NEW INSIGHTS INTO THE ADAPTIVE MORPHOLOGICAL CHARACTERISTICS

OF THE ROOT OF NATURAL Calamus platyacanthus Warb. ex Becc.

IN TAMDAO NATIONAL PARK

Abstract. Calamus platyacanthus Warb. ex Becc is a valuable non-timber forest product, which has been deploited to the edge of exhaustion in nature. When studying its distribution in Tam Dao National Park, we found Calamus in both dirt and rocky mountains, places with steep slope. This article describes the difference of morphological and structural characteristics of the roots of Calamus plants growing in those two relatively distinct environments. The result showed that the roots of Calamus express the general characteristics of monocots; there was no secondary growth; the vasculation of the pillar demonstrated many differences to that of a general monocot. Lignification time and the size of the structural compositions were also variable.

Keyword: Calamus, roots, vascular system, lignification

1. Introduction

The rattan Calamus platyacanthus, a high economic value non-timber forest product (NTFP), has been used widely in Vietnam. Calamus source is mainly from nature and it has been spontaneously exploited, which affected greatly on the quantity and the regeneration capacity of seedlings. Many places used to have relatively large amount of C. platyacanthus, but in these years it has been difficult to find the presence of this species [1]. C. platyacanthus in particular and Calamus species in general used to grow widely in Tam Dao National Park, however, they are rarely seen these days. Some with high economic value like C. platyacanthus has even become very rare. The natural regeneration capacity of the seedlings is not high, with few seedlings reaching 1m distribute in the area. Natural C. platyacanthus in Tam Dao National Park can be found in two types of terrain: rocky slopes and dirt mountain slits with steep slope. The roots of C. platyacanthus is an underground organ, hence the physical and chemical characteristics of soil and biological competition are major ecological factors affecting the growth, the morphology as well as the structure of the roots. This study demonstrates the morphological and structural characteristics of the roots, which helps them grow in both two distinct earth conditions: forest earth, with high humidity, and little earth mixed with rock, with low humidity, which can serve as the scientific basis for the development of C. platyacanthus genetic resources in the nature.

Of the total area of 368.85 km2 of Tam Dao National Park, Calamus plants grow mostly in places with high humidity, slope around 30o – 45o with very low density. The plants grow in clumps, each clumps includes only 1-2 mature shoots and a few juvenile shoots formed by asexual reproduction. After the fallen of mature fruits, seedlings grow scatteredly around their mother plant on yellowish-red feralit soil on low mountains or soil mixed with exposed rock. This type of soil develops on sour crystallized rock, thin topsoil, light mechanical compositions,

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thin humus layer, over 75% exposed rock, distributed at Tam Dao mountain slope at the height of 400 – 700 m [3]. However, places where Calamus plants grow have relatively heavy mechanical compositions (Table 1).

Table 1: Characteristics of the soil where C. platyacanthus plants grow

No. Characteristic UnitRocky

mountain

Dirt mountai

n

1 pH(KCl) 5,29 3,92

2 Humidity % 3,091 5,895

3 OM % 6,721 4,549

4 Nts % 0,336 0,256

5 P2O5 ts % 0,124 0,103

6 K2Ots % 0,906 1,079

7 P2O5 dt mg/100g 3,73 3,55

8 K2Odt mg/100g 58,93 30,95

9 Particles

2-0,02 mm % 51,52 21,03

0,02-0,002 mm % 37,79 55,52

<0,002 mm % 10,69 23,45

The analysis shows that C. platyacanthus is distributed in sour (rocky mountain) to very sour soil (dirt mountain); humus, N, P, and K content in the soil in 2 areas are quite high. Soil of dirt mountain was heavy soil, the capacity of holding water and other nutritions was good, suitable for the growth of plants; soil of rocky mountain was sandy light soil, airy, convenient for the growth of roots but the capacity of holding water was not as good as dirt, plus high ratio of rock – up to 75% [3] which could lead to drought, especially in dry season. Thus, soil where C. platyacanthus was distributed has certain different characteristics comparing to that of the National Park in general.2. Materials and Methods

Materials: Roots of Calamus platyacanthus growing on rocky and dirt mountains in Tam Dao National Park.

