Abstract
The anatomical characteristics of the culms and culm bases of ssanggol- and min-bamboo, which are mutated species of Phyllostachys bambusoides, were investigated using optical microscopy to understand the material properties. The vascular bundles in the culms and culm bases of both species were type I. There were a few abnormal vascular bundles in the culm base of ssanggol-bamboo, and more developed fiber bundles in the inner part of min-bamboo. A wavy surface was observed in the pith cavity of the ssanggol-bamboo culms. In the inner part, the vascular bundles of min-bamboo culms showed a regular diagonal arrangement, whereas those of ssanggol-bamboo were distributed randomly. In the culm base, ssanggol-bamboo had a significantly higher proportion of parenchyma and vessels, whereas min-bamboo had a higher fiber proportion. Both species had comparable proportions of cells in their culms. The vascular bundle density of min-bamboo was significantly higher than that of ssanggol-bamboo, whereas ssanggol-bamboo showed a significantly larger vessel diameter than min-bamboo. Ssanggol-bamboo had a shorter parenchyma cell length and larger parenchyma cell width than min-bamboo, whereas the min-bamboo culm showed a longer fiber length than that of ssanggol-bamboo. Furthermore, in the culm base, the fibers of ssanggol-bamboo were longer.
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A Comparative Study on Anatomical Characteristics of the Mutations of Phyllostachys bambusoides: Ssanggol- and Min-bamboo
Go Un Yang,a+ Byantara Darsan Purusatama,b+ Jong Ho Kim,a Denni Prasetia,a Alvin Muhammad Savero,a Eun Ji Ko,c and Nam Hun Kim a,*
The anatomical characteristics of the culms and culm bases of ssanggol- and min-bamboo, which are mutated species of Phyllostachys bambusoides, were investigated using optical microscopy to understand the material properties. The vascular bundles in the culms and culm bases of both species were type I. There were a few abnormal vascular bundles in the culm base of ssanggol-bamboo, and more developed fiber bundles in the inner part of min-bamboo. A wavy surface was observed in the pith cavity of the ssanggol-bamboo culms. In the inner part, the vascular bundles of min-bamboo culms showed a regular diagonal arrangement, whereas those of ssanggol-bamboo were distributed randomly. In the culm base, ssanggol-bamboo had a significantly higher proportion of parenchyma and vessels, whereas min-bamboo had a higher fiber proportion. Both species had comparable proportions of cells in their culms. The vascular bundle density of min-bamboo was significantly higher than that of ssanggol-bamboo, whereas ssanggol-bamboo showed a significantly larger vessel diameter than min-bamboo. Ssanggol-bamboo had a shorter parenchyma cell length and larger parenchyma cell width than min-bamboo, whereas the min-bamboo culm showed a longer fiber length than that of ssanggol-bamboo. Furthermore, in the culm base, the fibers of ssanggol-bamboo were longer.
DOI: 10.15376/biores.18.2.3228-3243
Keywords: Min-bamboo; Phyllostachys bambusoides; Qualitative and quantitative anatomical characteristics; Ssanggol-bamboo
Contact information: a: Department of Forest Biomaterials Engineering, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 24341, Republic of Korea; b: Institute of Forest Science, Kangwon National University, Chuncheon 24341, Republic of Korea; c: Gugak Research Office, National Gugak Center, Seoul 06757, Republic of Korea;
* Corresponding author: kimnh@kangwon.ac.kr
+These authors contributed equally to this work
INTRODUCTION
Bamboo grows in the southern part of the Korean peninsula. It is a source of medicine, food, building material, pulp paper, and material for crafts and musical instruments (Kang and Lee 2011).
Korean traditional musical instruments include a wide range of types such as string, wind, and percussion. Among the traditional Korean musical instruments, daegeum, known as the large bamboo flute, has been used as an important primary instrument in traditional concerts because it creates the most authentic Korean sound. Moreover, the fascinating sound from traditional bamboo instruments such as Daegeum is well-received by all.
Ssanggol-bamboo, a mutated species of Phyllostachys bambusoides, has been used as the most suitable material for manufacturing Daegeum because of its thick bamboo culm and hardness (Kang and Lee 2011; Kim et al. 2018). Min bamboo is also a mutated species of P. bambusoides and is sometimes used as raw material for daegeum. However, there is no study regarding the quality of both bamboos to support the effective utilization of both bamboos for daegeum.
