Background

Due to their sessile nature, plants are frequently subjected to injuries caused by biotic and abiotic factors. These injuries, when left unattended, can compromise plant immunity, growth, and even survival (Hwang et al., 2017; Radhakrishnan et al., 2020). To overcome the adversities of wounding, plants have evolved a remarkable repertoire of regenerative responses ranging from wound healing in the form of local cell proliferation to complete replacement of amputated organs, such as root tip regeneration (Ikeuchi et al., 2016; Shanmukhan et al., 2020). Although numerous studies have probed the mechanisms underlying several regenerative responses in plants, investigations regarding the regeneration potential of aerial organs are limited (Iwase et al., 2011; Kareem et al., 2015; Durgaprasad et al., 2019). Thus, despite their higher susceptibility to injury than underground organs, there is a dearth of information on regeneration in the aerial organs of plants, particularly, the leaves. Although leaves play a crucial role in plant physiology, their regeneration potential has hardly been investigated (Kuchen et al., 2012; Radhakrishnan et al., 2020).

Leaves possess an elaborate network of vascular tissue with a central midvein that transports substances back-and-forth between the main plant body. Damage to the midvein calls for prompt repair, failing which the transport of substances, and consequently the growth of the leaf and its adjacent branches, are impaired (Sachs and Hassidim, 1996; Radhakrishnan et al., 2020). Recently, a new vascular incision assay in leaves has been developed to study wound repair and tissue restoration in response to injury. The assay revealed that the mechanically disconnected parental stands are reunited by regenerating vascular tissue that bypasses the site of injury. The assay was instrumental in understanding the molecular mechanism underlying vascular regeneration in aerial organs growing in the normal developmental context. Upon injury, a coherent feed-forward loop comprising cell fate determinants, PLETHORA (PLT) and CUP-SHAPED COTYLEDON2 (CUC2), activates local auxin biosynthesis, leading to vascular regeneration in growing aerial organs (Radhakrishnan et al., 2020). Although vascular regeneration necessitates the PLT-CUC2 regulatory axis, the extent of injury acted as a limiting factor (Radhakrishnan et al., 2020). Here, we show that the regenerative ability of leaf vasculature is determined by the size of the injury, age of the leaf explant, and position of the injury along the proximodistal axis of the leaf blade.

This easy-to-master, reproducible assay can be performed using readily available laboratory supplies. The convenience of performing real-time confocal imaging and other molecular techniques, such as quantitative real-time PCR, using the injured leaves makes the assay valuable for studying the molecular players and mechanisms regulating wound-induced response and regeneration in the normal developmental context. The method will also be useful for studying the interplay between mechanisms of vein patterning during development and that of vein regeneration.