Annual Progress Report on Malting Barley Research March, 2002

53
We have available the sugarcane badnavirus (ScBV) promoter which exhibits constitutive
expression in oat (Tzafrir et al., 1998). We obtained a plant transformation plasmid with
the ScBV promoter fused to the ß-glucuronidase (GUS) gene.
All of the plant transformation plasmids described above were obtained from either Drs.
Ann Blechl (USDA-ARS) or Richard Zeyen (University of Minnesota).
1.1.3 Selectable markers and reporter gene plasmids
pAHC25 contains the maize ubiquitin promoter driving the expression of the bar and
GUS genes. The pAHC20 plasmid contains the maize ubiquitin promoter driving
expression of the bar gene. Expression of bar in plant cells confers resistance to the
herbicide bialaphos. Expression of GUS confers a blue precipitate upon incubating the
tissue in X-Gluc (Jefferson et. al., 1987).
1.1.4 Explant sources
Barley spikes are harvested approximately 12-14 days after anthesis. Developing kernels
are sterilized according to Tingay et al., 1997. Immature embryos are excised from the
kernels and placed scutellum-side up on callus induction media. A few days later, the
root and shoot axis is removed from the embryo and the embryo halved, the two halves
being placed on fresh callus induction media. The embryo pieces are then left in the dark
at 24°C for approximately two weeks.
1.1.5 Biolistic transformation
Small callus pieces are placed on osmoticum media overnight in the dark at 24°C before
being bombarded with gold-coated DNA according to Wan and Lemaux (1994). All
bombardments contain a plasmid carrying the maize ubiquitin promoter driving the
expression of an AFP gene as well as the pAHC25 plasmid.
1.1.6 Selection and regeneration of transgenic plants
We have modified a method for selection and regeneration of transgenic barley
developed by L. Dahleen, USDA-ARS. After particle bombardment, the callus pieces are
transferred to callus induction media supplemented with 5 mg/l bialaphos. Selection of
embryogenic callus takes six weeks and takes place in the dark at 24°C. Callus pieces are
then placed on maintenance media for 4 weeks, supplemented with 5 mg/l bialaphos, and
placed at 24°C under dim light for 16 h/day. To regenerate plantlets, resistant
embryogenic callus are transferred to regeneration media supplemented with 5 mg/l
bialaphos, and incubated at 24°C under bright lights (16 h/day). This process takes 4
weeks. To induce rooting, plantlets will be placed on rooting media with 3 mg/l
bialaphos. After roots develop, plantlets will be transferred to soil and grown to maturity.
1.1.7 Characterization of transgenic plants
GUS activity will be monitored histochemically with X-Gluc substrate as described in
Jefferson et al., 1987. Antifungal gene expression will then be determined by RT-PCR.
Antifungal protein expression will be determined by Western blotting using our affinity
purified polyclonal antibodies. Further molecular characterization of the transgenic
plants will be conducted by RNA and DNA gel blot analysis. Expression analysis of
AFP genes will be conducted in the T0, T1 and T2 generations.