Screening for recombinants of Crambe abyssinica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration

Qi, Weicong; Tinnenbroek-Capel, Iris E. M.; Salentijn, E.M.J.; Schaart, Jan G.; Cheng, Jierong; Denneboom, Christel; Zhang, Zhao; Zhang, Xiaolin; Zhao, Han; Visser, R.G.F.; Huang, Bangquan; Loo, E.N. van; Krens, Frans A.

doi:10.1038/srep14033

Cited by 3 publications

(1 citation statement)

References 36 publications

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“…There is much controversy over genetically modified (GM) crop: the uncertainty of their safety is the main concern of governments, non-governmental organizations, and the general public [4]. A common concern is the marker genes used for selecting transformed plants, asserting that the food produced from GM plants could be harmful to human health due to potential toxicity or allergenicity [5].…”

Section: Introductionmentioning

confidence: 99%

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Jin

Guo

et al. 2019

Genes

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Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

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Section: Introductionmentioning

confidence: 99%

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Jin

Guo

et al. 2019

Genes

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Cisgenesis and Intragenesis as New Strategies for Crop Improvement

Moradpour

Abdullah

2017

Crop Improvement

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Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species.

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Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

Feng,

Liu,

et al. 2023

Phytopathol Res

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Oomycetes, such as Pythium species, contain numerous devastating plant pathogens that inflict substantial economic losses worldwide. Although CRISPR/Cas9-based genome editing is available, the selection markers available for genetic transformation in these species are limited. In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1), known to confer oxathiapiprolin resistance, was introduced into the CRISPR/Cas9 system for in situ complementation in Pythium ultimum. We targeted PuLLP, which encodes a loricrin-like protein, and showed significant downregulation when the Puf RNA-binding protein-encoding gene PuM90 was knocked out. The PuLLP knockout mutants could not produce oospores, indicating a similar biological function as PuM90. The reintroduction of PuLLP into the knockout mutant using PcMuORP1 as a selection marker restored oospore production. Further comparisons with the conventional selection marker NPTII indicated that PcMuORP1 could be applied at a lower concentration and cost, resulting in a higher screening efficiency. Successive subculturing in the absence of selective pressure showed that PcMuORP1 had little long-term effect on the fitness of transformants. Hence, it could be reused as an alternative selection marker. This study demonstrates the successful implementation of the PcMuORP1 gene as a selection marker in the genetic transformation of Py. ultimum and reveals the loricrin-like protein PuLLP as a sexual reproduction-related factor downstream of the Puf RNA-binding protein PuM90. Overall, these results will help accelerate the functional genomic investigation of oomycetes.

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Screening for recombinants of Crambe abyssinica after transformation by the pMF1 marker-free vector based on chemical selection and meristematic regeneration

Cited by 3 publications

References 36 publications

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Cisgenesis and Intragenesis as New Strategies for Crop Improvement

Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

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