Characterization of sludges. Good practice for sludge utilisation in agriculture

2010 ◽  
Keyword(s):  
Good Practice

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.

Standardized characterization procedures: a necessary support to regulations

2016 ◽  
Vol 74 (1) ◽  
pp. 220-228 ◽  
Author(s):  
Ludovico Spinosa
Keyword(s):  
Good Practice ◽  
Technical Committee ◽  
Good Practices ◽  
European Committee ◽  
Sludge Characterization ◽  
Recent Developments ◽  
Eu Directives ◽  
Definition Of ◽  
The Eu

Sustainable procedures for sludge/biosolids management require the development of strategies to reduce the amount of sludge to be handled and to produce higher quality biosolids. Therefore, realistic and enforceable regulations adapted to local situations must be developed, defined outlet procedures established and good quality products guaranteed. To this end, the definition of standardized characterization procedures and guidelines for good practices becomes a necessary support to regulations. To contribute to the development of the EU directives dealing with sludge, the European Committee for Standardization (CEN) has established Technical Committee 308 (TC 308) whose scope is the standardization of methods for sludge characterization and the production of guidelines of good practice. In this paper, the most recent developments of the CEN/TC 308 work for the chemical, biological and physical characterization of sludge/biosolids are discussed in relation to the main different treatment and outlet options.

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