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Maize line MON89034 expresses two Bt-toxins encoded by the genes cry1A.105 and cry2Ab2 from Bacillus thuringiensis that confer resistance against certain lepidopteran pests such as fall armyworm (Spodoptera sp.), black cutworm (Agrotis ipsilon), european corn borer (Ostrinia nubilalis) and the corn earworm (Helicoverpa zea).

PV-ZMIR245

A second T-DNA, designated as T-DNA II, contains the nptII (neomycin phosphotransferase II) expression cassette. The nptII gene cassette that produces the NPTII protein consists of the promoter (P-e35S) from the cauliflower mosaic virus (CaMV) 35S RNA followed by the 3' nontranslated region of the nopaline synthase (T-nos) sequence from Agrobacterium tumefaciens which terminates the transcription and directs polyadenylation.

During transformation, both T-DNAs were inserted into the genome. The nptII gene confers resistance to Kanamycin and similar antibiotics and was used as the selectable marker for isolation of transformed cells and regeneration of transgenic plants. Once transgenic plants had been regenerated, the selectable marker gene was no longer needed and conventional breeding was used to isolate plants that only contain the cry1A.105 and the cry2Ab2 expression cassettes (T-DNA I) and did not contain the nptII expression cassette (T-DNA II), thereby, producing marker-free transgenic lines, one of which was selected and designated as MON89034.

The Cry2Ab2 coding sequence was modified for optimal expression in plants.

Southern blot analyses demonstrated that the DNA inserted into the corn genome is present at a single locus and contains one functional copy of the cry1A.105 and the cry2Ab2 expression cassettes. All genetic elements are present in the inserted DNA as expected with the exception that the e35S promoter, which regulates expression of the cry1A.105 gene, has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border sequence in MON 89034. No backbone plasmid DNA or nptII sequences were detected. PCR and DNA sequence analyses provided the complete DNA sequence of the insert and confirmed the organization of the elements within the insert. Furthermore sequence analysis indicated that MON 89034 no longer has the duplicated enhancer elements compared to the original e35S promoter in PV-ZMIR245, possibly due to a recombination event that resulted in its deletion.

Utilizing a vector with two T-DNAs is the basis for an effective approach to generate marker-free plants. It allows for the TDNA with the traits of interest (T-DNA I) and the T-DNA encoding the selectable marker (T-DNA II) to be inserted into two independent loci within the genome of the plant. Following selection of the transformants, the inserted T-DNA encoding the selectable marker can be segregated from progeny through subsequent traditional breeding and genetic selection processes, while the inserted T-DNA containing the trait(s) of interest is maintained resulting in an LMO that marker-free and contains only the Cry expression cassette.