Regeneration of fertile transplastomic Arabidopsis thaliana plants from leaves
摘要
In the model plant Arabidopsis thaliana L., the ability to obtain chloroplast transformants at a high frequency is dependent on transformation of spectinomycin-hypersensitive plant lines, in which the ACC2 nuclear gene was inactivated. However, when leaves were used for transformation, the regenerated plants were sterile, presumably due to the polyploid nature of the tissue. To obtain fertile plants at a high frequency, a steroid inducible BABY BOOM (BBM) gene was introduced into the nuclear genome of spectinomycin-hypersensitive Ws-acc2 Arabidopsis plants. The BBM protein, fused to a glucocorticoid receptor domain, remains in the cytoplasm until the steroid dexamethasone enables its movement into the nucleus, where the BBM transcription factor activates its target genes involved in embryogenesis. Arabidopsis Wassilewskija (Ws) acc2 plants were transformed with the steroid inducible BBM gene. Three independently obtained and non-segregating Ws-acc2-BBM lines showed consistent production of viable seed from plants regenerated from leaf tissue. When chloroplast transformation experiments were performed, sixteen spectinomycin-resistant callus cultures were obtained. After removal of dexamethasone, ten callus cultures regenerated into plants and seven produced viable seed. Molecular characterization identified one chloroplast transplastomic event; the rest were spontaneous rrn16 mutants. In contrast, bombardment of Ws-acc2 leaf tissue without the steroid inducible BBM gene yielded fourteen spectinomycin-resistant callus cultures and none of the regenerated plants produced viable seed. The newly developed Ws-acc2-BBM lines described here couple spectinomycin-hypersensitivity with improved fertility and enabled recovery of viable seed from spectinomycin-resistant plants six mo after bombardment.