Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells.

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation.

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 2. Assessing natural variability.

Proteomics is currently tested as a complementary tool for the safety assessment of genetically modified (GM) crops. Understanding the natural variability of the proteome is crucial for the interpretation of biological differences between transgenic and nontransgenic parental lines. The natural variation of seed protein profiles among a set of 12 Arabidopsis thaliana ecotypes was determined by utilizing two-dimensional electrophoresis (2DE). The total number of different resolved protein spots found among the 12 ecotypes was 931 with a range of 573 (Mt-0) to 653 (Condara) in any one ecotype. Although the ecotypes were grown side-by-side in an environmentally controlled growth chamber, almost half of the resolved spots varied with respect to their presence/absence, and 95% of the spots present in all ecotypes varied in spot quantity (2-53-fold). In the evaluation of unintended effects of genetic modification, it is concluded that the experimental design must account for existing natural variability, which, in the case of the expressed proteome, can be substantial.

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of gentically modified crops. 3. Assessing unintended effects.

The current procedures to assess the safety of food and feed derived from modern biotechnology include the investigation of possible unintended effects. To improve the probability of detecting unintended effects, profiling techniques such as proteomics are currently tested as complementary analytical tools to the existing safety assessment. An optimized two-dimensional gel electrophoresis (2DE) method was used as a proteomics approach to investigate insertional and pleiotropic effects on the proteome due to genetic engineering. Twelve transgenic Arabidopsis thaliana lines were analyzed by 2DE, and their seed proteomes were compared to that of their parental line as well as to 12 Arabidopsis ecotype lines. The genetic modification of the Arabidopsis lines, using three different genes and three different promoters, did not cause unintended changes to the analyzed seed proteome. Differences in spot quantity between transgenic and nontransgenic lines fell in the range of values found in the 12 Arabidopsis ecotype lines or were related to the introduced gene.