A urea-type cytokinin, 2-Cl-PBU, stimulates adventitious bud formation of Eucalyptus urophylla by repressing transcription of rboh1 gene

An efficient regeneration protocol is the prerequisite of genetic modification of eucalyptus. However, adventitious bud differentiation from calli of eucalyptus is difficult because of rapid callus browning. By comparing three cytokinins in tissue culture of Eucalyptus urophylla, it was found that N-phenyl-N'-[6-(2-chlorobenzothiazol)-yl] urea (2-Cl-PBU), a home-made urea-type cytokinin, induced more green callus than N-phenyl-N'-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ) or 6-benzylaminopurine (BAP). Only green calli were regarded as organogenic calli because they were the only one capable of differentiating adventitious buds in subsequent procedures. Six physiological indexes of green calli, including the contents of hydrogen peroxide, superoxide anion radicals, cellulose and lignin, and the activities of peroxidase and superoxide dismutase, were higher than that of white calli. Furthermore, the transcription levels of four respiratory burst oxidase homolog (rboh) genes declined significantly in organogenic green calli, especially that of EgrRboh01 (rboh1). Among the three treatments by 2-Cl-PBU, BAP and a combination of 2-Cl-PBU and BAP, the primary callus induced by BAP showed the highest rboh1 transcription level, 12.7-20 times higher than that by 2-Cl-PBU or by the combination of BAP and 2-Cl-PBU. The lowest rboh1 transcription level was observed in the calli induced by 2-Cl-PBU. The results indicated that 2-Cl-PBU restrained callus browning and promoted adventitious bud differentiation of E. urophylla by repressing the transcription of rboh1 gene.