Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995 Page: 3 of 4
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the manifestation of other recessive chlorate resistance mutations, i.e., those not affecting the
NIA2 gene. The other known recessive chlorate resistance mutations are either on different
chromosomes (chI2 and cnx) or do not show high linkage (chll on chromosome 1; Doddema
et al. 1978; Koorneef et al. 1983; Koorneef 1990 - NIA] gene, Cheng et al. 1988). In
addition to the NIA2 gene, the RLD genotype provided the extremely efficient regeneration
and transformation competence. Therefore, the transfer of the NIA2 deletion into RLD
background is in progress. A final cross with Columbia WT gives the hemizygote. G5RS is
being maintained as a sustained root culture which can be transformed directly (Czako et al.
1993) or it can be first converted into a totipotent cell suspension culture (Mathur et al.
1995), which gives transformants at very high frequency, up to 80% of plated colonies
(Columbia ecotype, Wenck, unpublished).
The root culture is fully totipotent and genetically stable in long term culture and/or
storage). These tools are all very important prerequisites for a homologous gene targeting
recipient system utilizing a heterozygous plant. In addition, the embryogenic green mass
regenerating from these root cultures proved to be a high yield source of totipotent
protoplast cultures. More than 60% of the protoclones regenerated multiple shoots (Wenck
and Marton, 1995).
4. Construction of homologous gene replacement vectors and transformation of the NIA2
The core gene targeting vector, pMXY-16 (Fig. 2, in appendix) was constructed from the 5
kb SacI fragment of the Arabidopsis NIA2 gene (pAt60, Crawford et al. 1989) inserted into
pGA768 (pGA768, G. An, unpublished). The kanamycin resistance gene cassette (nptll, from
pGA472, An et al. 1986) was inserted into the unique XhoI site 100 bp downstream of the
NIA2 translation start site. The resulting construct, pMXY-16, contained a 2 kb NIA2
fragment near the right border of T-DNA and a 3 kb NIA2 fragment near the left border.
The TATA box and the translation start codon as well as the introns are shown in the map.
Transcription of nptll is in reverse orientation to NIA2 (Fig. 2). Plasmid pMXY-18, a control
vector, contained only nptll between the T-region borders.
The ectopic introduction of NIA2 sequences (by the pMXY-16 vector) caused
extremely frequent silencing of the NIA2 gene copy in the transgenic plants (Table 1,
appendix). More interestingly, RLD cultures, where the ectopic copy of NIA2 interacts with
the (normally at least) two cognate copies, produced only about 3% NR deficient mutants
among the kanamycin resistant transgenic plants. This mild cosuppression is in contrast with
the extreme, nearly complete (91%) cosuppression observed in the hemizygotes indicating
that it is related to the relative copy numbers of the incoming and cognate genes and to the
gene replacement construct itself. Identification of the NIA2 regions responsible for this
silencing is part of another ongoing project.
5. Negative selection using chimeric marker genes and their incorporation into the
homologous gene targeting vector
In order to circumvent the need for selection for chlorate resistance, which is of dubious
value due to the frequent silencing, we are going to capitalize on multiple negative selection
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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. (digital.library.unt.edu/ark:/67531/metadc668270/m1/3/: accessed November 14, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.