There have been a few studies regarding the acoustical properties of Phyllostachys bamboo. Kang and Lee (2011) reported that the ultrasonic properties of Phyllostachys bambusoides were different from wood because of the distinctive anatomical structure of bamboo tissue, and the ultrasonic velocity of the peripheral zone was higher than the inner zone. Deng et al. (2022) mentioned that the outer part of Phyllostachys pubescens showed higher acoustical converting efficiency than the inner part owing to a lower hemicellulose content, number of parenchyma cells, and porosity.
A few studies on the anatomical characteristics of Korean bamboo species have been conducted by Lee and Park (1987), So et al. (1999), Yoon (2010), and Jeon et al. (2018a, b). Lee and Park (1987) investigated the anatomical characteristics of culm, as well as the dimensional variation in the length of internodes and culm thickness in P. bambusoides, Phyllostachys edulis, Phyllostachys nigra var. henonis, and Phyllostachys nigra grown in Damyang district. So et al. (1999) observed the anatomical characteristics of the surfaces of P. bambusoides, P. nigra var. henonis, and Phyllostachys pubescens grown in Damyang district. The vascular bundles of all species were type I with reticulate vessels and tyloses (Lee and Park 1987; So et al. 1999). Yoon (2010) investigated the anatomical characteristics of 20-day-olds, 60-day-olds, 1-year-old, and 2-year-old moso bamboo (P. pubescens). Yoon (2010) reported that the vascular bundle sheath was completely formed in 60-day-old bamboo. Meanwhile, all tissues were completely formed and mature in 1-year-old bamboo. Jeon et al. (2018a) investigated the anatomical characteristics of three Korean bamboo species: P. pubescens, P. nigra, and P. bambusoides. The three Korean bamboo species had vascular bundle type I with tyloses in the intercellular space. Furthermore, the relative crystallinity and crystal width of P. bambusoides were the highest among the three species. Jeon et al. (2018b) reported significant differences in the qualitative and quantitative anatomical characteristics between moso bamboo samples that were 1-year-old and 2-year-old, or older. They confirmed that the size of the vascular bundle sheath, fiber length, and relative crystallinity had the lowest values in 1-year-old bamboo, and the quantitative anatomical characteristics between 2-year-old and 5-year-old bamboo were not significantly different.
To date, there has been no comparative study of the anatomical characteristics of ssanggol- and min-bamboo. To understand the material characteristics of both bamboo species, for their use as musical instruments, the qualitative and quantitative anatomical characteristics of the culms and culm bases were observed.
EXPERIMENTAL
Materials
Nine culms with 5 to 7 m height from each 4 to 5 years old ssanggol-bamboo and min-bamboo were harvested from Jangheung (34°80′ N, 126°83′ E) and Gimhae (35°32′ N, 128°80′ E), respectively, in Korea. The air-dried bamboo samples were divided into culm bases and culms (Fig. 1). Anatomical characteristics of the bamboo culm were observed in the third internode, which was approximately 20 cm above the ground. In the present study, the 3rd internode was used because the length of the internode is mostly constant from the 3rd internode of both bamboo species. The bamboo culm and culm base were divided into inner and outer part. The basic information for each sample is shown in Table 1 and Fig. 2.
Fig. 1. Culm and culm base of ssanggol- (a) and min-bamboo (b) used in this study (Fig. 1A). Black scale bar: 20 cm
Fig. 2. Split bamboo culm of ssanggol- (A) and min-bamboo (B). X: culm thickness, Y: pith cavity, D: culm diameter. Black scale bar: 10 mm
Table 1. Basic Information on Bamboo Samples
Optical Microscopy
Specimens with dimensions of 15 mm (L) × 4 to 9 mm (R) × 10 mm (T) from the inner and outer part of the bamboo culm and culm base were prepared. The transverse, radial, and tangential sections were observed using these specimens. The samples were soaked in a boiling mixture of glycerin and water (1:1). Thereafter, the three surfaces were sliced to a thickness of 15 to 20 µm using a sliding microtome (MSL-H, NOW, Tokyo, Japan). The slices were stained with each 1% (v/v) safranin and light green diluted in 99% ethanol and dehydrated with a series of alcohol (50%, 70%, 90%, 95%, and 99% ethanol) and xylene solutions (50%, 100% xylene) (Park et al. 1993). The permanent slides were prepared using Canada balsam. The qualitative and quantitative anatomical characteristics were observed under an optical microscope (Nikon ECLIPSE, E600, Tokyo, Japan) connected to an image analysis system (IMT i-Solution Lite, Vancouver, Canada).
