Southern analysis should include DNA isolated from nonmodified recipient, all or selected transformed lines, and the vector. Parental plasmid DNA (eg PUC 18) not
containing intended donor genes may be 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.
Probed for aroA
Probed for nptII
Probed for Ampand lac
Fig. 2—Southern blot analysis of DNA: Detection of aroA and nptII genes in the Banjaran. Line lanes 1 and 2 contain 10 µg DNA of Banjaran and Stoneville 825 DNA respectively digested with NdeI. Lane 3 is Sal1 digested pUC 18 DNA as a positive control for amp and lac sequences. Lane 4 is a HindIII digest of phage lambda DNA, used as molecular weight markers. All four DNA samples were electrophoresed on three different 1% agarose gels and transferred to nitrocellulose filters. One nitrocellulose filter was hybridized with 32P-labeled AroA probe, a second filter with the nptII probe and a third one with the amp+lac probe. A similar hybridization pattern was obtained when Southern blots were hybridized with either AroA or nptII probes. The PstI fragment (amp+lac) hybridized to pUC 18 DNA but not to the plant genomic DNA. This data is consistent with the presence of a single insertion of the genes between the LB and RB incorporated in the genome of the transgenic plant.
B. Mendelian Inheritance
The primary transformants that expressed the NPT II marker gene and the EPSP synthase gene were allowed to self- pollinate, and the seeds were collected. These seeds (T1) were planted in a single 25-foot row. Seedlings were thinned to a density of two plants per foot. Seedlings were sprayed with one application of glyphosate at a rate of 8 oz/acre. Symptoms of bleaching or necrosis appeared 8 to 10 days after application and were compared to the symptoms of nontransformed plants that received an identical herbicide application. The number of resistant and sensitive plants in three separate rows was counted (Table 2). If primary transformants were expressing the EPSP gene from a single insertion site (genetic loci), the expected segregation would be 3:1. The segregation ratio totals for all 3 rows was 110:40, which fits the Mendelian prediction of 112½:37½ (2 = 0.22) well. The nontransformed plants were fully susceptible to glyphosate.
SAMPLE PETITION—Herbicide-Tolerant Plants