The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system

Vain, Philippe; Harvey, Alison; Worland, Barbara; Ross, S. M.; Snape, J. W.; Lonsdale, David

doi:10.1007/s11248-004-2808-5

Cited by 26 publications

(30 citation statements)

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“…Copy number in barley plants transformed with pDM805 was generally higher [24] than in tobacco plants transformed with pGreen [23]. Additional virulence genes in the pGreen binary system significantly altered plant transformation efficiency in rice but had no effect on transgene copy number, percentage of expressing lines or expression level [37]. However, the lack of selection of phosphinothricin-resistant plants in combinations transformed with pGreen, where bar was also under the 35S promoter, which is strong for dicotyledonous plants, might suggest a positive correlation of copy number with the level of resistance.…”

Section: Discussionmentioning

confidence: 99%

Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression

Nadolska‐Orczyk

Pietrusińska

Binka-Wyrwa

et al. 2007

Cellular and Molecular Biology Letters

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This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression. In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%) was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and, depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein.

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Section: Discussionmentioning

confidence: 99%

Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression

Nadolska‐Orczyk

Pietrusińska

Binka-Wyrwa

et al. 2007

Cellular and Molecular Biology Letters

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“…In earlier studies (Vain et al, 2004), the addition of the wild-type virG gene (virGwt) in the vector backbone sequence nearly doubled the overall performance of the pGreen/pSoup vector system when considering transformation frequency, absence of backbone sequence integration, and expression of unselected transgenes. Therefore, some pCLEAN vectors were constructed (i.e.…”

Section: Overview Of the Pclean Vector Systemmentioning

confidence: 99%

“…The EcoRV fragment of pSLJ261 enclosing the NOS promoter:HPT:NOS terminator (Jones et al, 1992) was cloned into the blunted PacI-digested pCLEAN-S161 to give pCLEAN-S166. The PstI fragment of pCLEAN-S48 containing the wild-type virG (Vain et al, 2004) was cloned into the PstIdigested pCLEAN-S161 to result in pCLEAN-S167 (GenBank accession no. EU186086).…”

Section: Construction Of Pclean-g and Pclean-s Vectorsmentioning

confidence: 99%

“…Vectors have been engineered to improve stable or transient transformation efficiency with the addition of virulence (vir) genes (Hiei et al, 1994;Vain et al, 2004), suppressors of gene silencing (Hellens et al, 2005), or overdrive sequences near the border sequences (Podevin et al, 2006). Vectors aiming at the transformation of particular plant species, such as cereal crops (Hiei et al, 1994;Wang et al, 1998), have also been designed.…”

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confidence: 99%

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The pCLEAN Dual Binary Vector System forAgrobacterium-Mediated Plant Transformation

et al. 2007

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The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.Plant transformation technologies are fundamental to state-of-the-art plant molecular genetics and crop improvement through genetic engineering (Vain, 2006). Over the past 30 years, the development of novel transformation vectors has been seminal to many breakthroughs in plant transgenesis (for review, see Vain, 2007). In the 1980s, the engineering of Agrobacterium tumefaciens Ti plasmids-namely, the removal of oncogenes and the use of a chimerical gene(s)-led to the production of the first fertile transgenic plants (Zambryski et al., 1983) and the development of binary vectors for plant transformation (Hoekema et al., 1983; Bevan, 1984). Additional types of vectors were developed for direct transfer of DNA into the plant nuclear genome (Paszkowski et al., 1984) and later on into the plastome (Svab et al., 1990). Vector development is particularly important for Agrobacterium-based technologies as binary vectors must comply with, and best exploit, the natural mechanisms and interactions between Agrobacterium and plant cells (Gelvin, 2003). In recent years, the further understanding of T-DNA integration into the plant nuclear genome, combined with an increasing demand for precise and efficient transformation technologies, has created a new opportunity to develop plant transformation vectors with improved characteristics.Since the 1980s, binary vectors for Agrobacteriummediated transformation have been optimized to contain a wide range of selectable marker and reporter genes (Rogers et al., 1987; Becker et al., 1992;Jones et al....

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“…Following construction, the vector was transferred into Agrobacterium to produce a strain suitable for introducing genes into target plants. The mid-1990s saw the development of the 'super-binary' system which enabled researchers to enhance the transformation efficiency by employing additional virulence genes [17,18]. This was achieved by inserting a DNA fragment containing virB, virC and virG genes from pTiB0542 into a small T-DNA carrying plasmid.…”

Section: Vector Systems For Transformationmentioning

confidence: 99%

Genetic Transformation –Agrobacterium

Curtis¹,

Davey

Anthony

2010

Plant Cell Culture

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The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system

Cited by 26 publications

References 31 publications

Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression

Diploid potato (Solanum tuberosum L.) as a model crop to study transgene expression

The pCLEAN Dual Binary Vector System forAgrobacterium-Mediated Plant Transformation

Genetic Transformation –Agrobacterium

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