Structure and histochemistry of the stem of Dracaena cambodiana Pierre ex Gagnep

Dracaena cambodiana Pierre ex Gagnep is an important plant resource for producing dragon's blood and one of most popular ornamental trees in China. For a better understanding of the physiological function of the stem, the structural characteristics and main substance histological location of the stems of D. cambodiana were studied. The structural characteristics of the different developmental stages of stems of D. cambodiana were observed and described detailly. And then a schematic diagram of the mature stem was created. Histochemical staining showed that two kinds of polysaccharides distributed in parenchymal cells. Saponins distributed mainly in ground tissue and phenolic compounds distributed mainly in the thick cell walls. An abundant of calcium oxalate raphide bundles were identified in cortex and primary tissue. Finally, the role of the above results in the taxonomy of Dracaena species and in their strong adaptability was discussed.

However, there are more than 60-190 species in the genus Dracaena, some of which are shrubby, some are arborescent (Jupa et al., 2017;Maděra et al., 2020).The anatomical structure characteristics of different Dracaena stems has their own specificity.For example, the growth rings are vague and difficult to identify in D. fragrans, but obvious in D. hookeriana (Cheadle, 1937).In addition, the histological localization of the main substances in the stem of Dracaena remains unclear.Therefore, in order to better understand the physiological function of the stem, more anatomical and physiologically oriented studies of the Dracaena are needed (Maděra et al., 2020).D. cambodiana Pierre ex Gagnep, an arborescent monocot in the family Asparagaceae, is mainly distributed in Hainan province in China and some other Southeast Asian countries (Liu et al., 2017;Zheng et al., 2012).It is one of two plant resources for producing dragon's blood in China (Liu et al., 2021;Zheng et al., 2012) and one of most popular ornamental trees in south China (Zheng et al., 2012)

| Optical microscopic imaging
Healthy stems in different developmental stages were pruned into 0.3 cm (length) Â 0.5 cm (width) Â 0.5 cm (height) small blocks and fixed in the 4% paraformaldehyde solution.The blocks were sectioned to the thickness of 80 μm using a freezing microtome (Leica CM1900, Germany).Then sections were observed under a microscope (Nikon 80i, Japan) and photographed by a camera (Liyang WN-HP, China).To observe sieve tube, the frozen sections of the transverse, radial, and tangential sections of first-class stems were stained using watersoluble aniline blue, then observed and photographed under the microscope with ultraviolet (UV) light (330-380 nm) as the excitation light sources.Three stems were observed in each developmental stage.

| Scanning electron microscope
The healthy D. cambodiana stems were fixed in formalin-acetic acidalcohol (FAA) fixing solution for over 24 h and then pruned into <5 mm blocks along the transverse, radial, and tangential directions.They were dehydrated using gradient alcohol (50%-70%-90%-100%-100%) till bone dry.After gold coating, the samples were observed under the scanning electron microscope (SEM) (JCM-6000plus, Japan).Three stems were observed in each developmental stage.The lengths of 30 raphide bundles in each stem were measured.

| Material dissociation and observation
Three stems were selected in each developmental stage, which were pruned into long-strip samples (0.3 cm Â 0.3 cm Â 5 cm) and boiled in water for 60 min.The boiling water was replaced by a mixture of glacial acetic acid and 30% hydrogen peroxide (1:1 vol/vol).Next, the samples were heated in a water bath at 100 C for 2-3 h.After cooling, the samples were centrifuged and rinsed three times in water.

| X-ray imaging
The second stems were cut into 1-2 mm sections along the transverse and tangential directions.To identify the kind of raphide bundles, the sections were then treated with water, 5% hydrochloric acid or 10% acetic acid.Finally, the treated sections observed under the Xray imaging system (Faxitron MultiFocus, United States).

| Schematic diagram making
A schematic diagram of D. cambodiana stem was created using Adobe Illustrator CS5 based on the images and statistical data.

