scholarly journals Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lorenza Dalla Costa ◽  
Stefano Piazza ◽  
Valerio Pompili ◽  
Umberto Salvagnin ◽  
Alessandro Cestaro ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gene Transfer ◽  
Genomic Dna ◽  
Fruit Trees ◽  
Plant Genome ◽  
Stable Gene ◽  
Binary Vectors ◽  
Exogenous Dna ◽  
Left Behind ◽  
Dicotyledonous Plants

AbstractGenome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

scholarly journals Improvement of adventitious organogenesis for regeneration of transgenic plants in blackberry

Genetika ◽  
2015 ◽  
Vol 47 (2) ◽  
pp. 599-608 ◽  
Author(s):  
Alena Gajdosová ◽  
Tatjana Vujovic ◽  
Miroslava Súkeníková ◽  
Gabriela Libiaková
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Regeneration ◽  
Gene Transfer ◽  
Transgenic Plants ◽  
Plant Genome ◽  
Ms Medium ◽  
Regeneration System ◽  
Promising Technique ◽  
Rubus Fruticosus ◽  
Foreign Dna

The introduction of foreign DNA into the plant genome by Agrobacterium tumefaciens is a promising technique of targeted gene transfer which depends on good working regeneration system. The aim of the work was to elaborate the system for efficient adventitious organogenesis and transgenic plant regeneration in Rubus fruticosus L. using explants from mature plants. Regeneration of putative transgenic shoots took place from flag explants cultivated vertically on MS medium with 1 mg l-1 TDZ and 0.02 mg l-1 IBA followed by transfer on MS medium with 1 mg l-1 BAP, 0.02 mg l-1 IBA and 0.1 mg l-1 GA3 supplemented with 10-15 mg l-1 hygromycin after transformation by A. tumefaciens strain LBA 4404 carrying plasmid pCambia 1304. Four putative transgenic plants of cv. 'Cacanska Bestrna' were rooted and acclimatized.

scholarly journals Integrated approach for the molecular characterization of edited plants obtained via Agrobacterium tumefaciens-mediated gene transfer

2021 ◽  
Author(s):  
Lorenza Dalla Costa ◽  
Daniela Vinciguerra ◽  
Lisa Giacomelli ◽  
Umberto Salvagnin ◽  
Stefano Piazza ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gene Transfer ◽  
Molecular Characterization ◽  
Good Practice ◽  
Integrated Approach ◽  
Digital Pcr ◽  
Plant Genome ◽  
Practical Case ◽  
Knock Out

AbstractAgrobacterium tumefaciens-mediated gene transfer—actually the most used method to engineer plants—may lead to integration of multiple copies of T-DNA in the plant genome, as well as to chimeric tissues composed of modified cells and wild type cells. A molecular characterization of the transformed lines is thus a good practice to select the best ones for further investigation. Nowadays, several quantitative and semi-quantitative techniques are available to estimate the copy number (CN) of the T-DNA in genetically modified plants. In this study, we compared three methods based on (1) real-time polymerase chain reaction (qPCR), (2) droplet digital PCR (ddPCR), and (3) next generation sequencing (NGS), to carry out a molecular characterization of grapevine edited lines. These lines contain a knock-out mutation, obtained via CRISPR/Cas9 technology, in genes involved in plant susceptibility to two important mildew diseases of grapevine. According to our results, qPCR and ddPCR outputs are largely in agreement in terms of accuracy, especially for low CN values, while ddPCR resulted more precise than qPCR. With regard to the NGS analysis, the CNs detected with this method were often not consistent with those calculated by qPCR and ddPCR, and NGS was not able to discriminate the integration points in three out of ten lines. Nevertheless, the NGS method can positively identify T-DNA truncations or the presence of tandem/inverted repeats, providing distinct and relevant information about the transgene integration asset. Moreover, the expression analysis of Cas9 and single guide RNA (sgRNA), and the sequencing of the target site added new information to be related to CN data. This work, by reporting a practical case-study on grapevine edited lines, explores pros and cons of the most advanced diagnostic techniques available for the precocious selection of the proper transgenic material. The results may be of interest both to scientists developing new transgenic lines, and to laboratories in charge of GMO control.