In the present study, the vascular bundle was identified according to Grosser and Liese (1971). The authors classified the basic vascular bundle types of bamboo into 4 types. Types I and II show a central vascular strand (vessels, phloem, and an intercellular space) with supporting tissue as a fiber bundle. The fiber bundle at intercellular space is bigger in type II than in type I. Tyloses are observed in the intercellular space of type I, while those are absent in type II. Type III shows a central vascular strand and a fiber strand, whereas type IV has a central vascular strand and two fiber strands.
The percentage of cell types on the cross-section was determined to be the proportion of the area of each cell type, such as parenchyma, vessel (vessel, phloem, and intercellular spaces), and parenchyma, to the total area of 4 mm2, using optical micrographs at 4x magnification (Maulana et al. 2022). The cell proportion on the cross-section was examined in 10 areas of 4 mm2 using the inner part of the bamboo culm and culm base. The vascular bundle density was the number of vascular bundles per 4 mm2 and the measurement was performed in 40 areas of 4 mm2 on the cross-section. The tangential vessel diameter on the cross-section was measured in 50 vessels. The parenchyma cell dimensions on the radial surface, such as length and width, were measured in 50 cells.
For the fiber length, specimens with dimensions of 1 mm wide and 20 to 30 mm long in the longitudinal direction were prepared near the pith cavity and near the epidermis of the bamboo culm and culm base. The specimens were immersed in Schultze’s solution for 24 h and then heated at 70 °C for 48 h. This was performed using a heating block while stirring at 2-hour intervals (Park et al. 1993). The samples were washed with distilled water until they were neutralized, and the fiber length was measured from 50 fibers.
Statistical Analysis
The significant differences in the quantitative anatomical characteristics between species, and between culm and culm base were statistically examined using analysis of variance (ANOVA) and post-hoc Duncan’s multiple range tests. The statistical analyses were performed using SPSS software (SPSS ver. 24, IBM Corp., Armonk, NY, USA).
RESULTS AND DISCUSSION
Qualitative Anatomical Characteristics
Macroscopic structure of the culm and culm base
The longitudinal surfaces of the bamboo culms and the transverse surface of the bamboo culm and culm base are presented in Figs. 3 and 4, respectively. Ssanggol-bamboo has two types of sulci along the direction of the axis on its surface one deep and one shallow. A base of branch commonly occurred between those sulci in ssanggol-bamboo culm and culm base. In contrast, min-bamboo only has a shallow sulcus on its culm surface showing repetition in 180° rotations for each internode, which commonly occurred next to the base of a branch in the culm. Sulci were absent in the culm base of min-bamboo. McClure (1945) and Renvoize (1995) mentioned that a shallow groove or sulcus from base to the tip of the internode in Phyllostachys aureosulcata and Phyllostachys aurea commonly occurred above branching nodes.
Fig. 3. Culm of ssanggol- (A) and min-bamboo (B) with sulci (black arrow) on the internode and base of a branch in the node (white arrow). Black scale bar: 4 cm
Fig. 4. Transverse surface of culm (A) and culm base (B) of ssanggol- (1) and min-bamboo (2). The black arrows point to the sulci. Black scale bar in A and B: 1.5 cm and 2.5 cm, respectively.
Cell structure and arrangement on the three surfaces
Optical micrographs of the cross-section in the culm base and culm of ssanggol- and min-bamboo are shown in Fig. 6. There were different distances from the epidermis to the vascular bundles between the culm base and culm in both species. The distance from the epidermis to the outermost vascular bundles was 500 to 1,000 μm, whereas, in the culm, the distance was less than 100 μm.
