Tesis doctoral de Anna Coll Rius
To ensure the safety of consumers and the environment, genetically modified (gm) food and feed are submitted to strict legislation in many countries. The eu legislation establishes an authorisation procedure requiring: molecular characterisation of the transgene, compositional comparative analysis, food/feed safety assessment, environmental risk assessment and post-marketing environmental monitoring plan. The use of profiling approaches to evaluate the possible occurrence of unintended effects derived from the insertion and/or expression of the transgene has been proposed as an interesting complementary tool for safety assessment. the objective of this thesis was to evaluate the variability associated to the insertion and expression of transgenes in plants, using as example mon810 maize i.E. The only gmo event that is authorised for commercialisation and cultivation in the eu. We aim to complement the existing targeted approaches by providing more unbiased information on the basis of profiling techniques. we initially used microarray hybridisation to compare the transcriptomic profiles of two mon810 and non-gm near-isogenic variety pairs. To avoid the effect of factors unrelated to the transgenic character, in vitro cultured plantlets were analysed. Analysis of ~1/3rd maize transcripts revealed transcriptomic differences of 1.7 and 0.1% in aristis bt/aristis and pr33p67/pr33p66, respectively. In contrast, maize varieties obtained by conventional breeding differed in 4% analysed transcripts. No sequence could be identified that was consistently regulated in five commercial variety pairs analysed. Consequently, just a reduced number of genes were differentially expressed in mon810 and non-gm varieties; and their varietal dependence (in terms of both, numbers and identities) pointed towards the lack of direct effects of mon810 transgene. due to the agricultural importance of this crop and to its high use in catalunya (where above 50% planted maize is mon810), we evaluated if these differences were kept in plants of the same varieties, at the identical development stage and grown in real agricultural fields according to conventional farming practices in the region. Reverse transcription-real-time pcr (rt-qpcr) based analysis of around 10% sequences regulated in plantlets grown in vitro showed they had similar expression levels in mon810 and non-gm varieties cultured in natural conditions. This further suggested mon810 and near-isogenic varieties are substantially equivalent, although we could not rule out the existence of other transcriptomic differences. we then used microarray technology to evaluate unintended effects of mon810 transgene in maize varieties grown under real field conditions, taking into consideration the variability among diverse varieties obtained by conventional breeding and the effects of different farming practices. Helen bt and helen leaves differed in less than 0.13% transcripts both under conventional and low-n fertilisation conditions; and most differentially expressed sequences were regulated in a variety specific manner. Chronic low-n stress in agricultural fields resulted in less than 0.17% differentially expressed sequences in maize leaves. However, they were mostly equally regulated in mon810 and non-gm varieties. Taken together, our results showed that natural variability among conventional varieties and farming practices explained most gene expression variability between mon810 and non-gm plants grown in agronomic fields. The transgenic character only explained 9.7% of the observed variability. we finally complemented the transcriptomics approach with a proteomic analysis of maize in an economically relevant scenario. Mon810 and comparable non-gm grains were grown in agricultural fields and analysed at the development stage typically used for forage (~75% maize is used for forage in the eu). Proteomic patterns of two variety pairs (pr33p67/pr33p66 and dkc6575/tietar, the most representative in the two fao maturity groups grown in the region) had less than 1.19% differentially accumulated proteins (at accumulation ratios below 1.8-fold), and they were not the same in different variety pairs. in conclusion, from the transcriptomics and proteomics perspectives, mon810 maize varieties seem to be substantially equivalent to their non-gm comparators. Thus, the production of gm plants with minimal unexpected effects is possible.
Datos académicos de la tesis doctoral «Variability associated to the insertion and expression of transgenes in plants«
- Título de la tesis: Variability associated to the insertion and expression of transgenes in plants
- Autor: Anna Coll Rius
- Universidad: Girona
- Fecha de lectura de la tesis: 10/09/2010
Dirección y tribunal
- Director de la tesis
- María Pla De Sola Morales
- Tribunal
- Presidente del tribunal: emilio Montesinos segui
- Jesús García cantalejo (vocal)
- esther j. Kok (vocal)
- josep María Casacuberta suñer (vocal)