TITLE:
A Versatile Liquid Culture Method to Control the in Vitro Development of Shoot and Root Apical Meristems of Bamboo Plants
AUTHORS:
Most Tanziman Ara, Taiji Nomura, Yasuo Kato, Shinjiro Ogita
KEYWORDS:
Bamboo, Digital Imaging, Node Culture, RAM, SAM, SLCE
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.11 No.2,
February
28,
2020
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.