The bacterial cytosine deaminase gene used as a conditional negative selection marker in transgenic rice plants

Dai, Shaotao; Carcamo, R.; Zhang, Z.; Chen, S.; Beachy, Roger N.

doi:10.1007/s002990100390

Cited by 12 publications

(8 citation statements)

References 27 publications

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“…The deamination of 5‐FC by FCY into cytotoxic 5‐FU has been used in the model plant Arabidopsis as a negative selection marker under the constitutive and strong viral 35S promoter (Perera et al , ; Kobayashi et al , ). Similar results have been reported in Oryza sativa (rice) (Dai et al , ) and Solanum lycopersicum (tomato) (Hashimoto et al , ). These studies have shown that 5‐FC incorporation triggers plant autonomous toxicity, whereas other negative markers such as iaaH (using IAM as a precursor) or NR (using chlorate as a substrate) can be toxic for neighboring plants through diffusion of their toxic product in the culture medium.…”

Section: Discussionsupporting

confidence: 88%

Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

et al. 2019

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Recent advances in the study of plant developmental and physiological responses have benefited from tissue-specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue-specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY-UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell-autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5-FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell-specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type-specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development. Tissue-specific genetic ablation 733Histochemical analysis and microscopy GUS staining was performed as previously described (P eret et al., 2007). Plants were cleared for 24 h in 1 M chloral hydrate and 33% glycerol. Seedlings were mounted in 50% glycerol and observed

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Section: Discussionsupporting

confidence: 88%

Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

et al. 2019

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“…Presence of >4.3 kb hybridizable bands with ASAL probe, in EcoRI digested DNA of transformants, suggests the independent nature of transgene integration in the primary transformants of cotton (Figure 2). It has been established that multiple copies of transgene(s) often result in the co-suppression and gene silencing [22], [23]. Single copy integration of transgene(s) is essential to achieve predictable patterns of inheritance and to eliminate the problem of gene silencing in the transgenic plants [24].…”

Section: Discussionmentioning

confidence: 99%

Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

et al. 2013

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Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1–2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects.

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“…A mutant rice gene, feedback‐insensitive α subunit of anthranilate synthase OsSAID , has been used as selectable agent in rice based on its ability to detoxify tryptophan analogue 5‐methyltryptophan (Yamada et al , 2004). Similarly, new negative or conditional selection systems have been developed for rice transformation (Dai et al , 2001a; Upadhyaya et al , 2000b; Oreifig et al , 2004). A new positive/negative selection system employing a marker gene, dao1 from yeast Rhodotorula gracilis has been described (Erikson et al , 2004).…”

Section: Rice Transformationmentioning

confidence: 99%

“…Similarly, new negative or conditional selection systems have been developed for rice transformation (Dai et al, 2001a;Upadhyaya et al, 2000b;Oreifig et al, 2004). A new positive/negative selection system employing a marker gene, dao1 from yeast Rhodotorula gracilis has been described (Erikson et al, 2004).…”

Section: Novel Reporter and Selection Marker Genesmentioning

confidence: 99%

Recent advances in rice biotechnology—towards genetically superior transgenic rice

Bajaj

Mohanty

2005

Plant Biotechnology Journal

210

109

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SummaryRice biotechnology has made rapid advances since the first transgenic rice plants were produced 15 years ago. Over the past decade, this progress has resulted in the development of high frequency, routine and reproducible genetic transformation protocols for rice. This technology has been applied to produce rice plants that withstand several abiotic stresses, as well as to gain tolerance against various pests and diseases. In addition, quality improving and increased nutritional value traits have also been introduced into rice. Most of these gains were not possible through conventional breeding technologies. Transgenic rice system has been used to understand the process of transformation itself, the integration pattern of transgene as well as to modulate gene expression. Field trials of transgenic rice, especially insect-resistant rice, have recently been performed and several other studies that are prerequisite for safe release of transgenic crops have been initiated. New molecular improvisations such as inducible expression of transgene and selectable marker-free technology will help in producing superior transgenic product. It is also a step towards alleviating public concerns relating to issues of transgenic technology and to gain regulatory approval. Knowledge gained from rice can also be applied to improve other cereals. The completion of the rice genome sequencing together with a rich collection of full-length cDNA resources has opened up a plethora of opportunities, paving the way to integrate data from the large-scale projects to solve specific biological problems.

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The bacterial cytosine deaminase gene used as a conditional negative selection marker in transgenic rice plants

Cited by 12 publications

References 27 publications

Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

Recent advances in rice biotechnology—towards genetically superior transgenic rice

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