scholarly journals Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation

AMB Express ◽  
2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Andrea Brenna ◽  
Barbara Montanini ◽  
Eleonora Muggiano ◽  
Marco Proietto ◽  
Patrizia Filetici ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gene Transfer ◽  
Tuber Borchii

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.

Development of Auxotrophic Agrobacterium tumefaciens for Gene Transfer in Plant Tissue Culture

2004 ◽  
Vol 20 (3) ◽  
pp. 890-896 ◽  
Author(s):  
J.I. Collens ◽  
D.R. Lee ◽  
A.M. Seeman ◽  
W.R. Curtis
Keyword(s):  
Tissue Culture ◽  
Agrobacterium Tumefaciens ◽  
Gene Transfer ◽  
Plant Tissue Culture ◽  
Plant Tissue

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.

Effect of ethylene on Agrobacterium tumefaciens-mediated gene transfer to melon

2000 ◽  
Vol 119 (1) ◽  
pp. 75-79 ◽  
Author(s):  
H. Ezura ◽  
K. -I. Yuhashi ◽  
T. Yasuta ◽  
K. Minamisawa
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gene Transfer
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