A disarmed Agrobacterium tumefaciens plant transformation system
including the double-border, binary vector PV-ZMAP595 was used to
transform LH59 embryos producing MON 87460.
The disarmed Agrobacterium tumefaciens transformation vector
PV-ZMAP595 contains both left and right T-DNA border sequences
facilitating the transformation. It was constructed using standard
molecular biology techniques. The T-DNA region of this vector
contains cspB and nptII expression cassettes and it is the portion
that is integrated to maize genome during transformation
event.
The genetic elements of PV-ZMAP595 intended for insertion into the
maize genome comprised between the T-DNA borders are, from the
right border region, promoter and leader from the rice actin gene
(P-Ract1), a non-translated intron from the rice actin gene
(I-Ract1), the cspB coding sequence (CS-cspB) and a polyadenylation
sequence from the transcript 7 gene (T-tr7). These elements
together constitute the cspB expression cassette which is followed
by the nptII expression cassette. The latter is flanked by two loxP
sites and constitutes of a transcriptional promoter (P-35S), the
nptII coding sequence (CS-nptII), and a polyadenylation sequence
from the nopaline synthase gene (T-nos).
MON 87460 contains one copy of the insert at a single insertion
site hosting both cspB and nptII intact expression cassettes. No
additional elements from the transformation vector PV-ZMAP595,
linked or unlinked to the cspB and nptII expression cassettes, were
detected in the genome of MON 87460. Additionally, backbone
sequence from the plasmid PV-ZMAP595 was not detected.
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