Annual Progress Report on Malting Barley Research March, 2002

52
there is a question about whether there is sufficient genetic variation for FHB resistance
in these programs.
To increase FHB resistance in barley, my laboratory is developing transgenic barley
carrying antifungal protein (AFP) genes. Our long-term goal is to develop a large set of
transgenic barley lines carrying a diverse array of AFP genes.
This novel germplasm will be screened for resistance to scab as well as against other
fungal pathogens in the glasshouse. However, glasshouse screening of transgenic barley
lines takes some time, with screening typically not occurring before T2 generation plants.
Therefore, we aim to develop in vitro assays which will allow us to determine whether or
not the desired antifungal effect is likely to be produced by our transgenic barley lines in
the glasshouse and field.
There are a limited number of useful promoter sequences for barley transformation.
Therefore, there is a need to develop and test new promoter sequences for use in barley
transformation. The sugarcane badnavirus (ScBV) promoter exhibits a high level of
constitutive expression in oat (Tzafrir et al., 1998). We have continued to investigate the
usefulness of the sugarcane badnavirus promoter in barley.
1. PLANT TRANSFORMATION AND CHARACTERIZATION
1.1 Materials and Methods
1.1.1 Plant material
Previously, we were using the cultivar Golden Promise in our transformation system and
were able to produce several lines of barley over-expressing a barley RIP gene.
However, at the beginning of this funding period, we decided to transform the cultivar,
Conlon. Conlon exhibits good agronomic and malting quality characteristics. Also,
screening of Golden Promise against FHB was known to be notoriously difficult. Thus,
we felt our efforts were better concentrated on developing a transformation system in our
laboratory for the variety Conlon. Consequently, our efforts have largely invested in this
area and our previously produced lines of Golden Promise carrying RIP have received
little attention.
Conlon stock plants are grown in growth chambers at 20°C under a 16-h light period and
at 18°C for an 8-h dark period.
1.1.2. Available antifungal protein genes and promoters
Currently, we have the following antifungal protein genes available: alfalfa acidic ß-1,3glucanase,
barley chitinase, Arabidopsis thaliana PR5, rice chitinase, oat thaumatin-like
protein 1, oat thaumatin-like protein 4, wheat α-thionin, wheat thaumatin-like protein 1,
barley Type I ribosome inactivating protein (RIP) gene, and the barley class-II ß-1,3glucanase
gene. In general, the mode of action for all of these AFPs is to degrade or
disrupt either the cell wall or membrane of F. graminearum. The exception is the RIP
gene, which encodes a protein that inhibits fungal protein synthesis. We are using the
constitutive promoter from the maize ubiquitin gene to drive expression of the AFP
genes.