| Histochemical staining
Histochemical staining of polysaccharides mainly referred to a previous study (Liu et al., 2019).The frozen sections of healthy D. cambodiana stems were stained using a periodic acid of Schiff (PAS) staining kit (Solarbio G1281, China) and an alcian blue-PAS (AB-PAS) staining kit (Solarbio G1285, China), with certain modifications.The stained sections were observed and photographed under the optical microscopic imaging system.Histochemical staining of saponins was done mainly referred to a previous study (Teng et al., 2009), with some modification.Specifically, healthy D. cambodiana stems were frozen and sliced into sections 80 μm thick.The sections were stained in a freshly prepared mixture of 5% vanillin in glacial acetic acid and perchloric acid at a volume ratio of 1:1 for 10 min.After developing, they were observed and photographed under the optical microscopic imaging system.
Then, the sections were treated by FAA fixing solution for 20 days to remove saponins as the negative control.
Histochemical staining of phenolic substance was done mainly by reference to the practice of previous studies (Jin & Kwon, 2009;Liu et al., 2019).The frozen sections of healthy D. cambodiana stems were observed and photographed under the optical microscopic imaging system after staining by Millon's reagent or 5% NaOH solution or phloroglucinol-HCl solution.
The granular substances observed in frozen sections were identified as follows: The sections were stained by iodine-potassium iodide staining and PAS and AB-PAS staining.Then the sections were observed and photographed under the optical microscopic imaging system.

Pierre ex Gagnep stems
The D. cambodiana stems were mainly divided into four parts: cork, cortex, monocot cambium, and ground tissue (Figure 2a).According to its developmental processes, the ground tissue was divided into two parts: the loose ground tissue in the center of D. cambodiana stems was primary tissue (Figure 2a), and the compact one outside the primary tissue was secondary tissue (Figure 2a).In the relatively mature first-class stems, secondary tissue was easily distinguished from primary tissue (Figure 2a).On the transverse sections of first-class stems, the secondary tissue accounted for about 30% of the ground tissues, while primary tissues accounted for 70% (Figure 2a).A few secondary tissues had differentiated in some second-class stems (Figures 2b and   4e), while most of the secondary tissue in the third-class stems had not differentiated yet (Figures 2c and 4f).
In short, the younger the stem, the smaller the proportion of secondary tissue, while the larger the proportion of primary tissue.With

| Microstructural characteristics of five parts of D. cambodiana stems
To further reveal the structural feature of D. cambodiana stems, the five parts were observed detailly through optical microscopy, SEM, and dissociation, respectively.
As the outermost layer of the stems, the cork was about 20 cells in thickness, where the brown cell walls were apparently thickened (Figures 3a and 4a).The cortex, which was below the cork, consisted of 25-40 layers of parenchymal cells, and most such parenchymal cells were circular (Figure 3b).Many raphide bundles were observed in the parenchymal cells of the cortex (Figures 3b and 4b).
Monocot cambium, located between cortex and ground tissue, consisted of 10-15 layers of cells, most of which were rectangular and tightly arranged, with a small volume (Figure 5).As observed in  Specifically, two or three cells inside each cambium formed mother cell clusters of vascular bundles first (Figure 5a) through multiple radial divisions.Then, these cell clusters of vascular bundles gradually differentiated into fibers, sieve tubes, and tracheids.Finally, mature vascular bundles were generated via cell enlargement and cell wall thickening (Figure 5a-c).
Located at the stem center, the primary tissue contained sparse collateral vascular bundles (Figures 3d and 4b,e,f).Secondary tissue, around the primary tissue, contained crowd amphivasal vascular bundles (Figures 3c and 4c).In addition, the parenchymal cells in secondary tissue had thicker wall than those in the primary tissue, and such cells were radially and orderly arranged around amphivasal bundles in a strip shape (Figures 3c,d and 4c).

