Annual Progress Report on Malting Barley Research March, 2002

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1.2 Results
Most of this year was spent establishing a transformation protocol for Conlon in our
laboratory. So far, 1500, 1105 and 40 callus pieces have been bombarded with DNA
encoding tlp-1, chitinase and glucanase. Numbers of regenerating callus pieces showing
the presence of green shoots are shown in Table 1. Many of these plantlets are due to be
moved onto rooting media on March 7 th 2003. Thus, we are very close to having plants
in soil and beginning the characterization work. Particle bombardments are continually
on-going in our laboratory.
Table 1: Conlon transformation tissue culture
Gene Total number of
regenerating plantlets
Number of unique
regenerating events
Bombarded callus pieces
not yet at regeneration
stage
Tlp-1 164 101 n/a
Chitinase 131 123 675
Glucanase n/a n/a 40
2. ANTIBODY PRODUCTION
Polyclonal antibodies were produced by Quality Controlled Biochemicals, Hopkinton,
MA. From the translated sequences of our barley glucanase and barley RIP genes, we
were able to choose one peptide sequence which was then synthesized, purified by HPLC
and its sequence verified by mass spectrometry. Each peptide was then conjugated to a
carrier protein and used to immunize two rabbits. For each animal, a pre-immune bleed
was taken, followed by four production bleeds at various intervals after a total of five
immunizations.
For our barley chitinase and our wheat tlp-1 proteins, two peptides were synthesized for
each antibody produced and used together for the immunization of each of the two
animals. The proteins were less immunogenic and thus, two peptides were chosen to
increase the likelihood that the animals would produce antibodies.
Each bleed was tested in our laboratory against the purified peptide and detection of the
protein by the antibodies was successful. The bleeds were then affinity purified to
produce purified polyclonal antibodies.
These antibodies will be used to detect the corresponding proteins in the transgenic
barley plants.
3. IN VITRO FUNGAL GROWTH ASSAYS
3.1 Materials and Methods
We have tried several different methods in an attempt to develop an in vitro assay which
would allow us to test whether or not a transgenic plant was likely to produce an
antifungal effect in glasshouse screening of F. graminearum.
Firstly, we tried to infect plant leaves floating on water in a Petri dish, by placing drops
of F. graminearum spore suspensions on the leaf surfaces. However, we could not get
reproducible fungal hyphae growth on plant leaves.