the sMAR at the 5' side of the reporter gene. However, variation of the detected transgene expression was significant in all transformed plant populations, irrespective of the construct used.

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A new double right border binary vector for producing marker-free transgenic plants

BMC Research Notes ◽

10.1186/1756-0500-6-448 ◽

2013 ◽

Vol 6 (1) ◽

pp. 448 ◽

Cited By ~ 11

Author(s):

Jonathan M Matheka ◽

Sylvester Anami ◽

James Gethi ◽

Rasha A Omer ◽

Amos Alakonya ◽

...

Keyword(s):

Transgenic Plants ◽

Binary Vector ◽

Right Border ◽

Marker Free

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Construction of Two Vectors for a Bypass of Monocotyledon Plants Photorespiration

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.340.351 ◽

2011 ◽

Vol 340 ◽

pp. 351-356

Author(s):

Xue Liang Bai ◽

Dan Wang ◽

Ning Ning Liu ◽

Li Jing Wei ◽

Ye Rong Zhu ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Transgenic Plants ◽

Binary Vector ◽

Transit Peptide ◽

Expression Vectors ◽

Chloroplast Transit Peptide ◽

E Coli ◽

Plant Expression ◽

Glycolate Dehydrogenase ◽

Save Energy

In order to modify the photorespiration of monocotyledonous crops, we aimed to construct vectors that will be used to introduce a bypass to the native photorespiration pathway. Firstly, we cloned the encoding sequences of glyoxylate carboligase (GCL) and tartronic semialdehyde reductase (TSR) fromE. coli, glycolate dehydrogenase (GDH) fromArabidopsis thalianaand chloroplast transit peptide (cTP) from rice. Then we constructed a universal vector pEXP harboring the encoding sequence of cTP for targeting a protein into chloroplast. By insertion of these three encoding sequences into the universal vector pEXP, we obtained the expression cassettes for GCL, TSR and GDH, respectively. Finally, we inserted the cassettes for GCL and TSR in tandem into the binary vector pCAMBIA 1301, and for GDH into another binary vector, pPGN, to obtain our plant expression vectors pCAMBIA 1301-TG and pPGN-GDH, respectively. These two expression vectors possess different selection resistance and can be used to transform monocots together, to introduce the bypass pathway of photorespiration. By this way, the transgenic plants can recycle glycolate, the by-product of photosynthesis in C3plants, within the chloroplast, simultaneously, save energy and avoid the loss of ammonia, which will contribute to improved growth.

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GENETIC TRANSFORMATION AND REGENERATION OF TRANSGENIC SWEETPOTATO PLANTS.

HortScience ◽

10.21273/hortsci.30.3.435f ◽

1995 ◽

Vol 30 (3) ◽

pp. 435f-435 ◽

Cited By ~ 2

Author(s):

Marceline Egnin ◽

C.S. Prakash

Keyword(s):

Agrobacterium Tumefaciens ◽

Transgenic Plants ◽

Genetic Transformation ◽

Binary Vector ◽

Blot Hybridization ◽

Pcr Amplification ◽

Ms Medium ◽

Petiole Explants ◽

Efficient Delivery ◽

Foreign Genes

This study aimed to optimize factors for the efficient delivery of foreign genes into sweetpotato using Agrobacterium tumefaciens and develop transgenic plants. Disarmed Agrobacterium C58 carrying a binary vector pBI 121C2H with gusA, nptll, and the nutritional protein asp-l genes was used to cocultivate (4 days) petiole explants of the sweetpotato genotype P1318846-3. Pre-incubation of petioles for 3 days on MS medium with 2,4-D (0.2 mg·liter–1) before infection resulted in higher transformation. Putative transgenic shoots were obtained by transfer of petioles to MS medium with TDZ (0.2 mg·liter–1) and kanamycin (80 to 140 mg·liter–1). The PCR amplification of gusA, nptll, and asp-1 genes in the 37 putative transgenic shoots showed that six plants contained the three genes. However, none of these plants showed histochemical expression of the gusA gene. The introduced gene may have been methylated resulting in the lack of its expression. DNA blot hybridization studies are underway to verify the presence and integration of the transgenes.

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