Risk Assessment of Genetically Modified Crops by Direct Tracking Pollen Movement and Testing Crop Genetic Load Using Directly Transformed \u3cem\u3eBrassica rapa\u3c/em\u3e with Bt \u3cem\u3ecry1Ac\u3c/em\u3e and \u3cem\u3egfp\u3c/em\u3e Genes

One concern with crop biotechnology is that there might be crop to weed transgene flow, which could result in more invasive and competitive weed populations. Transgene expression, introgression of crop genes, and other ecological factors may alter the fitness or productivity of weed populations. The Brassica napus (crop) to Brassica rapa (weed) model to assess transgene flow and consequences has been widely used. In this study, weedy accessions of Brassica rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (GFP)- coding transgenes using Agrobacterium to develop plants to be subsequently used in risk assessment research. Regenerated transgenic B. rapa lines were characterized by progeny analyses, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assays. GFP expression level and Bt protein concentration were significantly different among independently transgenic B. rapa events. Seed yield of transgenic B. rapa events was compared to B. rapa × B. napus introgressed hybrids in greenhouse and field experiments as comparative tools to evaluate the genetic load of introgressed crop genes in weedy populations. In a greenhouse study, the biotypes expressing the Bt transgene were significantly different from insect susceptible plants and insect resistance was the predominant factor in productivity under diamondback moth (Plutella xylostella) herbivore pressure. No significant differences were observed, however, in vegetative growth or reproductive yield between the transgenic B. rapa lines and crop-weed hybrids under field conditions. Directly transformed transgenic B. rapaplants were an essential positive experimental control to begin to assess genetic load of crop genes in crop-weed hybrid populations. This is the first report of the direct transformation of a weedy plant. Transgene movement via pollen is an important parameter for understanding and evaluating possible out-crossing capacities of transgenic crop varieties. Here, we describe the movement of oilseed rape (Brassica napus L. cv. Westar) pollen expressing a genetically encoded fluorescent tag. Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a GFP variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature and was shown to be an effective tool to monitor pollen movement in proof-of-concept studies in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by fans. Under field conditions, GFP transgenic pollen grains were detected up to 15 meters from the source plants. No significant difference was detected under field conditions for pollen frequency among distances 0, 5, 10, and 15 m from the source plant plot with no consistent wind effects on the number of pollen grains detected on pollen traps. No significant differences between transgenic pollen and non-transgenic pollen were detected for pollen dispersal under field conditions