Progress Report RTD-Project GMOCARE
Title: New methodologies for assessing the potential of unintended effects in genetically modified crops
Project: GMOCARE (QLK1-1999-00765)
Period: February - September 2000
Co-ordinator: Dr. Hub Noteborn, RIKILT, The Netherlands
Method development for identification of unintended effects due to genetic modification.
The GMOCARE project comprises eleven workpackages, which cover four sub-projects as follows:
Sub-project 1: Selection, production and verification of plant species (WP1, WP2, and WP3)
Sub-project 2: Benchmarks in GM-, mutated and traditional plants and establishment of base-line properties and statistical values (WP4 and WP10))
Sub-project 3: Demonstrating the effects of genetic modification and target lead identification of possible unintended effects using genomics, proteomics and metabolomics (WP5, WP6. WP7, WP8 and WP9)
Sub-project 4: Dissemination of new methodologies, knowledge of unintended effects and databases (WP11)
In general (WP1 - WP11)
The work has proceeded according to the planned schedule. The main topic of the first interim-progress meeting was the finalisation of those factors of importance to be considered in defining the overall strategy and methods used in GMOCARE such as: 1) plant species, 2) type of transformant (i.e. which metabolic modifications, indications for the occurrence of unintended effects and value in terms of consumers), 3) definition of control lines and 4) impact of environmental conditions.
To integrate work on metabolomics, proteomics and genomics it is considered to be essential to provide a priority listing so that the best candidates are well covered. Secondly, to obtain an imprint of frequency of possible unintended effects in transgenesis, it will be essential to perform extensive work on different phenotypes from one 'genetic modification' event. This prioritisation and selection of crop plant lines has been achieved now based on the criteria mentioned before (i.e. list has been completed). In addition, the consortium decided that more extensive metabolic profiling work may identify additional key candidate lines but that will require more intensive proteomic/genomic inputs. This latter criterion will be dealt with on a case by case basis.
In the next 6 months period participants will, by default, exchange the materials and information on a virtually continuous basis.
WP1: Provision of a wide range of molecularly characterised transgenic potato lines
- Started month 0;
- An application was made in January 2000 to the UK Advisory Committee on Releases into the Environment (ACRE; Application 00/R23/7). ACRE responded in March, posing several questions.
In addition, ACRE are insisting on a two-year post-release monitoring schedule. This does not equate to previous experience where a one-year period was acceptable when leaving the site bare fallow in the succeeding year. Plant production has been arranged in pots under containment using TYGAN netting, grown for seed (for 2001) and in a randomised design for experimental usage;
- Tubers from all plant lines were harvested on 14 September 2000 and are currently being washed and air-dried prior to sampling/storage. DNA has been extracted from experimental lines using leaf material harvested in 2000. Primer sets have been designed for insert and vector backbone analyses;
- Month 6-12: 3 types of Desiree transformants will be further characterised as scheduled
Mal1 (defective glycoprotein processing); SAMPAT (polyamine metabolism); W2GBSS (starch content)
WP2: Provision of a wide range of molecularly characterised transgenic and mutated Arabidopsis and tomato lines with added values
- Started Month 0;
- Production started of transgenic tomato lines, 35S crtI = phytoene desaturase from Erwinia, PG crtB = phytoene synthase from Erwinia, Anti-Psy-1 = antisense phytoene synthase-1, b-Lcy = sense lycopene b-cyclase and HMGR = sense cytosolic HMG-CoA reductase;
- Fruits from four lines currently available frozen from previous harvests with parent line controls have been transferred to other participants;
- Month 6-12: produce and examine further the transformants: 35S crtl (2-fold increase of b-carotene and lutein); crtB (increased carotenoids); anti-Psy (dramatically reduced carotenoids); beta-Lcy (2.5-fold increase of b-carotene) and HMGR (increased end-product phytosterols).
- 7 types of transformants will be developed and studied as follows: parental line, 3 CHS antisense (single copy) and 3 CHS (multi copy);
- sizes of T-DNA and vector backbone integrated in the plant genome in the different lines were analysed by PCR; specific primer pairs were designed; number of copies integrated in the modified lines have been analysed by Real-Time; binary vectors with DFR in antisense and sense under control of an ethanol inducible promoter are under construction;
- Month 6-12: continue to characterise selected 2nd generation transformants; construction of binary vector with antisense/sense constructs of DFR under control of an ethanol inducible promoter followed by transformation.
WP3: Provision of a wide range of molecularly characterised transgenic potato and mutated Nicotiana lines overexpressing amino acids
- Production started of transgenic potato lines transgenic Desiree DHDPS lines; 20 lines with patatin promoter/sense DHDPS gene/nos polyA (altered lysine content);
- Materials have been transferred other participants;
- Month 6-12: studies focussed on target line Desiree DHDPS (lysine content)
- Production started of transgenic/mutated Nicotiana lines including control wild type, mutants and crosses (altered phenotypes);
- Month 6-12: Focus on 4 types of mutants: RAEC-1 (mutated dhdps-r1), RLT-70 (mutated aklys 1), hybrid RLT-70/RAEC-1 and parental line.
