Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995 Page: 1 of 4

FINAL REPORT S-71
Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer
technology. (#DE-F602-92ER20073--
PI: Liszl6 Mdrton, Associate Professor,
Department of Biological Sciences, University of South Carolina, Columbia, SC 29208
Genetic manipulation of plants often involves the introduction of homologous or partly
homologous genes. Ectopic introduction of homologous sequences into plant genomes may
trigger epigenetic changes, making expression of these genes unpredictable. Expression of
randomly introduced non-homologous transgenes is also unpredictable because of the so
called position effect. Random insertion of transgenes is also mutagenic for the recipient
cells. Problems caused by random integration of the transforming sequences could be avoided
by homologous gene targeting (HGT). The main objective of this project, therefore, was to
examine the feasibility of using Agrobacterium-mediated gene transfer (AMGT) for HGT
in plants.
CONCISE SUMMARY OF OUR PROGRESS
1. Identification and preliminary characterization of AMGT-related mutagenesis, which
seems to be the result of a process analogous to bacterial error prone repair.
2. Discovery of frequent, long T-DNA transfer, which can be utilized for introducing
extreme long T-DNA-s required for the multiple negative selection based HGT.
3. Development of the Arabidopsis tissue culture and cell genetic background for molecular
biology such as totipotent sustained root cultures, protoplast cultures and facile
transformation.
4. Completion of the core Nia2 homologous gene targeting vectors.
5. AMGT experiments using vector pMXY16 yielded a large number of transgenic plants
which are being analyzed. Ectopic introduction of the Nia2 sequences led us to
recognize a system where full gene silencing can be obtained in nearly all of the
transformants.
6. Factors involved in gene silencing can be identified in this easy test system. 5' and 3'
truncations of the Nia2 gene and inversion of the nested selectable marker in plasmid
pMXY16 have been done to test the effect of modified transcription and mRNA
stability.
7. New negative selection markers and their characterization at the level of transformation.
a. Domestication of diphtheria toxin A gene by seed-specific expression under control
of the vicilin promoter, and identification of a transient inhibitory effect on
transformation.
b. Development of the HSVtk conditional lethal negative selection marker for
transformation.
c. Development of Pseudomonas and Rhodococcus aromatic oxidases for negative
selection.
MAS TEA
DISTRIBUTION OF THIS DOCUMENT IS UNLJMITO
PcC-

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Marton, L. Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995, report, February 1, 1996; United States. (https://digital.library.unt.edu/ark:/67531/metadc668270/m1/1/ocr/: accessed April 21, 2019), University of North Texas Libraries, Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.

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