Recent advances in
            <i>Medicago</i>
            <scp>spp</scp>
            . genetic engineering strategies

Confalonieri, Massimo; Sparvoli, Francesca

doi:10.1002/9781119409144.ch149

Cited by 2 publications

(3 citation statements)

References 128 publications

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“…1). The co-transformation technique involves the insertion of two T-DNAs into different genomic loci for segregation (Confalonieri & Sparvoli, 2019;Kausch et al, 2019;RamanaRao & Veluthambi, 2010). This technique offers unique advantages for the production of transgenic plants, i.e., it allows the simultaneous insertion of multiple genes of interest into a plant genome without many selectable marker genes, regardless of gene sequence (Miki & McHugh, 2004).…”

Section: Technique For the Co-transformation And Segregation Of Marker Genesmentioning

confidence: 99%

“…This protocol has been successfully applied to the production of Bt cotton using the pollen tube pathway technique or the genetically transformed potato AV43-6-G7 through agrobacteria-mediated transformation. However, the main disadvantages of non-marker transformation techniques are the poor recovery rate of transformants, high labor, time and cost requirements, and the use of a large number of regenerated plants for PCR analysis (Breyer, Kopertekh & Reheul, 2014;Confalonieri & Sparvoli, 2019). In addition, this technique requires high transformation and regeneration efficiencies that are typically genotype-based, limiting its application to very limited organisms.…”

Section: Technique For the Co-transformation And Segregation Of Marker Genesmentioning

confidence: 99%

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

Dormatey

Sun

Ali

et al. 2021

PeerJ

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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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Section: Technique For the Co-transformation And Segregation Of Marker Genesmentioning

confidence: 99%

Section: Technique For the Co-transformation And Segregation Of Marker Genesmentioning

confidence: 99%

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

Dormatey

Sun

Ali

et al. 2021

PeerJ

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“…Innovative genetic engineering methods have been recently developed to modify specific sites in targeted genes and generate knock-out mutants (Sedeek et al, 2019 ; Confalonieri and Sparvoli, 2020 ). Of these, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system due to its high efficiency, simplicity and specificity has become a powerful and effective tool for targeted genome editing and research into gene function in plants (Doudna and Charpentier, 2014 ; Manghwar et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-Mediated Targeted Mutagenesis of CYP93E2 Modulates the Triterpene Saponin Biosynthesis in Medicago truncatula

et al. 2021

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In the Medicago genus, triterpene saponins are a group of bioactive compounds extensively studied for their different biological and pharmaceutical properties. In this work, the CRISPR/Cas9-based approach with two single-site guide RNAs was used in Medicago truncatula (barrel medic) to knock-out the CYP93E2 and CYP72A61 genes, which are responsible for the biosynthesis of soyasapogenol B, the most abundant soyasapogenol in Medicago spp. No transgenic plants carrying mutations in the target CYP72A61 gene were recovered while fifty-two putative CYP93E2 mutant plant lines were obtained following Agrobacterium tumefaciens-mediated transformation. Among these, the fifty-one sequenced plant lines give an editing efficiency of 84%. Sequencing revealed that these lines had various mutation patterns at the target sites. Four T0 mutant plant lines were further selected and examined for their sapogenin content and plant growth performance under greenhouse conditions. The results showed that all tested CYP93E2 knock-out mutants did not produce soyasapogenols in the leaves, stems and roots, and diverted the metabolic flux toward the production of valuable hemolytic sapogenins. No adverse influence was observed on the plant morphological features of CYP93E2 mutants under greenhouse conditions. In addition, differential expression of saponin pathway genes was observed in CYP93E2 mutants in comparison to the control. Our results provide new and interesting insights into the application of CRISPR/Cas9 for metabolic engineering of high-value compounds of plant origin and will be useful to investigate the physiological functions of saponins in planta.

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Recent advances in Medicago spp . genetic engineering strategies

Cited by 2 publications

References 128 publications

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

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

CRISPR/Cas9-Mediated Targeted Mutagenesis of CYP93E2 Modulates the Triterpene Saponin Biosynthesis in Medicago truncatula

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