Effect of promoter driving selectable marker on corn transformation

Prakash, Neha; Prasad, Vandana; Chidambram, Thillai P.; Cherian, Shoba; Jayaprakash, T. L.; Dasgupta, Santanu; Wang, Qi; Mann, Michael T.; Spencer, Tim; Boddupalli, Raghava S.

doi:10.1007/s11248-007-9149-0

Cited by 26 publications

(15 citation statements)

References 40 publications

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“…In some cases, transgene expression that is too strong might be disadvantageous. For example, high expression of a selectable marker gene has been reported to produce leakage of the protein that confers resistance to antibiotic to adjacent non-transformed cells, resulting in false-positive transformants (Prakash et al 2008). High expression of transgenes might be also harmful for the growth of embryogenic calli and plant regeneration, probably because of increased use of energy and resources for the expression of transgenes and its proteins (Potenza et al 2004;Zhou et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation

et al. 2014

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A plant-derived 0.3 kb constitutive promoter was obtained from AtTCTP expression analysis, and successfully applied to the expression of a selectable marker gene for production of transgenic creeping bentgrass plants. The isolation and use of an efficient promoter is essential to develop a vector system for efficient genetic transformation of plants, and constitutive promoters are particularly useful for the expression of selectable marker genes. In this study, we characterized a small size of the constitutive promoter from the expression analysis of Arabidopsis thaliana translationally controlled tumor protein (AtTCTP) gene. Histochemical and fluorometric GUS analyses revealed that a 303 bp upstream region from the start codon of the AtTCTP gene showed strong GUS expression throughout all plant tissues, which is approximately 55 % GUS activity compared with the cauliflower mosaic virus 35S promoter (35Spro). To examine the possible application of this promoter for the development of genetically engineered crops, we introduced pCAMBIA3301 vector harboring the 0.3 kb promoter of AtTCTP (0.3kbpro) that was fused to the herbicide resistance BAR gene (0.3kb pro ::BAR) into creeping bentgrass. Our transformation results demonstrate that transgenic creeping bentgrass plants with herbicide resistance were successfully produced using 0.3kb pro ::BAR as a selectable marker. Northern blot analysis revealed that the transgenic plants with 0.3kb pro ::BAR showed reduced but comparable expression levels of BAR to those with 35S pro ::BAR. Moreover, the transcription activity of the 0.3 kb promoter could be increased by the fusion of an enhancer sequence. These results indicate that the 0.3 kb AtTCTP promoter can be used as a plant-derived constitutive promoter for the expression of selectable marker genes, which facilitates its use as an alternative to the 35S promoter for developing genetically engineered crops.

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Section: Discussionmentioning

confidence: 99%

Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation

et al. 2014

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“…Particle preparation 'Cassette DNA fragment' at a rate of 1.0 or 10 or 100 ng or 'entire plasmid' at a rate of 100 ng was precipitated respectively on 1.8 mg gold particles (0.6 l diameter; BioRad, Hercules, CA) as described previously by Shiva Prakash et al 2008. Each of these DNA coated gold preparations was used for four shots (bombardments), giving an effective delivery of 0.25 or 2.5 or 25 ng of cassette DNA or 25 ng of entire plasmid DNA coated on 0.45 mg gold particles per shot.…”

Section: Constructs Used In Transformationmentioning

confidence: 99%

“…Particle delivery using the helium PDS-1000 device Bombardment was performed using the PDS-1000/He Particle Delivery System TM (Bio-Rad) as described previously (Shiva Prakash et al 2008).…”

Section: Constructs Used In Transformationmentioning

confidence: 99%

“…After the delivery of DNA coated gold particles either through the Helium or Electric discharge gun, callus initiation, selection and multiplication and regeneration of stable corn transformations were carried out as described previously (Shiva Prakash et al 2008). Although multiple plants were regenerated from callus derived from a single embryo in many instances, only one plant per embryo chosen randomly was transferred to soil and considered as one independent event for molecular analysis.…”

Section: Corn Transformationmentioning

confidence: 99%

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Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA

et al. 2009

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Transgene copy number is an important criterion for determining the utility of transgenic events. Single copy integration events are highly desirable when the objective is to produce marker free plants through segregation or when it is necessary to introgress different transgenes into commercial cultivars from different transgenic events. In contrast multi-copy events are advocated by several authors for higher expression of the transgene. Till recently, it was thought that employment of the particle gun for transformation results in the production of a high proportion of multi-copy events often with complex integration pattern when compared to Agrobacterium-mediated transformation. However, it has been demonstrated that usage of cassette DNA for bombardment in place of whole plasmids would result in simple insertion pattern of the transgenes. While investigating the effect of varying the cassette DNA amount on stable transformation, the frequency of occurrence of low copy events was observed to increase when lower doses of cassette DNA was employed for bombardment. Large scale experimentation with rigorous statistical analysis performed to verify the above observations employing Helium gun and the Electric discharge gun for gene delivery confirmed the above observations. Helium gun experiments involving production of more than 1,600 corn events consistently yielded single copy events at higher frequencies at lower cassette DNA load (46% at 2.5 ng/shot) as compared to higher cassette DNA load (29% at 25 ng/shot) across 18 independent experiments. Results were nearly identical with the Electric discharge particle gun device where single copy events were recovered at frequencies of 54% at 2.5 ng cassettes DNA per shot as compared to 18% at 25 ng cassette DNA per shot. The transformation frequency declined from 41 to 34% (Helium gun) and from 48 to 31% (Electric discharge gun) with reduction in cassette DNA quantity from 25 to 2.5 ng per shot. This reduction in the transformation frequency is more than compensated by the savings in time and effort involved in the production and screening of events if the desired outcome is single copy events. These results demonstrate the flexibility of the particle gun method for controlling the frequency of production of either low copy or high copy events by altering the quantity of cassette DNA used for bombardment. The transgene expression levels over generations in relation to its integration need further investigations.

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“…It has also been observed that other promoters are stronger than the 35S promoter when used for in vitro transformation of different monocot and dicot plants. Indeed, the Cauliflower Mosaic Virus (CaMV) 35S promoter was less effective in driving the GUS gene expression in tomato (dicot) and maize (monocot) plant tissues as compared to the maize promoters Gos-2, Enolase, Actin-2 (Shireen et al, 2002) and Actin-1 (Prakash et al, 2008). By using immature and mature embryos, shoot tips and embryogenic calli for in vitro transformation of S. bicolor, Tadesse et al (2003) observed that the strength of the CaMV 35S promoter ranked after those of the ubi1, act1D, adh1 promoters.…”

Section: Discussionmentioning

confidence: 99%

In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

Tiecoura¹,

Kouassi²,

N’Nan-Alla³

et al. 2015

Afr. J. Biotechnol.

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Effect of promoter driving selectable marker on corn transformation

Cited by 26 publications

References 40 publications

Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation

Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation

Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA

In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

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