Background

Monocot stems have a characteristic vascular network in which veins remain separate and independent. Despite its importance to supporting growth and development, the pattern of vascular networks in monocot stems has been poorly studied. Visualization of stem vascular networks is quite challenging because veins are deeply embedded in tissues. Conventional tissue sectioning can be applied to observe the networks, however, it is a laborious and time-consuming process which requires observation of hundreds of sections. In addition, the size of crop stems, which is much larger than the field of view under the microscopes, makes it difficult to capture the whole system.

Recently, we reported that maize transcription factor BEL1-like homeobox (BLH) 12 and BLH14 play important roles in the stem development and vein network formation (Tsuda et al., 2017). To obtain a comprehensive view of the vascular systems, we adopted the micro-CT scanning described previously and optimized it for maize stems (Metscher, 2009a and 2009b, Degenhardt et al., 2010, Staedler et al., 2013, Gignac et al., 2016). By combining this method with image analyses, we were able to reconstruct 3D models of inner stem structures in an efficient and reliable manner. This protocol can be used to visualize inner structures such as veins in various species and tissues.