Background

In plants, above-ground organs are continuously generated from stem cells in the shoot apical meristem (SAM). This process is best described in the dicot plant Arabidopsis thaliana (reviewed in Fuchs and Lohmann [1]; Holt et al. [2]; Hong and Fletcher [3]; Janocha and Lohmann [4]). The initial vegetative SAM produces only leaves but changes into a reproductive SAM after transition to flowering. A subset of cells within the SAM will eventually lead to the formation of gametes. Mature flowers and siliques are formed after bolting when the main stem emerges from the basal rosette and the SAM changes into an inflorescence meristem (IM). Following the fate of stem cells during these important transitions of the meristem is therefore of utmost importance to understand this developmental program.

Different approaches have delivered detailed information about meristem development [5–7]. However, most techniques require dissection, staining, or cell separation and are not compatible with the observation of dynamic processes. Live imaging of the SAM has overcome this limitation to some extent but is usually performed by detaching the shoot tip of a bolting plant, placing it in a microscopy-suited vessel filled with growth medium and submerging it in water, followed by removing flowers and buds before microscopy observation [8–11].

In an alternative setup, Grandjean et al. [12] recorded SAM development in intact plants using an inverted microscope, with the drawback of working against gravity. This was later overcome by Heisler et al. [13] and Tobin and Meyerowitz [14]. These authors proposed observing the SAM under physiological conditions, after removing surrounding flower buds from a plant prior to bolting without separating it from the plant body, keeping it completely underwater. However, this limited the observations to an early stage, and the complete submergence of the plant can cause hypoxia and corresponding side effects on the development [15]. These side effects could be reduced by placing only a water drop between the plant tip and the front lens of a water immersion objective [14], however, at the price of low stability and restricted time for the acquisition.

The protocol described here provides an innovative method to observe the SAM of bolting Arabidopsis plants without detaching it from the stem. An “easy-to-built” custom-made device allows the preparation of the main inflorescence of a soil-grown plant for subsequent observation with an upright confocal microscope. This system limits submergence to the very top and permits precise positioning of the exposed SAM in all dimensions. In addition, the observation of the living SAM can be extended over several days, while the plant grows in regular substrate, light, and oxygen supply. The setup allows further control and variation of environmental conditions like drought, salinity, light stress, hormone or drug application, and pathogen infections. The procedure can likely be modified to also study meristem development in species with similar size and growth architecture as Arabidopsis.