Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific beta-glucuronidase gene-(uidA; gus) expression driven by B-1- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bnl. PCR analysis using DNA from calli of 22 different lines transformed with B-1- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from To leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T-1 progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15. 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T-1 progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T-5 progeny in one line, T-4 progeny in one line, T-3 progeny in three lines and T-2 or T-1 progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B-1- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T-1 progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T-2 or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T-2 or later generations. We conclude that the B-1- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering.