RNA—Northern analysis is generally not required except
for virus-resistant plants. However, such analysis may be necessary for ribozyme, truncated sense, or antisense
constructs, when protein levels can not be provided.
PROTEINS—Expression levels of gene(s) of interest and marker genes in various tissues, developmental stages of plant, and experimental conditions (induced or noninduced) are required. Assays can be of enzyme activity. Serology, ELISA, and Western blots may also be used. Describing the source of the immunogen is critical for serological analysis.
For virus resistant plants, the amount of viral transgene RNA produced should be determined and compared to the amount of the RNA produced by the viral gene in an infected nontransgenic plant. Applicants should address whether the transgene RNA (or protein) is present in the same tissues as are infected during natural infections. In addition, provide the amount of both coat proteins (i.e. from the transgene and the naturally infecting virus) produced in the transgenic plant singly infected with the widely prevalent viruses in the U.S. that normally infect the recipient plant (contact APHIS for the list of these viruses). For comparison, provide the amount of both coat proteins produced in the nonengineered plant in mixed infections of the virus from which the coat protein gene was derived and the same widely prevalent viruses used in the single infection study. Provide description of symptoms of infected plants in all cases
C. Expression of Inserted Genes
The production of the desired proteins in Banjaran was confirmed by immunoassay. Banjaran has been modified by the insertion of the aroA gene imparting the glyphosate resistance trait. In addition to glyphosate resistance, Banjaran expresses the selectable marker protein, nptII. As measured by immunoassay, the aroA and nptII proteins were expressed at low and relatively consistent levels in Banjaran across all field sites (see data reports 94–000–02, 94–000–03). The mean from all tests reported in 10 states at 50 sites showed that Banjaran contained in leaf and seed tissue respectively approximately 12.6 µg and 17.3 µg 5-enolpyruvyl-3-shikimate phosphate synthase protein/gram fresh weight of tissue (fwt), and 6.9 and 3.3 µg nptII protein/gram fwt. At the one field site at which expression was evaluated over time, the EPSP protein levels varied less than five fold in young leaf tissue collected over the growing season with the highest levels observed early in the season (see data report 94-000-05).
The above data was based on leaf and seed. However, the levels of EPSP and nptII proteins in whole plant tissue were much lower, on a fresh weight basis, than in leaf or seed tissues. EPSP is present in whole Banjaran plants from 1.1 to 1.7 µg/g fwt of the whole plant respectively; NPTII protein levels are 14.6 µg/g/fwt for Banjaran plants. These measured concentrations were used to estimate the amount of EPSP and NPTII protein that could enter the environment due to post- harvest incorporation of 5000 mature Banjaran plants into the soil. Predicted values are 23.4 g EPSP protein/acre and 183 g NPTII protein/acre, Nectar and pollen collected from Banjaran contain very low levels of EPSP protein. The expression of EPSP protein in pollen collected from Banjaran greenhouse- grown plants were 37.8 ng/g fresh wt, respectively. The EPSP protein levels in nectar were 0.88 ng/g fresh wt collected from Banjaran plants. Thus, pollen and nectar produced by Banjaran present a low source of potential EPSP protein exposure to nontarget organisms.
SAMPLE PETITION—Herbicide-Tolerant Plants