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A plant transformation vector, pCLKSCLA25 (EU327498), was developed to contain eight cloning sites and the inducible self-excision
system which provided an effective approach to eliminate the selectable marker gene(s) from transgenic plants. Upon induction
by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding the selectable marker neomycin phosphotransferase and cre itself. The excision efficiency was 41% in transgenic tomato regenarants. The stilbene synthase gene (vst1) from Vitis vinifera L. was cloned into pCLKSCLA25. The expression of vst1 gene contributed to the accumulation of trans-reveratrol from 3.4 to 8.7 μg/g fresh wt in different marker-free transgenic tomato lines.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Ya. B. Neskorodov A. L. Rakitin A. M. Kamionskaya K. G. Skryabin 《Plant Cell, Tissue and Organ Culture》2010,100(1):65-71
Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses
the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting
were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants.
Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta? at 3 l/ha. 相似文献
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Young Im Choi Eun Woon Noh Hyo Shin Lee Mu Seok Han Jae Soon Lee Kwan Sam Choi 《Journal of Plant Biology》2005,48(4):351-355
A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments.
So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance.
However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic
plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation.
The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium
containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance
to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve
as an excellent selectable marker for plant transformation. 相似文献
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Alternative selectable markers for potato transformation using minimal T-DNA vectors 总被引:4,自引:0,他引:4
Barrell P.J. Yongjin Shang Cooper P.A. Conner A.J. 《Plant Cell, Tissue and Organ Culture》2002,70(1):61-68
Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance. 相似文献
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Aisling Dunne Jodi Maple‐Grødem Daniela Gargano Richard P. Haslam Johnathan A. Napier Nam‐Hai Chua Rosalind Russell Simon Geir Møller 《The Plant journal : for cell and molecular biology》2014,80(6):1131-1138
The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin‐based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3‐ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over‐expression of isopentenyl transferase. Over‐expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic‐based selection systems, and may be used in combination with a negative selectable marker as part of a two‐step selection system to obtain homoplasmic plant lines. 相似文献
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Lu Lu Xingrong Wu Xiaoyan Yin Jonathan Morrand Xinlu Chen William R. Folk Zhanyuan J. Zhang 《Plant Cell, Tissue and Organ Culture》2009,99(1):97-108
We report an Agrobacterium-mediated transformation system that can generate marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] from a public line [P898012] using standard binary vectors with bar as a selectable marker. Eight co-cultivation conditions were examined for their effect on transformation. The average transformation
frequencies were 0.4 and 0.7% for pZY101-TC2 and pZY101-SKRS, respectively, derived from binary vector pZY102 and containing
bar and target gene(s) in separate T-DNA regions. A low selection pressure (2.5 mg l−1 DL-phosphinothrithin, PPT) was deployed during callus induction in combination with rapid selection to generate plants from
80 independent events, all but three of which were fertile and set seed. PCR and Southern analyses showed that 36 out of 80
events contained both bar and the target gene(s) (an average co-transformation frequency of 45%). Seedlings of the T1 generation transmitted T-DNAs
with target gene(s) and bar gene independently, generating a fraction of progeny with only the target gene(s). 相似文献
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We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large
number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by
nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both
histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency
in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks
associated with the use of these traditional selection marker genes can be eliminated. 相似文献
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Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker 总被引:6,自引:0,他引:6
Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series
of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used
to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of
this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas.
Received: 26 October 1999 / Accepted: 28 December 1999 相似文献
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Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata 总被引:3,自引:0,他引:3
Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin
acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved
with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing
GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots
but at a lower frequency (1–2%).
