Analysis Of Hormone-regulated Regenerated Inflorescence Development In Arabidopsis

Various signals can be involved in the transition from vegetative growth to reproductive growth in plant. Plant hormones play important roles in this process. To study the developmental mechanism of hormone-regulated regenerated inflorescence, we established a regeneration system of Arabidopsis inflorescence induced by cytokinin and auxin in vitro. In this regeneration system, pistil was used as explant to induce the formation of callus,and then the callus was transferred to another kind of media for inflorescence induction. The frequency of inflorescence regeneration was about 60%.Semi-quantitative RT-PCR analysis was performed to determine the expression patterns of genes involved in inflorescence development. The results showed that the expression of AP1, AP3, AG, TFL1, LFY, WUS and CLV3 was induced after callus was transferred to the media for inflorescence induction. In contrast, the expression of AP2 and TFL2 was constitutive during inflorescence regeneration.The expression patterns of TFL1 and LFY during inflorescence regeneration were analyzed by using pLFY::GUS and pTFL1::GUS transgenic plants. We found that TFL1 expression mainly located in the cells with inflorescence meristem identity and in the mature regenerated inflorescence. And the expression of LFY was found in the callus cells differentiating to floral primordium and in the mature regenerated inflorescence.CLV3 and WUS genes were involved in regulating inflorescence development in plants, so the expression patterns of CLV3 and WUS during the regeneration of inflorescence were determined. The results indicated that WUS was firstly expressed in a group of cells located in the inner of callus, named it organization center, and then, the expression of CLV3 was detected in the cells of layer 1-3 above the organization center. With the development of inflorescence primordium, the expression of CLV3 and WUS was focused in the inflorescence meristem. After the expression of WUS was inhibited by anti-gene strategy, the regeneration of inflorescence was reduced obviously. Thus, these results suggest that the expression of WUS, earlier than that of CLV3, is essential for the inflorescence formation.The generation of stem cell was inhibited after the polar transportation of auxin was interrupted by adding NPA, an inhibitor of auxin polar transportation, in media. And the frequency of inflorescences formation was reduced significantly after adding NPA. These results suggest that auxin polar transportation is very important for the generation of stem cell and inflorescence formation in vitro.These results indicate that inflorescence and floral meristem identity genes are induced during the regeneration of inflorescence. Further study indicates that organizing center cell and stem cell formation is necessary for the regenerated inflorescence development.