Fig. 5. Optical micrographs of the cross-section of ssanggol- (A, C, E, and G) and min-bamboo (B, D, F, and H). Outer (A, B) and inner (C, D) part of the culm base. Outer (E, F) and inner (G, H) part of culm. White arrows indicate abnormal vascular bundles. Black arrow indicates multiple metaxylem vessels. Scale bars for A, B, C, D, G, and H: 500 µm. Scale bars for E and F: 200 µm
In the outer part of the culm base, ssanggol-bamboo showed type I vascular bundles, but there were more or less different shapes (white arrows in Fig. 5A) of type I vascular bundles compared with those of min-bamboo. Min-bamboo mainly showed normal type I vascular bundles. In the inner part of the culm base, both species showed type I vascular bundles, which were randomly arranged.
The pith cavity of the ssanggol-bamboo culms had a wavy surface, whereas the min-bamboo had a flat pith cavity surface (Figs. 5 G and H). In the inner part of the bamboo culm, the vascular bundle of the min-bamboo showed a more regular diagonal arrangement than that of the ssanggol-bamboo. It was also observed that the vascular bundle was smaller and denser in the outer part than in the inner part of both species. The typical type I vascular bundle in ssanggol- and min-bamboo culms are shown in Fig. 6, which consisted of vessels, phloem with thin-walled sieve tube, intercellular space with tyloses, and fiber bundles.
Grosser and Liese (1971) observed 52 bamboo species of 14 genera distributed throughout Asia and reported that the vascular bundles became denser toward the epidermis in all species. Additionally, vascular bundle type I is commonly observed in Phyllostachys bamboo, which is in line with the results of the present study. Ito et al. (2015) reported that the distance between the vascular bundles became narrower toward the epidermis of rhizomes, culms, and roots of P. pubescens, which is consistent with the results of this study. Therefore, it is considered that the number of vascular bundles generally increases toward the epidermis, and the vascular bundles also become dense, regardless of the bamboo species. The vascular bundle types of both species in this study were consistent with those found in P. bambusoides, P. nigra, and P. pubescens (So et al. 1999; Jeon et al. 2018a).
Fig. 6. Optical micrographs of typical type I vascular bundles on cross-sections of ssanggol- (left) and min-bamboo (right) culm. FB: fiber bundle, MX: metaxylem vessel, Ph: phloem with thin-walled sieve tube, Pa: parenchyma cells. The black arrows indicate intercellular space with tyloses. Scale bars: 100 µm
Optical micrographs of the radial and tangential surfaces in the culm base and culm of ssanggol- and min-bamboo are shown in Figs. 7 and 8, respectively.
In the outer part of the radial and tangential surfaces, the culm base of ssanggol-bamboo mainly presented a small square shape for the parenchyma cells, while those of min-bamboo showed a small square shape and a rectangular shape. In the inner part, the culm bases of both species showed square-shaped parenchyma cells. Additionally, in the radial section of both species, the parenchyma cells tended to be smaller toward the pith and epidermis area and had a dense distribution.
In the outer part of the bamboo culm, the parenchyma cells of both species were rectangular and had a regular arrangement in the axial direction. In the inner part of the culm, the parenchyma cells of both species had square and rectangular shapes. In the radial section of both species, the parenchyma cells became smaller toward the pith and epidermis area and showed a compact distribution. Additionally, tyloses were observed in the intercellular spaces of both species (Figs. 7G and H).
The shape of the parenchyma cells on the tangential section of both species was comparable to that on the radial surface, which is in line with the results of Jeon et al. (2018a,b). The authors reported that the tangential surfaces of P. pubescens, P. nigra, and P. bambusoides had anatomical characteristics that were similar to those of the radial surface. Markedly, the radial and tangential surfaces of P. pubescens showed a small and circular shape for the parenchyma cells (Jeon et al. 2018b). Grosser and Liese (1971) also reported that elongated and almost cube-like parenchyma cell shapes were mainly observed on the radial surface of 52 bamboo species.
Fig. 7. Optical micrographs of the radial section in ssanggol- (A, C, E, and G) and min-bamboo (B, D, F, and H). Outer (A, B) and inner (C, D) part of culm base. Outer (E, F) and inner (G, H) part of culm. Epi: epidermis area, P: pith area, FB: fiber bundle, Pa: parenchyma cells. Scale bars: 200 µm