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New CAMBIA vectors for Plant Transformation

C. S. Prakash Prakash at Acd.Tusk.Edu
Fri Oct 31 16:26:32 EST 1997

>From November ISB News Report (http://www.nbiap.vt.edu)

Plasmid vectors are essential for developing transgenic plants. These
tiny DNA circles are the unsung workhorses of biotechnology, helping
scientists shuttle genes into plant cells that are then regenerated
into transgenic plants. Current plasmid vectors have many limitations,
and thus scientists at the Center for the Application of Molecular
Biology to International Agriculture (CAMBIA) in Canberra, Australia
have developed improved vectors which they are making freely
available to academic researchers. Richard Jefferson, who is the head
of CAMBIA, says that the new CAMBIA vectors are highly efficient and
useful in developing transgenic plants and are extremely versatile for
plant molecular biology research.

Jefferson started CAMBIA to develop "enabling technologies" of
relevance to international agriculture. The GUS gene system, which he
developed in the mid-eighties at Cambridge and made widely available to
researchers even before publication, has made a tremendous impact on
plant molecular biology. Transformed plant cells expressing the GUS
gene appear dark blue in color when stained with a substrate and thus
can be easily identified.

The new upgraded CAMBIA vectors consist of 18 plasmids and have several
modular features: choice of selection in plants (hygromycin or
phosphinothricin resistance), choice of selection in bacteria
(chloramphenicol or kanamycin resistance), multiple cloning sites close
to the right border (ensuring no loss of the introduced gene),
blue/white screening in E. coli, presence of an intron sequence (to
suppress GUS expression in the bacteria), high copy number plasmid
replication, Kozak consensus sequence for improved expression, minimal
extraneous DNA sequences (such as spurious AG or terminator sequences)
and stability of plasmids under non-selection conditions.

Further, a protein of interest can be fused to the GUS protein and the
new protein purified with a single step because of the hexahistidine
tail engineered at the carboxyl end of GUS.  A promoter-less vector and
a reporter gene without start codon are also available. All vectors
have been tested successfully with tobacco and rice using particle
bombardment and Agrobacterium transformation methodologies.

Recently green fluorescent protein (GFP) from jellyfish has emerged as
a viable alternative to the GUS gene, as GFP could be visualized in
plant cells non-destructively. Jefferson cautions, however, that GFP
has several caveats such as its unsuitability for field use,
stochiometric detection which limits sensitivity, need for use of
expensive microscopy and the photo toxicity of the protein. Nonetheless,
he is also offering a codon-modified GFP (developed by Jim
Hasseloff of UK) in the CAMBIA vector along with GUS-GFP (or GFP-GUS)
gene fusions, thus providing scientists with a single system with
features of both reporter genes.

The original GUS gene was isolated from E. coli and has certain
drawbacks. To improve the plasmid vector system further, CAMBIA
scientists have cloned an improved GUS gene from another newly
described bacteria, Bacillus oz, and call this gene "BoGUS". The BoGUS
gene is a major improvement because it is secretory in nature. It is
also more robust with very high thermal stability, according to
Jefferson. BoGUS is not inhibited by its end products or detergents and
thus is active even in high concentrations of salt and organic
solvents. This gene is also being further improved through codon

Jefferson visualizes that the new BoGUS will facilitate a
non-destructive and non-disruptive assay of gene expression and will
facilitate the development of transgenic plants without antibiotic
selection or tissue culture. The new gene will require cheaper
substrates and may have other applications such as targeted cell death
to cause male sterility through special substrates. The new BoGUS gene
will be made available to researchers soon.

Academic researchers can obtain the CAMBIA vectors at no cost while
commercial institutions are required to license them. For more
information, send email to: cambia at cambia.org.au.  Richard Jefferson
can be reached at raj at cambia.org.au

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash at acd.tusk.edu

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C. S. Prakash, Ph. D.                      Prakash at acd.tusk.edu
Center for Plant Biotechnology Research    Ph: (334) 727 8023
College of Agricultural, Env and Nat Sci.  Fax:(334) 727 8067
Tuskegee University                        http://agriculture.tusk.edu/
Tuskegee, AL 36088, USA
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