Cytokinin-Regulated Carpel Regeneration In Arabidopsis

Cytokinin influences numerous aspects of plant development and plant physiology,including seed germination, etiolated seedlings turning green, chloroplasts specializing, theapical dominance, plant pathogen interactions, flower and fruit development and leaf aging.At the present, reproductive organs, such as petals, sepals, stamens, pistils and ovules, can beinduced in vitro by exogenous hormones using explants from wheat, rice, Dracae nafragranscv, Hyacinthus orientalis and so on. As a model plant, the regeneration of carpels inArabidopsis has not been reported.AG was firstly cloned as a flower development regulating gene. It encodes a transcriptionfactor, regulating the development of the stamen and carpel primordium. In dracaenafragrans cv and Hyacinthus orientalis, auxin and cytokinin regulate the development offlower organs by regulating floral organ identity genes. In this study, we explored themechanism of the carpel regeneration regulated by plant hormones. The main results are asfollows:We incubated pistil explants of Arabidopsis on callus-induction medium (CIM) with ahigh auxin/cytokinin ratio for callus induction. Then we transferred the callus ontoinflorescence-induction medium (IIM) with2.0mg/L cytokinin for inflorecence regeneration.When the concentration of cytokinin in the medium increased to20mg/L, carpel-likestructures formed at the position of flowerlet on the regenerated inflorescence stem. Weperformed qRT-PCR to detect the expression levels of relevant genes during both carpelregeneration and inflorecence regeneration process. As a result, the expression levels ofWUSCHEL (WUS) and AGAMOUS (AG) were found to be increased significantly at4days,while other genes, such as TERMINAL FLOWER1(TFL1) and CLAVATA3(CLV3), remainingthe same. Though WUS expression was induced by the high concentration of cytokinin, itcannot be detected in the carpel primordium during carpel regeneration, which indicates thatcytokinin could induce AG expression and carpel regeneration directly without WUSexpression. In order to investigate the mechanism of cytokinin regulating carpel regeneration withoutWUS expression, we analyzed the carpel regeneration frequency in the condition of WUS andAG knocked down and in the ARABIDOPSIS RESPONSE REGULATOR (ARR) mutantsarr1arr10arr11, arr1arr10arr12, and found that the expression level of AG wassignificantly decreased in antisense AG transgenic plants and arr1arr10arr11, arr1arr10arr12mutants, but only moderately decreased in antisense WUS transgenic plants comparedwith that of wild type. The carpel regeneration frequency corresponds to AG expression level.And in antisense WUS transgenic plants, the domain of cytokinin signal activity wasconsistent with the location of AG expression.Taken together, we demonstrate that high level of cytokinin could induce AG expressionthrough its signal transduction factors ARRs and resulted in carpel regeneration by inhibitingWUS expression.