| Structural analysis of vascular bundles
Vascular bundle is the more complex and remarkable structural feature in the stem of D. cambodiana.Therefore, their structural characteristics was further studied.The amphivasal vascular bundles in the secondary tissue were usually long elliptic in the transverse section (Figures 3c and 4c), slightly curved in the longitudinal direction, and the adjacent different vascular bundles can cross each other (Figure 3e,f).The amphivasal vascular bundle was mainly composed of tracheid, and more or some vascular parenchyma cells distributed in them (Figures 3c and 4c).The collateral vascular bundle was nearly circular in transverse section (Figures 3d and 4d-f), curved and can crossed in longitudinal section (Figure 8b,d).The outer ring of collateral vascular bundle was mainly fibers (Figures 3d and 4d).About 5-7 tracheids with a large aperture embedded in the bottom center of the collateral vascular bundle and vascular parenchyma cells distributed over them (Figures 4d and 7c).
There are several types of tracheary elements in the vascular bun- (Figure 7a,c) distributed near the center of collateral vascular bundle and amphivasal vascular bundle.The sieve tubes in a vascular bundle were longitudinally linked (Figure 7b,d), and some were transversely connected between adjacent vascular bundles (Figure 7b).

| Structural analysis of raphide bundles
Many raphide bundles were observed in the cortex (Figures 3b and   4a), and primary tissue (Figure 4e,f).Only a few of raphides were in the parenchymal cells in secondary tissue (Figure 3e).These raphide bundles appears as distinct white particles in X-rays (Figure 8a (Table 1).

| Determination granular substances
A few granular substances were observed in cortex of D. cambodiana (Figure 9a), and they turned yellow after iodine-potassium iodide staining (Figure 9b), indicating the possible existence of proteins in these granular substances.These substances became dark purple after PAS staining, indicating polysaccharides were their main component T A B L E 1 Length of raphide bundles in different tissues of the first stem.(Figure 9c).They became light purple after AB-PAS staining, indicating the absence of viscous proteins (Figure 9d).Through the analysis of the staining results, granular substances were identified to be a complex of polysaccharides and proteins.

| Histochemical staining of polysaccharides
After PAS staining, the thick and brown cell walls of the cork turned into distinct reddish-brown (Figure 10a).The substances in the parenchymal cells in the cortex exhibited distinct purple (Figure 10a).The cell walls of tracheid in amphivasal bundles became light purple, while the parenchymal cells embedded in the amphivasal bundles were stained as purple (Figure 10c).In primary tissue, fibers and tracheids in collateral bundles were stained as light purple, while parenchymal cells and sieve in vascular bundles were distinct purple (Figure 10d).Moreover, parenchymal cells surrounding vascular bundles were stained as light purple (Figure 10c,d).
Based on the staining results above, it was speculated that the polysaccharides were different between in the parenchymal cells such as cortex, sieve tube, and in the thicker cells such as fiber and tracheid.
To further distinguish these polysaccharides, the stems were stained by AB-PAS.The AB-PAS staining results reveal that the polysaccharides in the thick cell wall were different from those in the thin cell walls (Figure 11).The result was as follows: The cortex (Figure 11a,e), parenchymal cells and sieve tubes in the center of

| Histochemical staining of saponins and phenols
Based on the chromogenic reaction between saponins with the mixture of vanillin/glacial acetic acid/perchloric acid, saponins in stems could be located.Almost no saponins was observed in cork and cortex (Figure 12a), however, a dark-red stain in ground tissues was observed (Figure 12b), revealing that saponins mainly exist in ground tissue.Based on the brick red chromogenic reaction between Millon's reagent and phenolic compounds, almost no phenolic compounds were observed in parenchymal cells of cortex (Figure 12c).The cell wall of fiber and tracheid in the vascular bundles was strongly dyed as brick-red (Figure 12d), and the surrounding parenchymal cells in secondary tissue were light brick-red (Figure 12d).This chromogenic reaction showed that phenolic compounds mainly existed in the cell walls of fiber and tracheid in the vascular bundle and less in the parenchymal cells of the secondary tissue, but neither in the parenchymal cells of cortex nor primary tissue.The chromogenic reaction between phloroglucinol-HCL solution further showed that the phenolic compounds contained in the stems are mainly lignin (Figure 12e,f).

