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800 N. Lindbergh Blvd.
St. Louis, MO 63167, USA
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).
The term Recipient organism refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas Parental organisms refers to those that were involved in cross breeding or cell fusion.
- Agrobacterium-mediated DNA transfer
Ti plasmid left border repeat
CaMV Enhanced 35S promoter
5' untranslated leader from chlorophyll a/b-binding protein
Heat shock protein 17.3 terminator
Transit peptide and first intron of Rubisco SSU
Nopaline Synthase Gene Terminator
Ti plasmid right border repeat
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
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
- Resistance to diseases and pests
- Lepidoptera (butterflies and moths)
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.