labeled and hybridized to Southern blots to demonstrate that only the intended sequences have been incorporated in the genome of the transgenic plant. Restriction enzymes to be used might include enzymes that do not cut within the transforming plasmid but will cut the “entire insert” into one fragment from the DNA of the transgenic plant.

In the case of an Agrobacterium-based transformation system, the applicant should determine if genes that reside outside the LB/RB are inserted in the genome of the regulated cultivar. If a complete copy of any of these genes is present, the applicant should determine whether it is expressed in the plant. For direct transformation systems, applicants should determine which sequences are inserted in transgenic plants and whether they are expressed. PCR analysis may be used to prove that only the targeted DNA has been incorporated. Sequencing of the transgene in plant and adjacent sequences is not required. Determination of the number of copies of integrated transgenes is not required, but the number of insertions may be used to support analysis of inheritance data.

If the inserted DNA sequence order is complex, as is often the case for plants engineered via direct transformation systems (e.g., electroporation, polyethylene glycol transformation of protoplasts, or particle bombardment techniques), the applicants should summarize the data by providing the following information for all the genes (whether under the direction of plant or bacterial promoters). Is there a complete copy of the gene present in the regulated article? Is the protein expressed in the plant? If multiple complete copies of a gene are present, applicants do not have to determine if each copy of the gene is expressed. Applicants should provide a table, like the one shown below [the user’s guide provides a table with hypothetical data], that summarizes the results and indicates where specific data is to be found.

Mendelian inheritance data and Chi square analysis for at least 2 generations are appropriate to demonstrate whether the transgene is stably inserted and inherited in Mendelian fashion. Such data are generally not necessary for infertile vegetatively propagated crops such as male-sterile potatoes.

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 cannot 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



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