For Agrobacterium-based transformation protocol, the applicant must indicate how Ti plasmid based vector was disarmed (i.e., all tumorigenic DNA was removed). Applicants can provide citations that describes the transformation procedure. However, any significant modifications of transformation, strain designation, etc. should be described.
For other methods of transformation, the applicant can
describe the sources of various components of the plasmid (or other DNA including possible carrier DNA)
and method of transformation by citation. However, any significant modifications in transformation, tissue
regeneration, etc. should be described.
The applicant must provide a detailed restriction map of
the plasmid that is sufficient to be used in the analysis of Southern data. Description of added restriction sites is helpful in interpretation of Southern data and should be
provided (see Fig. 1).
SAMPLE PETITION—Herbicide-Tolerant Plants
III. Description of Transformation System
The vector system (pVST1) used to transfer the genes to cotton plants is based on the Ti plasmid from Agrobacterium tumefaciens (Fig. 1). The DNA becomes incorporated into the plant cell chromosome (Klein et at., 1989). Although some of the DNA sequences used in the transformation process were derived from the plant pathogen A. tumefaciens, the genes which cause crown gall disease were first removed, and therefore the recipient plant does not have crown gall disease (Zambryski, 1988; Klee and Rogers, 1989). Once the introduced genes are inserted into the chromosome of the recipient, they are maintained in the same manner as any other genes.
The T-DNA, which includes the glyphosate resistance and nptII genes, was transferred into the genomes of individual cotton hypocotyl sections as described by Umbeck et al. (1987). Plants were regenerated as described by Trolinder and Goodlin (1987).
RB nos5' nptII nos3' CaMV35s aroA nos3' LB amp ori lac
Fig 1—pVST1: A linear schematic illustration of the plasmid vector (7.8kb) that was conjugated into Agrobacterium tumefaciens (Malik and Wahab, 1993). Various components of pVST1 have been described in Table 1. Map reflects the gene order but not the sizes. The plasmid does not have restriction sites for SpI and NdeI, but has one site for SmaI and NotI.