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PLANT GENETIC TRANSFORMATION, CELL SELECTION AND SOMATIC HYBRIDIZATION
AGROBACTERIUM-MEDIATEDSEEDTRANSFORMATIONOF
ARABIDOPSIS THALIANA
A.A.Tomilov, N.B.Levchenko, O.A.Ogarkova, V.A.Tarasov.
Laboratory o fGenom Variability, Vavilov Institute o fGeneral Genetics, Gubkin str. 3,
RussianAcademy ofScince, Moscow 117809, Russia. E-Mail:
Arabidopsis thaliana has many advantages as a model system fo r
studies in the molecular genetics of plant physiology and development such
as a short life cycle, small size, small genome size and well developed
classical genetics. In the perspective, collection of 50-100 thousands
independent transformants would contain enough inserts to ensure a 95%
probability that any single gene in the genome would contain enough inserts.
In planta transformation was first achieved in Arabidopsis thaliana by Feldman
and Marks, 1987. This efficient non-tissue culture method o f transformation
invo lve s in fec ting germ inating seeds o f A rabidopsis thaliana w ith
Agrobacterium tumefaciens and results in transformants that do not exhibit
somaclonal variation. The ultrasonic treatment o f seeds in water solution
containing aluminium oxid has been developed to provide the high yeld of
transformants. This allows to increase (up to 10 times) the frequency of
transformants: the yield of GUS-expressing plants amounts to 2...5% of
seeds of T2 generation. The binary vector system PLD-3 based on the modified
vector pBI-121 was used. The modification consist in inserting marker genes
fo r resistance to chloramphenicol to T-region between the genes encoding
NPT II and GUS. The presence in the T-region of the resistant genes
expressed both in plant and E.coli cells will essentially facilitates not only
genetic analysis and molecular mapping, but also cloning the mutant genes.
In the result the collection of T2 progeny of A. thaliana was created in
our laboratory. All selected plants had the antibiotic resistance. Histochemical
assay of the reporter GUS-gene expression o f the obtaind mutants was
made to select about 150 GUS-positive plant. Some of them had considerable
morphological alterations. The presence of insertions in the selected plants
nucleus genom was proved by PCR-analysis of genomic DNA using one
pair of 20bp primers for the GUS-gene within the T-DNA. The collection of
callus culture created from this transgenic plants is used for amplification of
overlapping T-DNA region of nucleus genom by means of PCR and/or cloning
o f the mutant genes in E.coli followed by sequencing of cloned genes.
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