Collecting soil sample: Soil samples were collected following the Diagon Principle in places where C. platyacanthus distributed. Soil samples are collected from level zero (facial soil) to 40 cm; collected one sample each 10 cm because the roots are distributed mainly from 10 to 25 cm. After collected, soil samples were mixed equally and brought back for process and analysis according to current Vietnam Standard (TCVN) (Vietnam Soils and Fertilizers Research Institut, 1998) [7].

Collecting root samples: Root samples were collected in different soil levels, cleaned,

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morphological photographed then preserved in 50o alcohol.

Root anatomy: Root samples were excised by razor blade into thin sections (60 – 100 µm), with the cutting sites at each 1 cm intervals along the roots.

Micro-section staining: (1) Soaked the sections in bleach solution 12% for 3 minutes; (2) Soaked the sections in HCl 3% for 2 minutes then rinsed with water; (3) Stained the sections with carmine solution 7.5% for 5 minutes then rinsed with water; (4) Stained the sections with methyl green solution 1% for 1 minute.

3. Results

3.1. Morphological characteristics and distribution of the root system

Roots of C. platyacanthus expressed general characteristics of monocots: fibrous root system – large roots were derived from the last stem [4, 5]; from which lateral roots were formed and dug deep in the earth. Different environments could affect the growth of the roots. Lateral root system of plants growing on dirt mountain grew deeper and wider than that of plants growing on rocky mountain, although rocky mountain possessed better humus content and airy degree (Fig 1A, C). Roots of plants growing on dirt mountain were common in the 20-25cm range while roots of plants growing on rocky mountain only reach 10-15cm or even more shallow if there was large rock presenting beneath. Soil characteristics and topsoil thickness were important factors which influence the depth of root system. In general, Calamus roots developed shallowly in topsoil, which was a general characteristic of monocots. Large roots originated from the stem were responsible for withholding mechanical force, lateral roots were responsible for absorbing nutrients. Few large roots (derived from the last stem) were found at the depth of 30cm and below, mainly were small roots at various degrees. These 2 types of roots were similar in tissue organization but different in size and tissue differentiation. Along with the secondary root system, the size of roots of plants growing on rocky mountain was also larger than that of plants growing on dirt mountain which increased mechanical strength. Plants growing on rocky mountain, with the average stem diameter of 3 – 4 cm and height of 3 – 5 m, often had roots with the average diameter of 0.3 – 0.5 cm; while that of plants growing on dirt mountain was 0.2 – 0.3cm.

Figure 1. Morphogy of C. platyacanthus’s roots growing on rocky mountain (A), dirt

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mountain (C) and young roots on rocky mountain (B), and dirt mountain (D). 1. Root tip; 2. Region of absorption

Roots of plants growing in two environments had similar colour, white when young and gradually turned brown and darker when mature but not brownish – black as other plants (Fig 1). Roots were cyclindrical-shaped, with little difference between diameter of absorption region and mature region.

3.2. Anatomical characteristics

There was barely any difference between the anatomical characteristics of the roots of C. platyacanthus growing on dirt mountain and rocky mountain, but root diameter and the depth of distribution. As the roots of general plants, the roots of C. platyacanthus was divided into root tip, root apical meristem, elongation region, absorption domain and mature region. In this research we focused on the structure, elongation region and mature region of the root tip, so that the entire anatomical structure related to important functions as penetrating into the soil, absorbing nutrients and withholding mechanical forces (anchroing plant to the soil).

3.2.1. Root tip

Root tip was responsible for protecting the root apical meristem and determining the direction of the roots. This was the first part to contact with the soil, hence the soil’s features would have direct effect on the morphology and structure of the roots. The soil on rocky mountain had light mechanical components, more humus than the soil on dirt mountain but due to high percentage of exposed rock, the penetrating ability of roots growing on rocky mountain was lower than that of roots growing on dirt mountain.