| DISCUSSION
The taxonomic boundaries of genus Dracaena and the identification and relationship among the genus are still debated (Maděra et al., 2020).Previous studies have showed that the morphological characteristics of the leaves are an effective ways to resolve these issues, such as calcium oxalate cuticular deposits (Pennisi & Mcconnell, 2001), shape of epidermal cells and stomata types (Klimko et al., 2018).Combined with the former research results, this study provides a way to identify Dracaena trees via the morphological characteristics of the stem.
The present results showed that the stems of D. cambodiana could be divided into five parts: cork, cortex, monocot cambium,  , 1937) and D. cinnabari (Hubálková et al., 2017).
It is reasonable to assume that the structural characteristics of the stem holds throughout the genera and could be an identifying criterion for Dracaena species.However, there are some slight differences in some specific structural features in the stem of Dracaena trees.
There are some cells with polyphenols in the cortex of D. draco stem (Jura-Morawiec & Tulik, 2015), which were not observed in this study.
In addition, the length of calcium oxalate crystals ranged from 83.60 to 135.74 μm in this study, while in the stem of D. cochinchinensis ranged from 45 to 90 μm (Fan et al., 2008).There is also some difference in the average length of tracheid: 3166 μm in D. cambodiana, 4950 μm in D. draco (Jura-Morawiec, 2017) and 2000 μm in D. fragrans (Cheadle, 1937).These slight differences may suggest the reason for their separation into different species.In conclusion, although there is some variation in some specific quantitative indicators, the stems of Dracaena have similar structural features.These results can provide reference for the controversial taxonomic problems of species.
Dracaena trees are remarkably long-lived plants and tertiary relict species (Maděra et al., 2020;Xu et al., 2022), which shows that they have a strong survival ability.The studies of the stem structure can uncover the ability to a certain extent.First, as showed in this study, the proportion of secondary tissue is positively associated with the stem maturity (diameter).Compared to primary tissue, more compact amphivasal bundles imbedded in the thicker walled conjunctive tissue.
The tracheid, distributed in the peripheral region of amphivasal vascular bundles, which displaced from each other or even interwoven with each other to form tracheid bundles (Figure 4d).As found in D. draco, tracheid considerable intrusive growth resulted in a rigid network with a braid-like arrange (Jura-Morawiec, 2017).Moreover, plenty of sieve tubes were found in the center of amphivasal vascular bundles (Figure 7a,b).These structural features demonstrate that amphivasal vascular bundles provide strong functions in mechanical support and material transport.What is more, the larger the diameter of the stem, the more amphivasal vascular bundles in it, indicating that it has the stronger functions.Second, this study showed that the younger the stem, the greater the proportion of primary tissue.In other words, the primary tissue is an inverted cone, which is protected by the secondary tissue (Zimmermann & Tomlinson, 1970).The primary tissue is mainly composed of parenchyma cells with high amounts of polysaccharides.This structure provides extraordinary ability to store and release water (Jupa et al., 2017;Maděra et al., 2020), which can help Dracaena trees survive in the drought period.Third, the saponins and calcium oxalate crystals in the stem might play an important role in coping with adverse environments.

| CONCLUSION
The structure and histochemistry of the stem of D. cambodiana in context of its different developmental stages were revealed in this study.
And then a schematic diagram of the mature stem was created based on the above results (Figure 13).This study provide reference for the controversial taxonomic problems of Dracaena species and enhance our understanding of the reason of strong survival ability.The cytological mechanism of dragon resin formation in the stem of D. cambodiana will be further studied in the future.

ACKNOWLEDGMENTS
. The different developmental stages stems of D. cambodiana was selected in this study, and their structure characteristics and distributions of main substance in different tissues were studied via anatomical methods and histochemical staining.This study aims to provide anatomical support for deeper insights into physiologically function of the stem of dragon trees. 2 | MATERIALS AND METHODS 2.1 | Plant materials D. cambodiana used in this study was planted in the nursery of Haikou R & D Center, Hainan Branch of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, with an age of 10 years and a height of 2-3 m.It was identified by the associate researcher Rongtao Li as D. cambodiana.The stems in different developmental stages from the healthy trees were sampled and classified as firstclass, second-class, and third-class branches (Figure 1; 45.7, 22.5, and 17.5 mm in diameter, respectively).