Received: 9 April 1997 / Revision received: 3 July 1997 / Accepted: 30 July 1997 相似文献
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The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their
public acceptance and future commercialization. In addition, their elimination may allow gene stacking by the same selection
strategy. In apricot, selection using the selectable marker gene nptII, that confers resistance to aminoglycoside antibiotics, is relatively effective. An attractive alternative is offered by
the MAT system (multi-auto-transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with an MAT vector has been attempted
in the apricot cultivar ‘Helena’. Regeneration from infected leaves with Agrobacterium harboring a plasmid containing the ipt gene was significantly higher than that from non-transformed controls in a non-selective medium. In addition, transformation
efficiencies were much higher than those previously reported using antibiotic selection, probably due to the integration of
the regeneration-promoting ipt gene. However, the lack of an ipt expression-induced differential phenotype in apricot made difficult in detecting the marker genes excision and plants had
to be evaluated at different times. PCR analysis showed that cassette excision start occurring after 6 months approximately
and 1 year in culture was necessary for complete elimination of the cassette in all the transgenic lines. Excision was confirmed
by Southern blot analysis. We report here for the first time in a temperate fruit tree that the MAT vector system improves
regeneration and transformation efficiency and would allow complete elimination of marker genes from transgenic apricot plants
by site-specific recombination. 相似文献
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Suchandra Deb Roy Mukesh Saxena Prasanna S. Bhomkar Mikhail Pooggin Thomas Hohn Neera Bhalla-Sarin 《Plant Cell, Tissue and Organ Culture》2008,95(1):1-11
Despite the advances in transgenesis, transformation technologies still rely on the introduction of a selectable marker gene
to identify cells and tissues that have integrated the gene of interest in their genome. The continuous presence of the marker
genes in the transgenics is often controversial as it can potentially have multiple undesirable impacts. The present study
employed the self-excising Cre-loxP system to generate marker-free Arabidopsis thaliana expressing the agronomically important glyoxalase I (glyI) gene from Brassica juncea to confer salt stress tolerance. A binary vector was constructed wherein the salt-inducible rd29A promoter was used to drive
the expression of the glyI gene and the transformants of A. thaliana were recovered using kanamycin resistance as the selectable marker. The neomycin phosphotransferase II (nptII) gene was flanked by the loxP sites followed by the introduction of a heat-inducible Cre-recombinase in between the loxP sites. The kanamycin-resistant transgenic lines of A. thaliana using this vector showed an ability to withstand stress imposed by 150 mM NaCl. The exposure of the T2 transgenic lines to
a mild heat shock (37°C) resulted in the recovery of salt-tolerant, kanamycin-sensitive T3 progeny. Molecular analyses of
the T3 transgenic lines following the heat shock treatment confirmed the excision of the nptII gene and the completion of their life cycle in the presence of 150 mM NaCl-induced stress. 相似文献
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Danfeng Long Xueli Wu Zhimin Yang Ingo Lenk Klaus Kristian Nielsen Caixia Gao 《In vitro cellular & developmental biology. Plant》2011,47(6):658-666
A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used
systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production
of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing
either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype.
The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin
and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively.
Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration
pattern with multiple insertions. 相似文献
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Haematococcus pluvialis is a unicellular green alga which produces a ketocarotenoid, astaxanthin which has pharmaceutical and nutraceutical applications
owing to its high antioxidant activity. Biotechnological approaches such as genetic transformation methods (Agrobacterium-mediated) and cloning strategies, are essential to improve/regulate this ketocarotenoid in Haematococcus. For this studies are necessary to improve Haematococcus through biotechnological means. In this connection, a suitable cocultivation medium for Haematococcus and Agrobacterium tumefaciens was standardized and different antibiotics were screened. Among the cocultivation media viz Z8, Bold’s Basal Medium, Tris Acetate Phosphate Z8 with 0.5% mannitol and BBM and Z8 with half strength LB TAP medium was found suitable for growth of both the alga Haematococcus and Agrobacterium. For the different antibiotics screened to identify the sensitivity of algae, hygromycin at more than 2 mg L−1 showed lethal effect which can be used as a selectable marker gene in genetic transformation, whereas cefotaxime and augmentin
showed no effect up to 2,000 mg L−1. The growth of algae was higher in solid selection medium when compared to the liquid selection medium. 相似文献
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Transgenic oat plants via visual selection of cells expressing green fluorescent protein 总被引:11,自引:0,他引:11
H. F. Kaeppler G. K. Menon R. W. Skadsen A. M. Nuutila A. R. Carlson 《Plant cell reports》2000,19(7):661-666
New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation.
The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic
cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed
in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic
lines.
Received: 11 May 1999 / Revision received: 31 August 1999 / Accepted: 2 September 1999 相似文献
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Ping Che Shujun Chang Marissa K. Simon Zhifen Zhang Ahmed Shaharyar Jesse Ourada Dennis O’Neill Mijael Torres-Mendoza Yinping Guo Kathleen M. Marasigan Jean-Philippe Vielle-Calzada Peggy Ozias-Akins Marc C. Albertsen Todd J. Jones 《The Plant journal : for cell and molecular biology》2021,106(3):817-830
Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies. 相似文献
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Lamblin F Aimé A Hano C Roussy I Domon JM Van Droogenbroeck B Lainé E 《Plant cell reports》2007,26(6):765-772
In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants,
we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into
fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective
medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root
on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L−1 sucrose and 10 g L−1 mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in
leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached
6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the
Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully
used for the recovery of flax transgenic plants under safe conditions for human health and the environment. 相似文献