Selection of Phalaenopsis amabilis L. Blume Orchid Resistance to Hygromycin

Examination of Phalaenopsis amabilis orchid resistance to hygromycin antibiotic is an important step to doprior to Agrobacterium-mediated genetic transformation in this orchids using Hygromycin phosphotransferase(HPT) gene as a selection marker in the T-DNA that harboring a desired gene to be transfered. We exposedthe plant on hygromycin containing medium. The experiment was conducted using 6 weeks old P. amabilisprotocorms. These protocorms were subcultured onto NP medium supplemented with various concentrationof Hygromycin (0, 5, 10, 20, 1nd 40 mg/l). The number of survival protocorms were examined every week for4 weeks after subcultured (WAS). The resistancy of hygromycin was calculated as ratio of death protocormsper total protocorms). The result showed that 10 mg/l hygromycin with 1 weeks of application caused deathclose to LD 50. This data indicate that P. amabilis resistance to hygromycin treatment on the appropriateconcentration 10 mg/l, and this concentration can be used for other purposes in orchid system.

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The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Metabolites ◽

10.3390/metabo9030049 ◽

2019 ◽

Vol 9 (3) ◽

pp. 49 ◽

Cited By ~ 1

Author(s):

Ana Molina-Márquez ◽

Marta Vila ◽

Javier Vigara ◽

Ana Borrero ◽

Rosa León

Keyword(s):

Genetic Transformation ◽

Selectable Marker ◽

Genetic Manipulation ◽

Plant Cells ◽

Phytoene Desaturase ◽

Carotenoid Content ◽

Great Promise ◽

Encoding Gene ◽

Average Transformation ◽

Selection Of

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

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Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium

Biology Methods and Protocols ◽

10.1093/biomethods/bpz001 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 1

Author(s):

Nancy N Nichols ◽

Ronald E Hector ◽

Sarah E Frazer

Keyword(s):

Green Fluorescent Protein ◽

Genetic Transformation ◽

Pure Culture ◽

Ecological Niche ◽

Fluorescent Protein ◽

Microbial Consortium ◽

Transformed Cells ◽

Antibiotic Selection ◽

Green Fluorescent ◽

Selection Of

Abstract Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

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Dual selection of a genetic switch by a single selection marker

Biosystems ◽

10.1016/j.biosystems.2006.07.006 ◽

2007 ◽

Vol 90 (1) ◽

pp. 115-120 ◽

Cited By ~ 31

Author(s):

Yoko Nomura ◽

Yohei Yokobayashi

Keyword(s):

Selection Marker ◽

Genetic Switch ◽

Selection Of

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Germ line genetic transformation in cotton (Gossypium hirsutum L.) by selection of transgenic meristematic cells with a herbicide molecule

Plant Science ◽

10.1016/j.plantsci.2004.12.024 ◽

2005 ◽

Vol 168 (5) ◽

pp. 1227-1233 ◽

Cited By ~ 28

Author(s):

Francisco J.L. Aragão ◽

Giovanni R. Vianna ◽

Silvia B.R.C. Carvalheira ◽

Elibio L. Rech

Keyword(s):

Genetic Transformation ◽

Gossypium Hirsutum ◽

Germ Line ◽

Gossypium Hirsutum L ◽

Meristematic Cells ◽

Selection Of

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ptxD/Phi as alternative selectable marker system for genetic transformation for bio-safety concerns: a review

PeerJ ◽

10.7717/peerj.11809 ◽

2021 ◽

Vol 9 ◽

pp. e11809

Author(s):

Richard Dormatey ◽

Chao Sun ◽

Kazim Ali ◽

Sajid Fiaz ◽

Derong Xu ◽

...

Keyword(s):

Transgenic Plants ◽

Genetic Transformation ◽

Herbicide Resistance ◽

Selectable Marker ◽

Selection Marker ◽

Marker Genes ◽

Selection System ◽

Efficient System ◽

Marker System ◽

Selectable Marker Genes

Antibiotic and herbicide resistance genes are the most common marker genes for plant transformation to improve crop yield and food quality. However, there is public concern about the use of resistance marker genes in food crops due to the risk of potential gene flow from transgenic plants to compatible weedy relatives, leading to the possible development of “superweeds” and antibiotic resistance. Several selectable marker genes such as aph, nptII, aaC3, aadA, pat, bar, epsp and gat, which have been synthesized to generate transgenic plants by genetic transformation, have shown some limitations. These marker genes, which confer antibiotic or herbicide resistance and are introduced into crops along with economically valuable genes, have three main problems: selective agents have negative effects on plant cell proliferation and differentiation, uncertainty about the environmental effects of many selectable marker genes, and difficulty in performing recurrent transformations with the same selectable marker to pyramid desired genes. Recently, a simple, novel, and affordable method was presented for plant cells to convert non-metabolizable phosphite (Phi) to an important phosphate (Pi) for developing cells by gene expression encoding a phosphite oxidoreductase (PTXD) enzyme. The ptxD gene, in combination with a selection medium containing Phi as the sole phosphorus (P) source, can serve as an effective and efficient system for selecting transformed cells. The selection system adds nutrients to transgenic plants without potential risks to the environment. The ptxD/Phi system has been shown to be a promising transgenic selection system with several advantages in cost and safety compared to other antibiotic-based selection systems. In this review, we have summarized the development of selection markers for genetic transformation and the potential use of the ptxD/Phi scheme as an alternative selection marker system to minimize the future use of antibiotic and herbicide marker genes.