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I G U R E 1 Stems of Dracaena cambodiana in different developmental stages.(a) First-class branch.(b) Second-class branch.(c) Third-class branch.Red triangular marks represent sampling positions.
the development and thickening of stem, the proportion of secondary tissue is increasing.In order to accurately show the thickness of secondary tissues, the diameters of 30 first class stems within 3-20 cm and the thickness of secondary tissues therein were analyzed in this study.The thickness of secondary tissues was calculated for each stems as: δ is the thickness of secondary tissue, D is the diameter of the stem.F I G U R E 2 Transverse section of Dracaena cambodiana stems.(a) Firstclass stems.(b) Second-class stems.(c) Third-class stems.1. Cork. 2. Cortex.3. Monocot cambium.4. Secondary tissue.5.Primary tissue.

Figure 5 ,
Figure 5, the vascular bundles in secondary tissues formed due to the continuous division and differentiation of monocot cambium.

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I G U R E 4 Transverse section of different stages of stems under scanning electron microscope (SEM).(a-d) Transverse section of firstclass stem.(a) Cork and raphide bundles (indicated by arrows) in cortex.(b) Junction between secondary tissue and primary tissue.(c) Amphivasal vascular bundles in secondary tissue, ellipse indicated parenchyma cells in amphivasal vascular bundle.(d) Collateral vascular bundles in primary tissue, ellipse indicated parenchyma cells in collateral vascular bundle.(e) Transverse section of second-class stem.(f) Transverse section of third-class stem.Co, cork; F, fiber; PT, primary tissue; ST, secondary tissue; T, tracheid.The bar is 100 μm for (a), (c), and (d); 1 mm for (b), (e), and (f).
dle.In collateral vascular bundle, three types of tracheids were observed, mainly scalariform tracheid (Figure6a,b) and reticulate tracheid (Figure6c), less spiral tracheid (Figure7d).In amphivasal vascular bundle, four types of tracheids were observed, mainly pitted tracheid (Figure6g,h), less in reticulate, scalariform and spiral tracheids (Figure 6e,f).In general, amorphous polysaccharides accumulated in the sieve area could form a callose that could be used to locate the sieve as they could exhibit green fluorescence under UV after being stained by aniline blue.The results showed a total of 2-4 sieve tubes F I G U R E 5 Monocot cambium in Dracaena cambodiana stems.(a) Mother cell clusters (indicated by arrows) of amphivasal bundles and monocot cambium of first-class stem.(b) Monocot cambium in second-class stem and collateral bundles during formation (indicated by arrows).(c) Monocot cambium in third-class stem and collateral bundle during formation (indicated by arrows).MC, monocot cambium.The bar is 100 μm for (a) and (b) and 50 μm for (c).F I G U R E 6 Kinds of tracheary elements (tracheids and fibers).(a) and (b) Scalariform tracheid in primary tissue.(c) Reticulate tracheid in primary tissue.(d) Fibers in primary tissue.(e) Spiral and scalariform tracheids in secondary tissue.(f) Junction of tracheids in secondary tissue.(g) and (h) Pitted tracheid in secondary tissue.The bar is 50 μm in (a), (c), (d), (e), (g), and (h); 20 μm in (b) and 100 μm in (f).
,b), which indicates that the main ingredient is calcium.After being treated by hydrochloric acid, raphides were not dissolved.After being treated by acetic acid, raphides were dissolved (Figure 8c,d).The above results demonstrated that the raphide bundles is calcium oxalate crystals.In addition, we found that there is no significant difference in the length of the raphide bundles among different class of stems.But the result showed the raphide bundles in the secondary tissue were the longest (135.74 μm), compared to the means of 83.60 and 112.09 μm in cortex and primary tissues, respectively, showing significant differences among different tissues in one stem

