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

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 (Bam-busa, 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), 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 1 st 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 Band U-excitation lights with RGB and HSB (hue, saturation, and brightness) digital imaging analysis.


Introduction
Bamboo plants that belong to the Poaceae family, which contains more than 1500 species, have been exploited for a range of uses, such as food, medicine, charcoal products, and housing materials, especially in Asia [1] [2]. It is well known that bamboo presents unique biological properties in its vegetative growth. A rhizome system is a key to maintain the lateral growth and form new culms with abundant nodes for longitudinal growth without secondary growth.
The system is mainly classified into two types of growth feature: one is the sympodial and the clump-forming type (e.g., Bambusa sp.), which is distributed in tropical regions, and the other is the monopodial and single culm-forming type (e.g., Phyllostachys sp.), which grows mainly in temperate and subtropical regions [3].
A tissue culture protocol of bamboo (Phyllostachys meyeri McClure, Pm) was successfully achieved by Ogita et al. (2008) specifically regarding in vitro germination of the caryopses and plant regeneration from nodal segments of both the germinated seedlings and tissue-cultured clone plants [4]. In vitro node culture stocks of (Bambusa multiplex Raeush, Bm) have also been established using the method for Pm, as previously reported. Hence, several clonal small plantlets of two major bamboo species that have a high ability to form multiple shoots were obtained in a regular period (ca. every 1 -2 months), as shown in Figure 1. The node portions of these culture stocks with apical and intercalary meristems were considered as excellent models for the morphological and histochemical response control of the shoot apical meristem (SAM) and root apical meristem (RAM) of bamboo. General tissue culture techniques for in vitro micropropagation of bamboos through enhanced axillary branching using node explants have been well reviewed, e.g. by Singh et al. (2013) [5]. Factors affecting success in micropropagation of bamboos such as medium, plant growth regulators, medium pH, carbon source, propagule size, and culture duration were summarized in this report. However, there is no detailed description of a versatile methodology to regulate in vitro growth of bamboo nodes. The aim of this study is to review a simple but versatile node culture system in a liquid culture environment to better understand unique biological properties of its vegetative growth and to, thus, reveal the relation between color variation in the outward regions of culm and node tissues and their suitability as explants.
The concept of the Plan-Do-Check-Act (PDCA) cycle in a plant tissue culture [6] is central for the present study. Generally, researchers should realistically consider the following requirements: explant selection, medium preparation, and culture environment setting in the P and D cycles. Based on a careful monitoring of size, shape, and color variation of the target cells and tissues by macroand micro-scopic observations, they can recognize the specific pattern of growth promotion and/or inhibition during the culture of the target explant. These inputs are considered highly useful and interesting for the optimization of a protocol, especially regarding the C and A cycles.
A main topic of the present study is the culture environment setting. Sood et al. (2002) tested the effects of agar-solidified medium and liquid medium on shoot multiplication, whereby root formation of Dendrocalamus hamiltonii was investigated; it was found that a liquid culture condition is more suitable for the tissue culture of this bamboo species [7]. Using node culture system, we checked the effects of solid and liquid media on the growth of bamboo node cultures of Bm and Pm, and found that better growth performance was observed in the liquid medium condition (Table 1). Based on this preliminary result, a 6-well microplate that contains 2 mL per well of a liquid medium, which provides a small-scale liquid culture environment (SLCE) for optimizing the culture protocol of the bamboo within a short period was focused in the present study.   [8]. In the previous studies, we focused on evaluating the autofluorescence intensity of target plant cells, which reflects the histochemical features of the plant cell wall [9], and accumulation of specific secondary metabolites [10]. We also used digital imaging analysis to measure the growth features in a protoplast co-culture assay [11].
Currently, in the field of life sciences, the concept and application of autofluorescence measurement have developed with the requirement of non-destructive detection of a target cell and tissue [12] [13] [14]. In the present study, the color variation of explants as the relative fluorescent intensity by assessing the autofluorescence property of the whole shoots under LED 365 nm illumination with RGB (red, green, and blue) digital imaging analysis using ImageJ software was focused. The detailed autofluorescence properties of the outward 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.

