Efficient recovery of transgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker gene expression

Tian, Lining; Wang, H.; Wu, Keqiang; Latoszek‐Green, Marysia; Hu, Ming; Miki, Brian; Brown, Daniel C.

doi:10.1007/s00299-002-0459-7

Cited by 22 publications

(15 citation statements)

References 16 publications

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“…It was reported that the tCUP sequence could direct transgene expression in both dicots and monocots, but the expression level of the reporter gene seemed lower in monocot cell suspensions than in dicot leaf tissue as measured by the numbers of GUS-positive spots [22]. Although enhanced tCUP derivatives had been used to drive the neomycin phosphotransferase II ( NPTII ) gene and selection for kanamycin resistant transformants in tobacco, cauliflower, alfalfa and Arabidopsis [26,27], the performance of these elements in rice was not known. We therefore examined its expression style and strength in rice and using it for selection of transformants.…”

Section: Discussionmentioning

confidence: 99%

“…The plant cryptic promoter tCUP conferred strong constitutive expression when fused to a reporter gene in both tobacco and Arabidopsis [22], and was also highly active in a wide range of dicot plants including alfalfa, canola, cauliflower, tomato and pea as well as in conifers [23-25]. The strong constitutive feasibilities of the tCUP promoter made it possible to drive the expression of a selectable marker gene for recovery of transformed plants through both organogenesis and embryogenesis [26], and proved to be a useful alternative to CaMV 35S as it does not interact with tested promoters [27]. Although the expression of tCUP is reported to be minimal in monocot plants such as wheat, barley, maize and oat [22,23,28], we considered that it would be interesting to study the possible expression and application of tCUP in rice.…”

Section: Introductionmentioning

confidence: 99%

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Enhanced transgene expression in rice following selection controlled by weak promoters

et al. 2013

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BackgroundTechniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent.ResultsIn this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos).ConclusionsThe use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhanced transgene expression in rice following selection controlled by weak promoters

et al. 2013

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“…The tobacco cryptic promoter (tCUP) (Foster et al ., 1999) contains cryptic regulatory elements equivalent to those responsible for the expression of plant genes (Wu et al ., 2003a). Subsequent research has shown that the enhanced tCUP gene expression system generates levels of β‐glucuronidase (GUS) activity exceeding that of the CaMV 35S promoter (Malik et al ., 2002), and provides equivalent levels of selection in kanamycin (Tian et al ., 2002). Replacement of CaMV 35S promoter by either nos or mas promoters can, in some cases, reduce the misexpression of the transgene (Jagannath et al ., 2001; Yoo et al ., 2005); however, the influence of chimeric and plant‐derived promoters on the tissue specificity of transgene expression has not been investigated rigorously.…”

Section: Introductionmentioning

confidence: 99%

Strategies to mitigate transgene–promoter interactions

Gudynaite‐Savitch

Johnson

Miki

2009

Plant Biotechnology Journal

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SummaryThe expression pattern of tissue-specific promoters in transgenes can be influenced by promoter/enhancer elements employed for the expression of selectable marker genes or elements found in DNA flanking the insertion site. We have developed an analytical system in Arabidopsis thaliana to investigate strategies useful in blocking or reducing nonspecific interactions. These experiments confirm that the DNA configuration and the insertion of spacer DNA aid in the appropriate expression of tissue-specific promoters. It is also demonstrated that the novel tobacco cryptic promoter (tCUP), when used to replace the cauliflower mosaic virus (CaMV) 35S promoter/enhancer, does not show nonspecific interactions. Furthermore, it is shown that insulators isolated from yeast and animals may have potential application in plants. Our results may allow the design of strategies that, individually or in combination, can be used to minimize nonspecific interactions and to design vectors for individual tissue-specific promoters.

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“…Transgenic alfalfa plants were obtained via Agrobacterium -mediated transformation using the method well established in our laboratory [37]. Plant transformation was confirmed by PCR using uid gene primers, histochemical analysis and Southern blot analysis (described above).…”

Section: Resultsmentioning

confidence: 99%

“…The tCUP is plant derived promoter which was identified by promoter-trapping technology [29]. This promoter can drive high levels of gene expression in different plant species and in different organs [37], [41]. Regardless of the promoters used, all of the plants developed from desiccated somatic embryos showed GUS expression and the expression levels were comparable to that developed from embryos without desiccation (Figure 3A, B).…”

Section: Resultsmentioning

confidence: 99%

Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

et al. 2013

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Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species.

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Efficient recovery of transgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker gene expression

Cited by 22 publications

References 16 publications

Enhanced transgene expression in rice following selection controlled by weak promoters

Enhanced transgene expression in rice following selection controlled by weak promoters

Strategies to mitigate transgene–promoter interactions

Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

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