Analysis Of Hormone-regulated In Vitro Regenerated Petal In Petunia

Floral development is the key event of plant development. One floral organ identity model (ABC model) and five genetic pathways controlling flowering time (Photoperiodic promotion pathway, Vernalization promotion pathway, Autonomous promotion pathway, GA promotion pathway, and Repression pathway) were proposed based on the studies in Arabidopsis. It was well known the GA plays important role in flowering and floral development, but roles of other hormones (e.g. auxin, cytokinin) remain unclear. Some in vitro organ-regenerated systems have been established in many plants, such as Dracaena fragrans cv, Hyacinthus orentalis L, Oryza sativa, Arabidopsis thaliana, etc. The results in these studies raised the hypothesis that floral development is controlled in concert by auxin and/or cytokinin.To investigate hormone-regulated petal development, in vitro Petal Regeneration System for Petunia (Petunia hybrida) was established. The 1/3 basal part of the petal was used as explant, and was put on the culture medium containing 0.2 mg/L 6-BA and 0.8 mg/L IAA. After hormone induced, petal-like organ regenerated from the explant directly, the majority of organs were petal-like sheets and corolla-like tubes in the abaxial surface and the others were petal-like protuberance in the adaxial surface. In order to examine the expression pattern of petal marker gene during the development of regenerated petal, we analyzed the expression of beta-glucuronidase (GUS) gene driven by the EPF1 promoter. The GUS signal was detected in the regenerated region and the mature regenerated petal of the explant. These results further confirmed that the regenerated organ was petal.Morphological observation and histological analysis indicated that hormones have regulated cell-dedifferentiation in the regenerated region of the explant. Semi-quantitative RT-PCR and mRNA in situ hybridization provided the molecular evidence that hormones enhanced the petal identity gene GREEN PETAL (GP) expression, and regulated commencing the floral meristem identity gene ABERRANT LEAF AND FLOWER (ALF) expression. Taken together, the results suggested that both GP and ALF expression are regulated by auxin and/or cytokinin. The roles of both genes during petal regeneration are different. ALF expression may indicate dedifferentiated cells own floral meristem characteristics, GP may determine the direction of cell differentiation.