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Genetic Transformation of Musa acuminata cv. Vaibalhla (AAA) using Agrobacterium tumefaciens

Science & Technology Journal ◽

10.22232/stj.2017.05.02.09 ◽

2017 ◽

Vol 5 (2) ◽

pp. 120-131

Author(s):

Lalremsiami Hrahsel ◽

◽

Adreeja Basu ◽

Lingaraj Sahoo ◽

Robert Thangjam ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Genetic Transformation ◽

Basal Medium ◽

Male Flower ◽

Musa Acuminata ◽

Vacuum Infiltration ◽

Bacterial Suspension ◽

Immature Male ◽

Selection Of ◽

Selection Of Transformants

Agrobacterium-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was performed using Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA2301AtNHX1 plasmid. For the transformation efficiency assay, the un-and pre-cultured immature male flower explants were subjected to various methods of injury such as hypodermal needle injury, with and without sonication and vacuum infiltration. The explants were then inoculated in bacterial suspension by occasional shaking for 30 minutes. After inoculation, explants were co-cultivated for 3 days in dark condition at 22° C in a liquid MS basal medium supplemented with 100 µM acetosyringone. For selection of transformants, the treated explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP (2 mg/L), NAA (0.5 mg/L) and ascorbic acid (75 mg/L) along with antibiotics kanamycin (100 mg/L), cefotaxime (300 mg/L) and augmentin (300 mg/L).The putative transformation was analyzed using histochemical GUS assay. 14 and 21 days pre-cultured male flower explants subjected to 4-5 needle point injury resulted in 93.33% and 100% putative transformation respectively. 30s sonication combined with 5 minutes of vacuum infiltration for 7, 14 and 21 days pre-cultured immature male flower explants gave 73.33%, 66.66% and 71.42% putative transformation respectively.

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Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker

Journal of Genetics and Genomics ◽

10.1016/j.jgg.2011.03.005 ◽

2011 ◽

Vol 38 (4) ◽

pp. 173-179 ◽

Cited By ~ 6

Author(s):

Young Geol Yoon ◽

Michael Duane Koob

Keyword(s):

Genetic Transformation ◽

Mammalian Cells ◽

Selection Marker ◽

Drug Resistant

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A novel homologous dominant selection marker for genetic transformation of Penicillium chrysogenum: Overexpression of squalene epoxidase-encoding ergA

Journal of Biotechnology ◽

10.1016/j.jbiotec.2010.09.941 ◽

2010 ◽

Vol 150 (3) ◽

pp. 307-311 ◽

Cited By ~ 11

Author(s):

Claudia Sigl ◽

Monika Handler ◽

Georg Sprenger ◽

Hubert Kürnsteiner ◽

Ivo Zadra

Keyword(s):

Genetic Transformation ◽

Penicillium Chrysogenum ◽

Selection Marker ◽

Squalene Epoxidase

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Specific gene targeting in Spiroplasma citri: improved vectors and production of unmarked mutations using site-specific recombination

Microbiology ◽

10.1099/mic.0.28123-0 ◽

2005 ◽

Vol 151 (8) ◽

pp. 2793-2803 ◽

Cited By ~ 23

Author(s):

Sybille Duret ◽

Aurélie André ◽

Joël Renaudin

Keyword(s):

Gene Targeting ◽

Arginine Deiminase ◽

Selection Marker ◽

Specific Gene ◽

Phosphotransferase System ◽

Gene Encoding ◽

Spiroplasma Citri ◽

Recombination System ◽

Targeting Vector ◽

Selection Of

In Spiroplasma citri, where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, oriC plasmids. To improve the probability of selecting rare recombination events without fastidious, extensive passaging of the transformants, a new targeting vector was constructed, which was used to inactivate the crr gene encoding the IIA component of the glucose phosphotransferase system (PTS) permease. Selection of recombinants was based on a two-step strategy using two distinct selection markers, one of which could only be expressed once recombination had occurred through one single crossover at the target gene. According to this strategy, spiroplasmal transformants were screened and multiplied in the presence of gentamicin before the crr recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the crr-disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in S. citri the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon γδ TnpR/res recombination system to produce unmarked mutations in S. citri was demonstrated. In an arginine deiminase (arcA-disrupted) mutant, the tetM gene flanked by the res sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative oriC plasmid. Due to oriC incompatibility, plasmid loss occurred spontaneously when selection pressure was removed. This approach will be helpful for constructing unmarked mutations and generating multiple mutants with the same selection marker in S. citri. It should also be relevant to other species of mollicutes.

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