Root tip was formed from root apical meristem, new root tip cells are always created to replace the old ones shed in the penetration. During growth, the outermost cells usually viscosify, the mucus covered the entire root surface to reduce the friction between the roots and the environment, as well as protect the apical meristem [5]. The degree of viscosification of roots growing on dirt mountain was different from that of roots growing on rocky mountain. With roots growing on dirt mountain, the mucus was plentiful but due to the tendency of growing outwards and frequent friction with the soil, it shed rapidly. The outermost cells of roots growing on rocky mountain shed less, mostly because the roots grew rising on rock or crept in thin soil layers rich with humus, the friction force declined so the accumulation of mucus increased on the root surface, then gradually dried and stayed to maintained and balanced the humidity for root apical meristem, prevented the intrusion of poisons, reduced the friction with the environment and protect the apical meristem while crepting in stone gaps. The difference in the accumulation of mucus on root tip and dead cells could be observed through the morphology of the root tip.

Root tip cells havd thick wall, arranged to create a firm block that increased the hardness of the tissue, formed a sharp, hard mass to penetrate conveniently. The amount and properties of root tip cells havd few differences between two environments. Thus, this was a

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general property of this species, affected lightly by external conditions.

3.2.2. Root hair zone and maturation zone

The anatomical structure of C. platyacanthus roots showed the similarities to which of monocots, there was few structural difference from growing region to absorption domain and mature domain. The general structure consisted of epidermis, cortex and vascular cylinder.

3.2.2.1. Epidermis

The epidermis consists of a large cell layer whose morphology was completely dissimilar to other cells, arranged in continuous rings around the roots (Fig 2C). Epidermal cell was 3 to 4 times bigger than parenchymatous cells that attached to it. Epidermal cell morphology was a classifying characteristic of C. platyacanthus. When the roots were young, epidermal cells lengthened into root hairs. Their appearance significantly increased the surface contact area of the roots, thereby increased the ability of absorbing water and nutrients. Sometimes root hairs were hard to be observed on large roots derived from the stem of plants growing on rocky mountain, especially ones growing on rock surface or growing shallowly beneath the humus layer in dry season. Due to the exposure to the air or too low humidity, the lifespan of root hair was shortened considerably.

Figure 2. Root anatomy. (A) A cross section in elongation region with distinct

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cortex and vascular cylinder. (B) Casparian strip. (C) Epidermis and exodermis which were lignified, and a parenchyma layer between two layers. (D) Casparian strip with

suberin lamella in the radial walls, which were then expended to other walls (E, F, G).

1 and 1.1 – perenchyma beneath the epidermis. 2 – Parenchymatous cells in the cortex was tightly packed in young root, circular secretory cells outward of this layer. 2.1 – Programmed dead parenchymatous cells were lysated and release an air space, other cells

gradually lignified. 3 - Suberin lamella in endodermal cell radial wall. 3.1 – Suberin lamella development. 4 – Pericylce. 5 – Protoxylem. 6 – Early formed metaxylem with unlignified

wall. 6.1 – Mature metaxylem, with lignified cells. 7 – Phloem.

Usually for most plants, beneath epidermis layer was exodermis layer, consisting of polygonal, thick-walled, early lignified or suberinnized cells to enhance the ability of protecting inner tissues. However, when observing a cross-section of C. platyacanthus roots, beneath epidermis was some parenchyma cell layers, thin cellulose wall cells first, and then sclerenchyma cells. The way parenchyma tissues distributed beneath epidermis of C. platyacanthus roots was quite similar to velamen layer of Orchidaceae. There has not been any discussion about the role of parenchyma yet, but maybe they could boost the capability of retaining water and reduced mechanical effects from outside. Epidermis and cortex lignified early, often at the same time as protoxylem and metaxylem (Fig 2C).

3.2.2.2. Cortex

The cortex accounted for a large proportion in the structure (Fig 2A) with scattered single secretory cells (Fig 2A). Secretory cells could be observed most clearly when the roots were still young. After the roots have maturelized, the appearrance of many air chambers made it difficult to observe these cells. The number of secretory cells was not stable and showed no difference between different environments.

In region of elongation, the cells of cortex tissue had thin cellulose wall and arranged thickly, leaving small empty spaces. As the roots aged, some parenchyma cell bands died, leaving some air spaces (Fig. 2B), at the same time began the lignification of parenchyma cells in the cortex. Air spaces in roots were responsible for storing air for physiological activities of the plant.

