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McCormac, Alex C.; Wu, Huixia; Bao, Manzhu; Wang, Yibing; Xu, Rui‐ji; Elliott, Malcolm C.; Chen, Dongfang

doi:10.1023/a:1018303102488

Cited by 53 publications

(8 citation statements)

References 30 publications

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“…To address this problem, we analyzed transient GUS expression in immature embryos co-cultivated with Vec10. These experiments ( Figure 2C ) confirm earlier observations (McCormac et al, 1998; Murray et al, 2004) showing that a large number of different cells is infected and at least transiently transformed by Agrobacterium in this procedure. Although clearly not all cells showing transient GUS expression give rise to stable transformants later, this assay is a reasonable proxy for transformation, and the data obtained with it suggest that a single immature embryo produces multiple independent transformation events.…”

Section: Resultssupporting

confidence: 91%

“…However, many more shoots can be obtained from a single co-cultivated immature embryo. Transient expression experiments (McCormac et al, 1998; Murray et al, 2004) including our own ( Figure 3B ) suggested that these could be in the hundreds, but only few of them may be stable transformants. The number of independent transformants obtained from a single embryo is unknown, but our preliminary analysis suggested that 2.25 are generated with a single immature embryo on average.…”

Section: Discussionmentioning

confidence: 86%

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Gene Targeting Without DSB Induction Is Inefficient in Barley

2017

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Double strand-break (DSB) induction allowed efficient gene targeting in barley (Hordeum vulgare), but little is known about efficiencies in its absence. To obtain such data, an assay system based on the acetolactate synthase (ALS) gene was established, a target gene which had been used previously in rice and Arabidopsis thaliana. Expression of recombinases RAD51 and RAD54 had been shown to improve gene targeting in A. thaliana and positive-negative (P-N) selection allows the routine production of targeted mutants without DSB induction in rice. We implemented these approaches in barley and analysed gene targeting with the ALS gene in wild type and RAD51 and RAD54 transgenic lines. In addition, P-N selection was tested. In contrast to the high gene targeting efficiencies obtained in the absence of DSB induction in A. thaliana or rice, not one single gene targeting event was obtained in barley. These data suggest that gene targeting efficiencies are very low in barley and can substantially differ between different plants, even at the same target locus. They also suggest that the amount of labour and time would become unreasonably high to use these methods as a tool in routine applications. This is particularly true since DSB induction offers efficient alternatives. Barley, unlike rice and A. thaliana has a large, complex genome, suggesting that genome size or complexity could be the reason for the low efficiencies. We discuss to what extent transformation methods, genome size or genome complexity could contribute to the striking differences in the gene targeting efficiencies between barley, rice and A. thaliana.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 86%

Gene Targeting Without DSB Induction Is Inefficient in Barley

2017

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“…Floral dip experiments with infiltration media containing Agrobacterium AGL1 harboring pBECKSred (McCormac et al 1997) were performed on Crocus spikes at different stages of microspore development including the late uninucleate, the binucleate, and trinucleate stages (Table 1). No transformation events, as determined by red embryos, screens for paromomycin resistance and NPTII ELISAs, were isolated in 3,000 T1 seeds derived from dipping of binucleate stage spikes or in 4,200 T1 seeds derived from dipping of trinucealate stage spikes (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…For example, these alleles are associated with decreased expression of nucleases in the carpal at anthesis (Zale, unpublished) and might increase the probability of floral transformation. Moreover, a red seeded wheat line was chosen because expression of maize Lc/C1 in white wheat lines is known to be detrimental to plant growth (McCormac et al 1997). Zuzanna spring wheat is a Czech variety and was a gift from Dr. Ludmila Ohnoutkova, The University of Tennessee, Department of Plant Sciences.…”

Section: Methodsmentioning

confidence: 99%

“…pDs(Hyg)35S contains the NPTII gene driven by the nos promoter; a maize Ds element and a hygromycin gene interrupting a spectinomycin excision marker (Fig. 1a) pBECKSred is a binary vector carrying the NPTII gene driven by nos promoter and the maize transcription factors Lc and C1 (McCormac et al 1997). Lc is driven by the 35S promoter; C1 is driven by the 35S promoter: Adh 1 intron1 (Fig.…”

Section: Methodsmentioning

confidence: 99%

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Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens

et al. 2009

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Hexaploid wheat, one of the world’s most important staple crops, remains a challenge for genetic transformation. We are developing a floral transformation protocol for wheat that does not require tissue culture. This paper presents three transformants in the hard red germplasm line Crocus that have been characterized thoroughly at the molecular level over three to six generations. Wheat spikes at the early boot stage, i.e. the early, mid or late uninucleate microspore stages, were immersed in an infiltration medium of strain C58C1 harboring pDs(Hyg)35S, or strain AGL1 harboring pBECKSred. pDs(Hyg)35S contains the NPTII and hph selectable markers, and transformants were detected using paromomycin spray at the whole plant level, NPTII ELISAs, or selection on medium with hygromycin. Strain AGL1, harboring pBECKSred, which contains the maize anthocyanin regulators, Lc and C1, and the NPTII gene, was also used to produce a Crocus transformant. T1 and T2 seeds with red embryos were selected; T1 and T2 plants were screened by sequential tests for paromomycin resistance and NPTII ELISAs. The transformants were low copy number and showed Mendelian segregation in the T2. Stable transmission of the transgenes over several generations has been demonstrated using Southern analysis. Gene expression in advanced progeny was shown using Reverse Transcriptase-PCR and ELISA assays for NPTII protein expression. This protocol has the potential to reduce the time and expense required for wheat transformation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-009-0696-0) contains supplementary material, which is available to authorized users.

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Asiatic Beans

Sahoo

Jaiwal²

2008

Compendium of Transgenic Crop Plants

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Vigna species of the subgenus Ceratotropis constitute an economically important group of cultivated and wild species, of which a rich diversity occurs in Asian subcontinent. Progress in genetic improvement in Vigna species has been made mainly by conventional breeding methods. These methods, in addition of being time‐consuming and labor‐intensive, are met with a wide range of problems including traits associated with low genetic variation, low survivability of interspecific hybrids, specific inheritance of some valuable traits such as yield, disease, and pest resistance, as well as harvesting characteristics. Advances in genetic transformation technology offer enormous opportunities for crop improvement complementing traditional breeding programs. In this review, we highlight the progress in in vitro plant regeneration and genetic transformation of Vigna species. Applications of the developed gene transfer methods in specific crops vis‐a‐vis the limitations associated with each crop and gene transfer method, and the opportunities for future improvement are discussed.

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Cited by 53 publications

References 30 publications

Gene Targeting Without DSB Induction Is Inefficient in Barley

Gene Targeting Without DSB Induction Is Inefficient in Barley

Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens

Asiatic Beans

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