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 macro- and 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.

Growth performance was summarized after 30 days in culture. Data represent the average values ± SD from 5 explants. Data for each column were analyzed by a Student’s t-test. **Significant at 0.05.

Another main topic of the present study is the autofluorescence measurement technique. Autofluorescence (primary fluorescence) is the fluorescence of naturally occurring substances, such as chlorophyll, collagen, and fluorite. Most plant and animal tissues show some autofluorescence when excited with ultraviolet light (e.g., light with a wavelength of approximately 365 nm) [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.

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.

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.

This study was supported in part by Grants-in-Aid for Scientific Research (C) (Grant No. 22580387 and 25450519 to S.O.) from the Japan Society for the Promotion of Science (JSPS). The author (M.T.A.) thanks to the Rotary Yoneyama Memorial Foundation, Japan Student Services Organization (JASSO), Hiroshima International Centre (HIC) and Daikyo Nishikawa Company Ltd., JAPAN for providing the scholarships to finalize this research work.

The authors declare no conflicts of interest regarding the publication of this paper.

Ara, M.T., Nomura, T., Kato, Y. and Ogita, S. (2020) A Versatile Liquid Culture Method to Control the in Vitro Development of Shoot and Root Apical Meristems of Bamboo Plants. American Journal of Plant Sciences, 11, 262-275. https://doi.org/10.4236/ajps.2020.112020