Plant Materials
Nodes of 11 different bamboo species that belong to seven major bamboo genera were cultivated using planter boxes in a greenhouse of Toyama Prefectural Uni- angulatus (Munro) Nakai (Ta); Chimonobambusa-C. marmorea (Mitford) Makino (Cm); Pleioblastus-P. simonii (Carriere) Nakai (Ps); and Sasa-S. kurilensis Makino et Shibata (Sk). Fresh nodes were collected from the branches of these bamboo plants and cultured in vitro according to a method previously reported [4]. In vitro node culture stocks of Bm and Pm maintaining at Prefectural University of Hiroshima, Japan were also used in the following experiments to understand the sequential developmental processes of SAM and RAM of node explants. Briefly, a half-strength MS (Murashige and Skoog) [15] liquid medium that contains 30 g/L sucrose was prepared as a standard medium, unless otherwise specified. The pH of the medium was adjusted to 5.7 before autoclaving. All the cultures were maintained at 25˚C, with a 16 h photoperiod under fluorescent illumination (65 μmol m −2 s −1 ).

Histochemical Analysis of Mitotic Activity in Node Portions of 11 Bamboo Species
As shown in Figure 2, the dormant lateral buds of all bamboo nodes resumed expansion and elongation within the first 7 days in the SLCE. The mitotic activity was calculated by a digital imaging analysis and found an increase of up to 1.2-to 3.8-fold in terms of the SG/SF ratio after 7 days in the culture ( Table 2).  Table 2. American Journal of Plant Sciences

Morphological Features of in Vitro Bamboo Shoots
Systematically maintained in vitro node culture stocks of Bm and Pm, as shown in Figure 1, were used in this experiment. Since gradual white-to-green tinge shoots were observed, we investigated the relation between color variation in the outward regions of culm and node tissues and their suitability as explants light. By checking the autofluorescence property of whole shoots under LED 365 nm illumination with RGB digital imaging analysis using ImageJ software, the color variation of explants as the relative intensity of the blue value was specified. To refer to a color scale, positional differences of the relative autofluorescence intensity were also identified.

Growth Performance of in Vitro Bamboo Shoots
As shown in Figure 4, we could distinguish types of shoots according to the plant height, i.e., short (less than 5 cm), medium (ca. 5 -10 cm), and tall (more than 10 cm)-specifically from a culture box of in vitro node culture stock. The number of nodes per shoot varied depending on the size of the shoots. Tall shoots that have ca. 5 nodes on average are suitable as explants. Three types of node portions-the first node, middle nodes, and the top meristem-were collected from tall shoots that were more than ca. 10 cm in height and independently cultured in the SLCE to define a bent for the explants. The first node showed better growth performance in terms of weight increment ( Figure 5). Further growth features of each node were also monitored (data not   shown) and it was concluded that the first nodes have superior bent for explants of the bamboo node culture. By selecting the first nodes as suitable explants, a quick survey to set a highly efficient culture condition became theoretically possible.
Without selecting the best node tissues, i.e., first nodes, the resulting responses of a culture tend to vary widely. We effectively reconfirmed the superior bent of the first node when a test survey that set a highly efficient culture condition was performed as follows. Different concentrations and a combination of cytokinins, i.e., 3 μM of TDZ, and 3 and 10 μM of BA, were used for in vitro SAM development, and different concentrations of auxin, i.e., 0.1, 3, and 10 μM of 2,4-D, were also evaluated for whether in vitro RAM development was promoted. As expected, clear growth promotion and/or inhibition could be seen in the SLCE, especially in terms of the day to bud break values (Table 3).

