Arabidopsis callus grafts: a system to study symplasmic intercellular macromolecular transport and gene silencing spread.

In the present study, we present callus grafting, comprising a method for reproducibly generating tissue chimeras from callus cultures of Arabidopsis thaliana. In this way, callus cultures of different genetic backgrounds may be co-cultivated such that cell-to-cell connectivity is achieved as a chimeric tissue is formed. To track intercellular connectivity and transport between non-clonal callus cells, we used transgenic lines expressing fluorescently tagged mobile and non-mobile fusion constructs. Using fluorescently-labelled reporter lines that label plasmodesmata, we show that secondary complex plasmodesmata are present at the cell walls of connected cells. We use this system to investigate cell-to-cell transport across the callus graft junction and show that different proteins and RNAs are mobile between non-clonal callus cells. Finally, we take advantage of the callus culture system to probe intercellular connectivity of grafted leaf and root calli and the effect of different light regimes of cell-to-cell transport. Taking advantage of the ability of callus to be cultivated in the complete absence of light, we show that the rate of silencing spread is significantly decreased in chimeric calli cultivated in total darkness. We propose that callus grafting is a fast and reliable method for analysing the capacity of a macromolecule to be exchanged between cells independent of the vasculature.

An Arabidopsis Callus Grafting Method to Test Cell-to-Cell Mobility of Proteins.

A callus is a semi-disorganized tissue that can be induced to develop from diverse tissues by the addition of exogenous hormones. The fast growth and ease of propagation have made callus cultures useful for creating a wide variety of different experimental systems.Here, we describe a detailed and simple procedure by which different, non-clonal calli from transgenic and wild-type A. thaliana plants can be co-cultured such that they form symplasmic connections via plasmodesmata (PD). We show that callus cultures can be used to study both PD formation and transport of macromolecules between non-clonal cells via PD in a tissue lacking a vasculature. Further, we include a simple protocol for a method by which calli can be sectioned to image cells and PD by confocal laser scanning microscopy.