scholarly journals Obtaining Transgenic Anthurium through Agrobacterium-mediated Transformation of Etiolated Internodes

1996 ◽  
Vol 121 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Fure-Chyi Chen ◽  
Adelheid R. Kuehnle
Keyword(s):  
Antibiotic Resistance ◽  
Agrobacterium Tumefaciens ◽  
Gene Transfer ◽  
Plant Development ◽  
Genomic Dna ◽  
Nurse Cells ◽  
Antibiotic Selection ◽  
Agrobacterium Mediated Transformation ◽  
Chimeric Genes ◽  
Dna And Rna

Several procedures were tested in development of a gene transfer protocol for anthurium. Etiolated internode segments of anthurium cultivars `Rudolph' and `UH1060' were co-cultured with Agrobacterium tumefaciens LBA4404 carrying the chimeric genes neo, for antibiotic resistance, and att encoding antibacterial attacin. Assays of genomic DNA and RNA from kanamycin-resistant `Rudolph' and DNA from `UH1060' plantlets, recovered as soon as 1 year after culture on selection media, indicated the presence of introduced genes, including neo and att, and transcription of att. Western analysis confirmed the expression of attacin protein in calli induced from laminae of regenerated kanamycinresistant `Rudolph' plantlets. Use of tobacco nurse cells during co-cultivation of internodes with Agrobacterium did not increase recovery of shoots under the regeneration conditions used. Improvements in culture and antibiotic selection conditions during plant development are suggested.

Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.)

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 716-728 ◽  
Author(s):  
Pavel Neumann ◽  
Marcela Nouzová ◽  
Jirí Macas
Keyword(s):  
Pisum Sativum ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Tandem Repeats ◽  
Genomic Library ◽  
Chromosome Morphology ◽  
Plant Genome ◽  
Genomic Repeats ◽  
Dispersed Repeats

A set of pea DNA sequences representing the most abundant genomic repeats was obtained by combining several approaches. Dispersed repeats were isolated by screening a short-insert genomic library using genomic DNA as a probe. Thirty-two clones ranging from 149 to 2961 bp in size and from 1000 to 39 000/1C in their copy number were sequenced and further characterized. Fourteen clones were identified as retrotransposon-like sequences, based on their homologies to known elements. Fluorescence in situ hybridization using clones of reverse transcriptase and integrase coding sequences as probes revealed that corresponding retroelements were scattered along all pea chromosomes. Two novel families of tandem repeats, named PisTR-A and PisTR-B, were isolated by screening a genomic DNA library with Cot-1 DNA and by employing genomic self-priming PCR, respectively. PisTR-A repeats are 211–212 bp long, their abundance is 2 × 104 copies/1C, and they are partially clustered in a secondary constriction of one chromosome pair with the rest of their copies dispersed on all chromosomes. PisTR-B sequences are of similar abundance (104 copies/1C) but differ from the "A" family in their monomer length (50 bp), high A/T content, and chromosomal localization in a limited number of discrete bands. These bands are located mainly in (sub)telomeric and pericentromeric regions, and their patterns, together with chromosome morphology, allow discrimination of all chromosome types within the pea karyotype. Whereas both tandem repeat families are mostly specific to the genus Pisum, many of the dispersed repeats were detected in other legume species, mainly those in the genus Vicia.Key words: repetitive DNA, plant genome, retroelements, satellite DNA, Pisum sativum.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 85-97 ◽  
Author(s):  
María Elena Arias ◽  
Esther Sánchez-Villalba ◽  
Andrea Delgado ◽  
Ricardo Felmer
Keyword(s):  
Gene Transfer ◽  
Sperm Quality ◽  
Computer Assisted ◽  
Exogenous Dna ◽  
Sperm Analysis ◽  
Functional Parameters ◽  
Transgenic Embryos ◽  
Fertilization Capacity ◽  
Motility Parameters

SummarySperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) orin vitrofertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

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