Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism
During selection for transformed tissue, in vitro-cultured barley material rapidly loses regenerability or gives rise to albino plants, and this has caused difficulty in developing successful transformation technologies for important North American barley cultivars. Callus from three spring cultivars, Golden Promise (GP), Galena (GL), and Harrington (HT), was initiated from immature scutellar tissue and grown on callus-induction medium containing 2.5 mg/l of the auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), 0.01 or 0.1 mg/l of the cytokinin, 6-benzylaminopurine (BAP), and 5.0 μM cupric sulfate. The addition of BAP and copper, compared to auxin alone, resulted in shinier, more compact and slightly brown-colored callus, which was more regenerable. When the highly regenerable structures were exposed to dim light and maintained on 0.1 mg/l BAP, they could be cultured for more than a year without a marked loss in regenerability or evidence of albinism. When GP tissues were initiated on auxin alone (2,4-d or dicamba) and transferred to 2,4-d, BAP and copper, as an intermediate step before regeneration, green shoot production increased 2.4 to 11.4 times for both transgenic and nontransgenic calli. Similar increases were found for nontransgenic GL and HT. This increase in regenerability, likely due to a change in the developmental state of the cultures, along with other changes in the transformation protocol, resulted in successful transformation of the previously recalcitrant GL and HT cultivars.