Table 2. The size of root anatomical components

Components Dirt mountain Rocky mountain

Vascular cylinder diameter 1164,2 ± 31,27 1384,2 ± 33,14

Cortex thickness (µm) 956,67 ± 30,04 884,17 ± 37,42

Root radius (µm) 1455 ± 47,94 1643,8 ± 44,19

Vessel diameter (µm) 107,67 ±12,6 121,83 ± 18,12

Number of metaxylem cells/vascular cylinder

37,6 ± 1,545 43,5 ± 2,271

Number of xylem bundles 35,5 ± 1,02 39,8 ± 1,71

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Thickness of sclerenchyma ring beneath epidermis (µm)

95,077 ± 1,809 100,37 ± 3,157

In the root absorption region, roots mainly absorbed water and soluble nutrients, hence aerenchyma system had not develop fully, parenchyma cells died slowly, hence, water delivery could be accomplished by both intracellular and cellular pathway. After tissues developed fully, root hairs shed away, laternal roots appeared, air space reached maximum size and separated by 1-2 parenchyma bands. The size of aerenchyma tissues varied according to living conditions and relied on the humidity of soil. Plants growing on rocky mountain or high airy low-humidity hills had smaller aerenchyma tissues (7-12%) than plants growing on dirt mountain with higher humidity (Table 2). After having reached the mature status, the size of air chambers hardly changed.

At the same time with the formation of air spaces, parenchyma cells of the cortex lignified to increase mechanical strength of the roots (Fig. 2B, G). Different root regions had different lignification rate: Slow in growing phase and absorption region, fast after root hair had shed away. At this time the roots mainly carried out mechanical functions that hold the plant tightly on the ground. Lignification started from the epidermis, to outer cells, endodermis, and parenchyma in the middle of the cortex. Lignification degree declined gradually from exodermis to epidermis, endodermis and cortex. Similar to the size of air spaces, lignification degree depended on living conditions. Plants growing on rocky mountain often lignified earlier than plants growing on dirt mountain (Fig. 3). Thus, cortical parenchyma was the position showed many changes that allowed plants to adapt to the environment.

Endodermis consisted of a cell layer larger than pericycle and outer parenchyma which had been suberinized to form typical Casparian strips around the vascular cylinder, controlling the amount of water and nutrients entering vascular system, acting as a semi-permeable membrane which prevent water to outflow from vascular tissue even in drought (Paula, 2007). The suberin lamella formed very early, even in region of elongation, starts from radial walls (Fig. 2D). The early presence of suberin lamella in radial walls ensured well control of water and nutrients flowing from cortex to vascular tissue through membranes of endodermis cells, preventing substances moved by apoplastic pathway. Besides, in this stage, suberin lamella had little effect on the growth capacity of cells because this was also when cells grew quickly in size, root diameter increased quickly. There was absolutely no difference in structural tissue organization as well as the development of Casparian strips of plants growing on rocky mountain and plants growing on dirt mountain.

Early lignification of endodermis was a general characteristic of many monocots. Because their roots did not have secondary growth as their stems did (Tomlinson, 2001), many root anatomical components lignified quickly to enhance mechanical strength of roots. Endodermis, pericycle and ground tissue lignified quickly, creating a strengthenned stele for living activities of C. platyacanthus.

3.2.2.3. Vascular cylinder

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Vascular cylinder was a central cylindrical portion of the root, consisting of pericycle, vascular tissue and ground tissue. The outermost boundary of vascular tissue was pericycle, composed by a small cell layer (Fig. 2D, 3B) lignified early at the same time as endodermis (Fig. 2G). The main role of pericycle was producing laternal roots and enhanced mechanical strength after the roots had been lignified. In terms of origin, percicylce belonged to primary structures formed from root apical meristem.

Vascular system consisted of alternating phloem and xylem bundles arranged in a fairly regular fashion, xylem diffrentiated radially as a special structure of monocotyledonous roots. The amount of vascular bundles did not change from the region of absorption to mature region (Fig 2A,F) although the size and lignification degree of cells increased as cells aged. Vessles in the middle of vascular cylinder appeared at the same time as vessel elements distribution near pericycle (Fig 3A), rather than appeared orderly from outside to inside. There were differences between vessel diameter, number of xylem bundles of plants growing on rocky mountain and dirt mountain (Table 2). Around vessel element was early lignified pith parenchyma to enhance mechanical properties (Fig. 3A, C, E).

