SummaryHvGAMYB is a transcription factor that was first identified in barley aleurone cells and shown to be upregulated by gibberellin (GA). Using RNA and immunoblot analysis we have shown HvGAMYB is also strongly expressed in barley anthers. Transgenic barley expressing a HvGAMYB:GFP fusion gene have been created and GAMYB expression in anthers analysed. GFP expression was clearly visible during early anther development in the nuclei of the epidermis, endothecium, middle layer and tapetum. Expression in the epidermis and endothecium persists until just prior to anther dehiscence, expression in the other two cell layers is visible until they are compressed and broken down as the microspores develop. Further evidence of a role for HvGAMYB in anther development was provided by the creation of transgenic barley overexpressing the HvGAMYB gene. Associated with the increase in HvGAMYB levels was a progressive decrease in anther size, particularly a decrease in anther length. Anthers also became increasingly lighter in colour. Anthers with fourfold more HvGAMYB protein than non-transgenic controls failed to dehisce and were male sterile, anthers with approximately three to fourfold endogenous GAMYB protein levels were smaller and paler but still shed normally. To investigate the hormonal regulation of HvGAMYB expression in anthers, HvGAMYB and SLN1 protein levels in anthers were analysed following application of GA 3 . As in cereal aleurone, HvGAMYB levels were found to increase and SLN1 levels decrease following GA 3 application suggesting a similar GA-signalling pathway to that in aleurone exists in anthers.
Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyte Acremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700-800 transformants/micrograms DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The beta-glucuronidase (GUS) gene, uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid and by enzyme assays of mycelial extracts. Several hph- and uidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass.(ABSTRACT TRUNCATED AT 250 WORDS)
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