Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform

Kleidon, Jennifer; Brinin, Anthony; Paul, Jean Yves; Harding, Robert M.; Dale, James L.; Dugdale, Benjamin

doi:10.1007/s11248-019-00179-6

Cited by 14 publications

(6 citation statements)

References 33 publications

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“…Apparently, Agrobacterium strains have the distinct capacity to deliver T-DNA into different hosts, even plants of the same species. In developing marker-free banana, LBA4404mediated transformation resulted in an average of two copies of the transgene in two cultivars, while AGL1 generated very high copy numbers: an average of 5 and 13 in these varieties, respectively [22]. Whether the Agrobacterium strain LBA4404 or EHA105 is less virulent than GV3101 to the strawberry cv.…”

Section: Discussionmentioning

confidence: 99%

“…'Calypso' [20]. The same vector and marker-removal strategy was later applied to generate marker-free apple [21] and marker-free banana [22]. In a model tobacco plant, a double T-DNA vector was changed to a head-to-head pattern for the RB and LB directions, which significantly increased the frequency of marker-free plants in a cotransformation system [23].…”

Section: Introductionmentioning

confidence: 99%

“…Droplet digital PCR (ddPCR) was developed to detect DNA by partitioning individual amplifications into separate droplets and detecting the endpoint amplification products [41]. This method has been demonstrated to be more sensitive and have a lower error rate than qPCR and has been successfully used to determine the transgenic copy number in banana [22], tobacco [42], and maize [43]. A comparative study using ddPCR and Southern blotting in an array of genetically modified crops established single duplex ddPCR as an accurate method for measuring transgenic insertion events [44].…”

Section: Introductionmentioning

confidence: 99%

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A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Zhao²,

et al. 2021

Plants

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Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Zhao²,

et al. 2021

Plants

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“…The obtained cisgenic plants were tested in field conditions for three years and showed a stable resistant phenotype (Krens et al ., 2015 ). Interestingly, the effectiveness of the same recombinase system was recently also tested in banana, inducing the excision of the green fluorescent protein, used as reporter gene (Kleidon et al ., 2019 ).…”

Section: Cisgenesis: Approaches and Potentials In Plant Protectionmentioning

confidence: 99%

Novel and emerging biotechnological crop protection approaches

Giudice

Moffa

Varotto

et al. 2021

Plant Biotechnology Journal

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Traditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for non-model plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation.

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“…To eliminate the marker gene, transgenic progeny in which the GOI T-DNA has segregated from the markergene T-DNA are selected (Ling et al, 2016;Pan et al, 2020). In the Cre/lox strategy (Bai et al, 2008;Nandy et al, 2015;Ow, 2016;Zhu et al, 2018;Kleidon et al, 2020), a marker gene flanked by lox sites was deleted after transformation via sitespecific recombination, and the GOI was retained by transgenic plants. Similarly, in another study a selection marker and a CRISPR/Cas9 expression cassette were placed between two gRNA target sites in a T-DNA sequence and deleted via double breakage at the target sites (Wang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System

Pan

et al. 2021

Front. Plant Sci.

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Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

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Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform

Cited by 14 publications

References 33 publications

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

A Strategy for the Production and Molecular Validation of Agrobacterium-Mediated Intragenic Octoploid Strawberry

Novel and emerging biotechnological crop protection approaches

Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System

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