The Use of Green Fluorescent Protein for Transgene Monitoring and Detection of Pollen Distribution and Gene Flow Patterns under Field Conditions.

There have been many recent and new possibilities for crop improvement since the development of techniques, which allow for the transfer of novel genes into host plant genomes. Fitness enhancing genes that confer disease, drought, and herbicide resistance to important food crops such as corn (Zea mays L.), soybean (Glycine max L.), canola (Brassica napus L.), and rice (Oryza sativa L.) will be invaluable tools to feed the exponentially growing human population world wide. This widespread use of transgenic crops has increased the risk of transgene escape into the environment. This risk becomes more significant when engineered crop species have wild relatives growing in close proximity. The potential of transgenes resulting in fitness-enhancement of weedy relatives warrants the need for an in vivo gene monitoring system suitable for use in the field so that the potential risks can be quantified and evaluated. Green fluorescent protein (GFP) has been demonstrated as an effective tool to monitor the expression and possible introgression of transgenes into crop-related wild species (Stewart, 1996; Harper et al., 1999; Halfhill et al., 2001). Therefore, GFP may be an ideal candidate for the detection of gene flow and pollen distribution patterns under field conditions. In this study, the pollen-specific LAT59 promoter was used to express a green fluorescent protein (GFP) gene in tobacco (Nicotiana tabacum L.). The result yielded transgenic tobacco plants, which express GFP protein only within pollen grains. GFP-tagged pollen was developed as a tool for tracking the movement of transgenic tobacco pollen under field conditions. A two-year field study was set up and monitored during the summer f 2001 and 2002. The goal of this research was to characterize spatial distribution patterns of transgenic pollen to gain knowledge about pollination mechanisms under field conditions in tobacco. However, a possible pitfall is that tobacco is normally a self-fertilizing crop so detection of pollen movement in this system may be difficult. Another aspect of this research was to estimate the frequency of out-crossing under field conditions. Transgenic tobacco engineered with the CaMV35s promoter to express GFP throughout the entire plant (WPGFP) was used. Pollen flow from a pollen-donor population of WPGFP tobacco plants to non-transgenic tobacco plants grown at specified distances and directions from the transgenics were assessed. Hybrid frequency was determined by screening the progeny from each wild type recipient plant for the GFP phenotype. Out-crossing in 2001 was 0.009% and 0.07% in 2002. Average out-crossing within the center plot in 2002 was 0.7%. The goal of this research was to determine the dynamics of actual out-crossing events in a field of tobacco. If this system is to be an effective monitoring tool, then GFP transgenic pollen viability and longevity must be tested. Anytime a foreign protein is introduced into a reproductive cell, there is a possibility of adverse effects on cellular regulation or disruption of cell development, which could be detrimental to a tobacco plant’s capacity to reproduce. Expression of GFP has been demonstrated in many different plant parts and species, and toxicity or adverse developmental events have not been observed (Pang et al., 1996; Leffel et al., 1997; Harper et al., 1999; Quaedvlieg et al., 1998; Tian et al., 1999; Molinier et al., 2000). Although GFP expression has not been found previously to have an effect on plant cells, a comparison of pollen tube germination frequencies to those of wild type tobacco pollen were conducted to determine the consequences of GFP expression. Data from these experiments did not suggest that expression of GFP had an effect on pollen fitness. Since GFP can be expressed in tobacco pollen under the control of the LAT59 pollen specific promoter, a system to monitor and detect pollen distribution and gene flow patterns can be developed on a large scale. This could reveal answers to many questions involving ramifications of the introgression of transgenic crop species into the environment