In field conditions, the top part of the root system will grow in minimal light and the lower part will even develop in darkness, while the shoot can be exposed to various different light conditions (Smith, 1982). However, under traditional agar-plate culture system (TPG, traditional plant-growing) for Arabidopsis plants, the root system grown in the presence of light and sucrose-added medium (Xu,2013). Light affects root organogenesis (growth rate and LR production), orientation and pigmentation (Usami et al., 2004; Sassi et al., 2012; Moni et al., 2015). Root biology studies have exposed roots to light without evaluating the additional effect that light might have on them, direct perception of light by the root can be considered a non-natural condition (Silva-Navas et al., 2015).Although hydroponic-cultured or soil-cultured Arabidopsis root are grown in darkness without sucrose addition, it is inconvenient to investigate root growth and response in situ, which can be facilitated in agar-plate system (TPG) (Xu et al., 2013). For all of these reasons, studies with growing system closer to the natural one for assaying plants are essential, both in the physiological and molecular levels in higher plant.
Several solutions to these undesirable lab conditions have been postulated, keeping living roots in darkness and bring them into the light only when necessary, including dark agar plugs (Sassi et al., 2012) and the D-root system consisting of plate inserts plus cover slips (Silva-Navas et al., 2015). It should be highlighted that sucrose is added into the agar medium in these systems so that sucrose additional effect is not taken into account. Although the D-root system undoubtedly allows root to grow in the dark, it has limitations. First, the method often requires a second methacrylate comb, which adds to the time of the protocol. Second, sucrose is added into the agar medium, it is not closer to the natural one for assaying Arabidopsis plants since roots are plant organs that typically lie below the soil without sucrose addition. Third, the method also requires a auxiliary box, which increases the cost of the device.
Here we describe a simple and effective step-by-step protocol for growing the A. thaliana root in an improved agar-plate culture system (IPG, improved plant-growing), the shoot is grown outside of plates to receive light for photosynthesis ,but plates (the roots inside the in vitro plate) is black to protect the root away from light and without sucrose addition. This improved method allows to study the root growth and responses of Arabidopsis plants under the most approximate natural growing conditions.