Figure 3. Lignification of root structural components. (A) Partial cross-section of absorbing region of roots growing on dirt mountain. (B) Development of Casparian strips

and many passage cells allowing water to pass through. (C, D) Cortex of plants growing on dirt mountain lignified lately but air spaces. (E) Cortex of plants growing on rocky

mountain lignified early.

1,2. Non-lignified cortical parenchyma; 3. Lignified cortical parenchyma; 4. Caspary strip and many passage cells; 5. Lignified pith parenchyma; 6. Non-lignified xylem.

Lignification of vascular tissue started from ground tissue at the center of vascular cylinder (Fig. 3A, C), started with around large vessel elements at the center, then spread to surrounding cells, exceptionally strong at parenchyma cell bands inside metaxylem (Fig. 3A, C). Lignification of cells of vascular cylinder of C. platyacanthus roots showed some dissimilarities to other typical monocots. Metaxylem vessel elements distributed in the centre

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with surrounding parenchyma cells lignified early, with fixed size, while outer cells near pericycle lignified much later. Thus, the size of cells increased gradually along the growth of the root as well as lignification from inside to outside (Fig 3A, C). The result was root diameter increased due to cell growth, hence the diameter of C. platyacanthus root hardly changed from the region of elongation to mature region (Fig. 1).

In spite of similar structure, lignification time and lignification degree of root structural components of C. platyacanthus depended closely on living conditions. Plants growing on rocky mountain with thin topsoil had weaker penetrating ability, leading to early lignification of parenchyma cells near vascular cylinder (Fig 3B, E). Plants growing on rocky mountain had a greater amount of vascular bundles than plants growing on dirt mountain, the size of vessel was also considerably larger (Table 2). These indicate plants growing in rocky mountain showed more developed vascular system, with strengthenned mechanical support cappacity, allowing plants to adapt to the rocky topographic condition and against mechanical affects, anchoring plants firmly to the rock and the soil, ensuring the mechanical support and transportation of the stem.

4. Conclusion

In both environments, immature C. platyacanthus roots appeared ivory white while mature roots were brown to dark brown, root diameter was slightly different in the regions of sbsorption and maturation. Roots of plants growing on dirt mountain penenstrated deeper, had smaller diameter and thinner root tips than those of plants growing on rocky mountain.

The anatomical structure of C. platyacanthus roots expressed general characteristics of monocots, consisting of epidermis, cortex and vascular cylinder with alternating xylem and phloem bundles. The cortex was lignified early and formed air spaces due to the death of parenchyma cells during development.

Lignification of vessels and other cells in vascular cylinder happened inside out gradually. Protoxylem and pericycle were lignified lastly.

MỘT SỐ DẪN LIỆU MỚI TRONG NGHIÊN CỨU ĐẶC ĐIỂM HÌNH THÁI GIẢI PHẪU THÍCH NGHI CỦA RỄ LOÀI SONG MẬT (Calamus platyacanthus Warb. ex Becc.) MỌC

TỰ NHIÊN Ở VƯỜN QUỐC GIA TAM ĐẢO

Song mật (Calamus platyacanthus Warb. ex Becc) là loại lâm sản ngoài gỗ có giá trị kinh tế cao hiện nay đã bị khai thác có thể dẫn đến cạn kiệt trong tự nhiên. Khi nghiên cứu sự phân bố của song mật ở Vườn quốc gia Tam đảo chúng tôi tìm thấy sự phân bố của loài cây này ở cả núi đất và núi đá, những nơi có độ dốc lớn. Bài báo này mô tả các đặc điểm hình thái, cấu tạo của rễ song mật sinh trưởng trong hai điều kiện tương đối khác biệt đó. Kết quả nghiên cứu cho thấy, cấu tạo rễ song mật mang những đặc điểm chung của thực vật Một lá mầm; cây không có sinh trưởng thứ cấp; sự hóa gỗ của trụ dẫn có nhiều điểm khác biệt so với cây Một lá mầm nói chung. Thời gian hóa gỗ, kích thước các thành phần cấu tạo có sự khác biệt giữa rễ cây sinh trưởng trên núi đất với núi đá.

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