Transformation of barley (Hordeum vulgare) using the wheat puroindoline gene

Abstract

Feed and malting barley are major crops in Montana. Harrington, a two-rowed spring malting barley variety, is the most cultivated variety in the state. Barley was the last of the world's major cereals for which transformation methods were developed because in vitro-cultured barley rapidly loses regeneration ability or gives rise to albino plants during selection for transformed tissue. Previous research used the variety Golden Promise because it regenerates well under research conditions, though it is not commercially used. Transformation could be an important method to improve varieties in North American barley cultivars. The puroindoline proteins (PINA and PINB) can be isolated from wheat endosperm and are basic cysteine and tryptophan-rich proteins that might play a role in defense against pathogens. The puroindolines show antifungal activity both in vitro and in vivo. Milk stage seeds were harvested to obtain embryos. Embryos were placed on induction media [2.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.01 mg/L 6-benzylaminopurine (BAP)] for 12 to 51days until calli formed. The biolistic method was chosen for transmitting the gene of interest into the calli, using a Biolistic PDS-1000/He Particle Delivery System (BioRad, Hercules, CA) with 900 psi rupture disks. Approximately 4,500 calli were bombarded with two plasmids; pinA driven by the maize ubiquitin promoter (Ubi1) which is expressed constitutively, and the hygromycin phosphotransferase (hph), which confers hygromycin B resistance and is driven by the CaMV 35S promoter for selection. The bombarded calli were selected on medium containing 30 mg/L Hygromycin B for 9 to 21 days, and subcultured to intermediate medium (1.0mg/L 2,4-D and 0.5 mg/L BAP), followed by regeneration media (1.0-3.0 mg/L BAP). Thirty hygromycin B resistant calli regenerated and 5 died in magenta box. Thirty five putative transgenic plants derived from 25 calli grew in soil and were harvested. Some plants tested by polymerase chain reaction (PCR) tested positive with primers for pinA and hph. Southern blots were performed to detect the presence of hph in T1 barley genomic DNA but were negative. Northern blots performed to detect the presence of pinA and hph transcribed RNA in the T1leaf tissue were also negative, showing that no stably transformed plants were obtained.