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I G U R E 7 Aniline blue staining of first-class Dracaena cambodiana stems.(a) Amphivasal bundles on transverse section of secondary tissue, where callose is marked light green (indicated by arrows).(b) Junction of amphivasal bundles on a radial section of secondary tissue, where the callose is marked light green (indicated by arrows).(c) Collateral bundles on a transverse section of primary tissue, where the callose is marked light green (indicated by arrows).(d) Collateral bundles on a radial section.F, fiber; T, tracheid.The bar is 50 μm.F I G U R E 8 Secondary-class stem of Dracaena cambodiana under X-ray.(a) and (c) Transverse section of secondary-class stem.(b) and (d) Radial section of secondary-class stem, arrows indicate vascular bundle.(a) and (b) Raphide bundles (white particle) mainly distributed in the cortex and primary tissue.(c) and (d) Disappearance of raphide bundles (white particle) after hydrochloric acid treatment.The bar is 1 mm for (a)-(d).

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I G U R E 9 Validation of granular substances and their composition in the cortex.(a) Granular substances in cortex under the scanning electron microscope.(b) Yellow color after iodine-potassium iodide staining.(c) Purple color after periodic acid of Schiff (PAS) staining.(d) Purple color after AB-PAS staining.The bar is 50 μm for (a) and 20 μm for (b)-(d).F I G U R E 1 0 Periodic acid of Schiff (PAS) staining of the first-class stem of Dracaena cambodiana.(a) Reddishbrown cork and purple parenchymal cells in cortex.(b)-(d) Light purple cell walls of tracheid and fiber in vascular bundles, and purple vascular parenchymal cells inside the vascular bundles after staining.The bar is 200 μm for (a) and (c), and 50 μm for (b) and (d).

1 1
Alcian blue-periodic acid of Schiff (AB-PAS) staining of first-and third-class stems of Dracaena cambodiana.(a)-(d) Transverse section of the first-class stem.(a) Cork is blue-brown, parenchymal cells in cortex is blue.(b) Junction between secondary tissue and primary tissue.(c) and (d) Local magnification of (b) showed that cell wall of tracheid or fiber in vascular bundles are purple, and parenchymal cells in the vascular bundles are blue after staining.(e) and (f) Transverse section of the third-class stem.(e) Cork is blue, parenchymal cells in cortex is blue after staining.(f) Tracheid and fiber in collateral bundles are light purple, and parenchymal cells are distinctly blue after staining.The bar is 200 μm for (a) and (b), and 50 μm for (c)-(f).vascular bundles (Figure 11c,d), and most parenchymal cells in primary tissue (Figure 11f) were wholly stained as distinct blue, indicating that the polysaccharides are acidic, which distributed in the protoplast of above parenchymal cells.The cell walls of tracheid and fiber in the vascular bundles, and the thicker cell walls of parenchymal cells outside the vascular bundles were stained as purple (Figure 11c,d), suggesting the existence of neutral polysaccharides (cellulose).

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Histochemical staining of saponin, phenol, and lignin in the first-class stems.(a) and (b) Vanillin/glacial acetic acid/ perchloric acid staining showing that saponin mainly distributed in ground tissue.(c) and (d) Millon's reagent staining showing that phenol mainly distributed in the tracheid or fiber in vascular bundle and less in parenchymal cells outside of the vascular bundle.(e) and (f) Acid phloroglucinol staining showing that lignin mainly distributed in tracheid or fiber in vascular bundle.The bar is 200 μm for (a)-(d) and 100 μm for (e) and (f).
secondary tissue and primary tissue.And crowd amphivasal vascular bundle imbedded in compact secondary tissue and sparse collateral vascular bundle distribute in loose primary tissue.Similar structural characteristics have been found in the stems of D. cochinchinensis (Fan et al., 2008), D. draco (Jura-Morawiec & Tulik, 2015) D. fragrans, D. goldieana (Cheadle

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I G U R E 1 3 Scheme showing three-dimensional structure of the stem of Dracaena cambodiana.
This research was supported by Hainan Provincial Natural Science Foundation of China (Grant No. 821QN352) and CAMS Innovation Fund for Medical Sciences (CIFMS) (Grant No. 2021-I2M-1-031).The authors also thank Sagene eBioart for their help of pattern diagram making.