Age, wound size,and position of injury -dependent vascular regeneration assay in growing leaves

Abstract

Recurring damage to the aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assaysforaerial plant parts such as the stem and inflorescence stalk are well established, those forleaf vasculature remain unexplored. Recently,we established a new vascular regeneration assay in growing leavesand discovered the underlying molecular mechanism. Here,we describe the detailed stepwise method forthe incision and regeneration assayused tostudy leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy,and other experimental approaches, we further show that the age of the leaf as well as the position and size of the injury determine the overall success rate of regeneration. This easy-to-master vascular regeneration assay is an efficient and rapid methodto study the mechanism of vascular regeneration in growing leaves. The assay can be readily combined with cellular and molecular biology techniques.