WP4: Provision of data related to phenotypic, performance and chemical characteristics of transgenic, mutated and control lines
- Started Month 0;
- Transfer to participants of potato material and freeze-dried powders of tubers available for analyses;
- Analysis of glycoalkaloids by HPLC, sugar balance (using HPAEC) and fatty acid profiles for selected potato lines;
- A novel, rapid protocol for total fatty acid analysis has been developed for freeze-dried powders and validated against fresh tuber extracts;
- Design of GC methods, identification of sugars, organic acids and amino acids initial set-up completed and reproducibility tests in progress;
- HPLC method developed for potato consistently showing peaks for 40 aromatic compounds (UV/DAD detection) and systematic investigation of potato lines and replicates under way;
- Month 6-12: reproducibility checks to be made, reference standards and available literature to identify compounds; data transfer methods developed for analysis of data by chemometrics; HPLC measurements to be completed on first batch of potatoes; LC-MS for identification of aromatic compounds; additional work requested at first interim-progress meeting by other participants (i.e. tomatine; glycoalkaloids and metabolite profiling by HPLC and GC of tobacco).
WP5: Differential gene expression analysed by genomics
- Started Month 0;
- Sequence analysis of the red and green tomato specific cDNA libraries;
- Hybridisation experiments with the subsequent stages of tomato ripening and hybridisation with (fragments of) selected cDNAs of tomato;
- Month 6-12: construct tomato array; determine redundancy of array; determine levels of gene expression in subsequent ripening stages of tomato; start to construct a potato array by subtraction of potato high from low glycoalkaloid-levels plus cDNA library of other participant; start to construct an Arabidopsis microarray using cDNA library of randomly selected differentially expressed genes.
WP6: Differential protein expression analysed by proteomics
- Started Month 0;
- Different protocols used in two participating institutes were compared and optimised to find a practical approach for sample delivery and compatibility with other analyses done within the project;
- Visit to DCHGR including training in the identification of proteins by MALDI peptide fragment analysis; protein patterns of various potato cultivars have been analysed including those seen during germination of the tubers;
- Month 6-12: Suitability of lyophilisation of plant materials; identification of landmark proteins by ESI-MS; proteome analysis of GM and non-GM plant materials; methods optimised for protein extraction and 2D electrophoresis suitable for use as analysis methods by others in the future.
WP7: Proteomics of IgE-reactive determinants
- Started Month 0;
- A strategy of glycoanalysis of glycoproteins post PAGE has been developed and demonstrated;
- Human sera containing specific IgE antibodies directed to rape seed has been collected and analysed for specific subclasses between allergic patients;
- A strategy of glycoanalysis of glycoproteins in plant tissue has been developed and demonstrated;
- Month 6-12: collection of flowers from wild type Arabidopsis; selection of Arabidopsis-specific human IgE; optimisation of N-glycan profiling of glycoproteins isolated from 2D-gels.
WP8: Metabolomics in vivo using 13C/31P NMR
- Started Month 0
- Preliminary cultures with potato pot plants have been done in special chambers and biomass fixed for 13C-NMR analysis;
- Start of NMR analysis;
- Month 6-12: utilisation of growth chambers for tomato plants including specific adaptations; analyses of 13C-labelled plant biomass of first plants tested; 31P NMR analysis.
WP9: Metabolomics in vitro using HPLC, LC- NMR, LC-MS
- Sample preparation and extraction procedures for NMR have been established and a partners fractionation procedure tested and modified to reduce running time'; comparable results achieved for tomato; trial experiments carried out on potato but method not yet well established;
- Methods for the automated acquisition and processing of NMR spectra; basic methods and parameters established;
- 2D NMR spectra recorded for fractions A, B, C of potato and tomato samples; analysis of GM potato and tomato samples by NMR and chemometrics;
- Month 6-12: NMR reproducibility check in progress on potato and tomato samples; principal components analysis; selection of final solvent conditions; inverse 2D NMR experiments on tomato fractions B, C, D; GC and HPLC work and possibly LC-MS/LC-NMR; fractionation procedure for potato; systematic measurement and chemometrics treatment of the large potato and tomato batches.
WP10: Statistical design and quality assurance of experiments
- Statistical design and analysis of the experiments have been examined including factors that affect the results;
- For exchanges of data agreement has achieved to harmonise the preparation of spreadsheets;
- Month 6-12: Integration of useful items in the statistical design and analysis.
WP11: Synthesis of methods and regulation impact of 'unintended effects' in GMOs
- Start at Month 6;
- Month 6-12: Review of the data gathered during the first year; integration of methodologies.