Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant

Merritt, Benjamin A; Zhang, Xudong; Triplett, Eric W.; Mou, Zhonglin; Orbović﻿﻿﻿, Vladimir

doi:10.1007/s00299-021-02760-y

Cited by 4 publications

(2 citation statements)

References 26 publications

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“…After PCR-based screening of regenerated shoots, efficiencies achieved were 20.41, 19.37, and 3.21% respectively. Recently, the study of Merritt et al (2021) reported glyphosate-resistant Duncan grapefruit plants, obtained by inducing native EPSPS mutations.…”

Section: Selection Markers New Reporter Genes and Marker-free Systems For Citrus Transformationmentioning

confidence: 99%

“…In citrus, the use of reporter genes based on the production of anthocyanin ( Dutt et al, 2016 , 2018b ; Huang et al, 2019 ) and systems to remove selectable markers have shown positive results ( Sugita et al, 1999 ; Zou et al, 2013 ). Merritt et al (2021) developed a citrus DNA glyphosate resistant-selection system by transforming Duncan grapefruit with a glyphosate-resistant mutated version of citrus EPSPS enzyme (TIPS EPSPS). They showed that a glyphosate treatment did not inhibit bud formation and rendered a 40% increment in transformation efficiency.…”

Section: Emerging Technologies: Cis and Intragenesis Trans -Grafting And Gene Editing In Citrusmentioning

confidence: 99%

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Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

et al. 2021

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Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

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Section: Selection Markers New Reporter Genes and Marker-free Systems For Citrus Transformationmentioning

confidence: 99%

Section: Emerging Technologies: Cis and Intragenesis Trans -Grafting And Gene Editing In Citrusmentioning

confidence: 99%

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

et al. 2021

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Identification and Characterization of Two Putative Citrus Phosphomannose Isomerase (CsPMI) Genes as Selectable Markers for Mature Citrus Transformation

Canton

Mahmoud

et al. 2022

Horticulturae

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Two Citrus sinensis (L.) phosphomannose isomerase (PMI) genes, CsPMI1 and CsPMI2, were evaluated as novel selectable markers in mature citrus transformation. Transgenic shoots produced after transformation of Kuharske rootstock with each PMI construct were selected on six treatments of mannose and sucrose. For CsPMI1, there were no significant differences among the various mannose and sucrose treatments for the mean number of positive shoots (PS), the mean transformation efficiency based on the number of shoots (TES), or the mean transformation efficiency based on the number of explants (TEE). However, for the CsPMI2 gene, the number of transgenics produced in two treatments (7.5 g L−1 mannose + 22.5 g L−1 sucrose and 15 g L−1 mannose + 15 g L−1 sucrose) was significantly greater than the sucrose control for TES at 4.2% and 3.7%, respectively. Moreover, TEE at 4.2% in the 15 g L−1 mannose + 15 g L−1 sucrose treatment, supported the TES value. Most of the transgenic lines demonstrated higher in vivo and in vitro enzyme assays compared with the wild-type control. CsPMI2 provided acceptable selection in mature citrus, and it will be applied in future intragenic research.

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Overview on Current Selectable Marker Systems and Novel Marker Free Approaches in Fruit Tree Genetic Engineering

Súnico,

Piunti,

Bhattacharjee

et al. 2024

IJMS

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Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation processes. They are especially important for fruit trees genetic transformation to individuate putatively genetically modified events, because most of the protocols used to genetic engineer these species are often unsuccessful or with low efficiency. Traditional selectable marker genes, mainly of bacterial origin, confer antibiotics/herbicides-resistance or metabolic advantages to transformed cells. Genes that allow the visual recognition of engineered tissues without using any selective agent, such as morphogenic regulators and reporter genes, are also used as selection tools to in vitro identify genetically modified regenerated lines. As final step, genetic engineered plants should be tested in field conditions, where selectable marker genes are no longer necessary, and strongly unpopular especially for the commercial development of the new products. Thus, different approaches, mainly based on the use of site-specific recombinases and/or editing nucleases, are being now used to recover marker-free fruit crops. This review describes and comments the most used and suitable selection tools of interest, particularly for fruit tree genetic engineering. Lastly, a spotlight highlights the biosafety aspects related to the use of selectable marker genes exploited for fruit species genetic engineering.

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Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant

Cited by 4 publications

References 26 publications

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies

Identification and Characterization of Two Putative Citrus Phosphomannose Isomerase (CsPMI) Genes as Selectable Markers for Mature Citrus Transformation

Overview on Current Selectable Marker Systems and Novel Marker Free Approaches in Fruit Tree Genetic Engineering

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