Autofluorescence Properties of the Outward Regions of Culm and Node Tissues
Given the relation between color variation in the outward regions of culms and nodes and their suitability as explants (see Figure 3), we attempted to identify the detailed autofluorescence properties of the target tissues using an inverted fluorescent microscope under B-and U-excitation lights with an RGB and HSB digital imaging analysis. As shown in Figure 6, the mature node region, especially near the first node, showed various strong fluorescence intensities. When B-excitation light was adapted, the images from the R and G modes of the RGB analysis and the hue and brightness modes of the HSB analysis were estimable.
When U-excitation light was used, the images from the G and B modes of the RGB analysis and the brightness mode of the HSB analysis were visible. Growth performances were summarized after cultured for 21 days. Data represent the average value ± SD from 5 explants. Figure 6. Autofluorescence of the outward regions of the tissues of culms and nodes under B-and U-excitation lights with an RGB and HSB (hue, saturation, and brightness) digital imaging analysis.

Discussion
A liquid medium condition has previously been suggested to be suitable for in vitro bamboo tissue cultures, especially in three major bamboo genera (Bambusa, Dendrocalamus, Phyllostachys) [4] [7] [17] [18]. In the present study, we investigated the effects of solid and liquid media on the growth of bamboo node cultures of Bm and Pm, and found that better growth performance could be observed in the liquid medium condition (see Table 1). Then, we focused on using a 6-well microplate that contains 2 mL per well of a liquid medium, which provides a small-scale liquid culture environment (SLCE). The underlying concepts of the SLCE are as follows: 1) to establish a simple and versatile liquid assay system to control morphological and histochemical responses of SAM and RAM of the bamboo node within a short period, and 2) to reveal the relation between color variation in the outward regions of culm and node tissues and their suitability as explants using techniques for autofluorescence measurement with a digital imaging analysis. For the first concept, we evaluated growth performance of nodes from 11 different bamboo species that belong to seven major bamboo genera (Bambusa, Dendrocalamus, Phyllostachys, Tetragonocalamus, Chimonobambusa, Pleioblastus, and Sasa), and concluded that the first node is the best explant for the bamboo culture system. As a test assay, we investigated the effects of plant growth regulators-both auxin and cytokinin-since it is well known that these chemicals interact in a complex manner to control many aspects of growth and differentiation [19]. The auxin-cytokinin interaction in the regulation of plant meristem development is overviewed in terms of biosynthesis, transport, and signaling control [20]. As shown in Table 3, we recognized that the combination of 10 μM of BA and 3 μM of TDZ was effective for in vitro SAM development during 3 weeks of culture. The addition of TDZ served as a trigger to induce multiple shoots. There are reports that TDZ is effective for enhancing micropropagation in cereal and grass plants [21] [22]. Moreover, the addition of 2,4-D effectively promoted in vitro RAM development. Interestingly, it was capable to monitor different bud breaking patterns (2 -10 days after culture) with various RAM developments through the test assay. The low concentration of 0.1 μM was effective for shoot and root development, while the high concentration of 10 μM induced early bud breaking with callusing. For the second concept, using macro-and microscopic fluorescence observation techniques, as described in the text, we estimated autofluorescence properties in the outward regions of bamboo node tissues by performing an RGB and HSB digital imaging analysis. The color of an object can be described by several color coordinate systems, referred to as color spaces. One of the most important decisions for imaging analysis is to select a color space, of which the most popular is RGB, often used in video monitors. HSV (HSB in ImageJ) is an alternative representation of the RGB color model designed to be more closely aligned to human vision and perception of color-making attributes [23]. As shown in Figure 6, we compared all the images obtained, and decided a suitable combination of B-and U-excitation lights with an RGB and HSB digital imaging analysis.

Conclusion
To the best knowledge, this is the first report that suggests a versatile node culture method in the SLCE to control morphological and histochemical responses of SAM and RAM in in vitro bamboo plants. Clear growth promotion and/or inhibition could be seen in the SLCE within a short period. For future studies, we are constructing detailed quantitative imaging analyses on the variation of the autofluorescence property by adapting a method [24] with our new culture system.