A Versatile Liquid Culture Method to Control the <i>in Vitro</i> Development of Shoot and Root Apical Meristems of Bamboo Plants

Abstract

We focus on controlling morphological and histochemical responses of the shoot apical meristem (SAM) and root apical meristem (RAM) of bamboo node by using a simple and versatile liquid culture system. First, nodes of 11 different bamboo species that belong to seven major bamboo genera (Bambusa, Dendrocalamus, Phyllostachys, Tetragonocalamus, Chimonobambusa, Pleioblastus, and Sasa) were cultured using 2 mL per well of a liquid medium in a 6-well microplate to form a small-scale liquid culture environment (SLCE). The dormant lateral buds of all bamboo nodes resumed expanding and elongating within 7 days in the SLCE. The dormant and active lateral buds were sectioned longitudinally and stained with Sytox green (SG) to monitor mitotic activity and counterstained with safranin (SF) to detect the inward region of the SAM region. Further, mitotic activity was calculated using a digital imaging analysis, which showed an increase of up to 1.2- to 3.8-fold in terms of the SG/SF ratio after 7 days in the culture. Moreover, we used in vitro node cultures of two typical bamboo species, the sympodial clump-forming type (Bambusa multiplex Raeush, Bm) and the monopodial single culm-forming type (Phyllostachys meyeri McClure, Pm), and noted the following: 1) since gradual white-to-green tinge shoots were observed, we investigated the relation between color variation in the outer regions of culm and node tissues and their suitability as explants. By checking the autofluorescence property of whole shoots under LED 365 nm illumination with an RGB (red, green, and blue) digital imaging analysis using ImageJ software, we specified the color variation of explants as the relative intensity of the blue value. 2) Since the obtained shoots of a 1-month-old culture box showed growth variation, we distinguished shoot types based on plant height, i.e., short (less than 5 cm), medium (ca. 5 - 10 cm), and tall (more than 10 cm). Tall shoots that have ca. 5 nodes on average were suitable for explant. 3) Three types of node portions—the first node (the base node near a rhizome tissue), middle nodes (upper nodes near the 1st node), and the top meristem—were independently cultured in the SLCE, and it was found that the first node showed the best growth performance. 4) By culturing the first node in the SLCE system, we performed a quick survey during the 3 weeks in the culture and found that a combination of 10 μM benzyl adenine and 3 μM thidiazuron was effective for in vitro SAM development, while the addition of 2, 4-D was effective for promoting in vitro RAM development. 5) The detailed autofluorescence properties of the outer regions of culm and node tissues were also identified using an inverted fluorescent microscope under B- and U-excitation lights with RGB and HSB (hue, saturation, and brightness) digital imaging analysis.