J. Jpn. Bot. 91: 218–225 (2016)

Fruit morphology and anatomy of chinensis ()

Balkrishna Ghimire, Go Eun Choi, Mi Jin Jeong, Hayan Lee, Kyung Mee Lee, Sung Won Son, Cheul Ho Lee and Gang Uk Suh*

Plant Conservation Division, Korea National Arboretum, Pocheon 487-829 KOREA *Corresponding author: [email protected]

(Accepted on March 5, 2016)

Comprehensive study on fruit morphology and anatomy of Persicaria chinensis was carried out to understand the ontogeny of pericarp and seed coat. The pericarp clearly differentiated into three histological zones: exocarp, mesocarp, and endocarp. The exocarp cells are much higher than wide in cross and longitudinal sections with sinuate anticlinal walls and narrow unbranched lumina. The young seed coat comprises of both testa and tegmen, however, mature one comprises 3−4 layers of discontinuous cells. The pericarp becomes hard and takes over the seed protection, while the seed coat is reduced and partly disintegrated or disappears during development. The gross surface appearance is of faintly reticulate pattern. This pattern consists of depressions in the periclinal walls of the exocarp cells and is bordered by ridges covered with numerous tubercles. The reticulate surface pattern with numerous tubercles and narrowly elongated-rectangular exocarp cells with undulating anticlinal walls in P. chinensis resembles other species of genus Persicaria particularly P. nepalensis, P. sieboldii and P. hastatosagittata.

Key words: Fruit anatomy, morphology, pericarp, Persicaria chinensis, seed coat.

Persicaria chinensis (L.) H. Gross, Himalaya to Southeast Asia, where numerous commonly known as Chinese knotweed, is an varieties have been recognized. In East Asia upright, perennial herb which can grow rapidly only the typical variety has been recorded except to varying height depending on the habitat and Southwestern China (Li et al. 2003). In Korea, P. on what it is scrambling over. It can tolerate chinensis is only found in Seogwipo-si, Jeju-do a wide range of environmental conditions, and the species has been designated as ‘critically including shade, high temperatures, high salinity endangered’ at national level (Korea National and drought (Webb et al. 1988). The species Arboretum 2009). is usually found in wet valleys, grassy slopes, Though the genus Persicaria was often mixed forest, thickets in valleys and mountain treated as a synonym of , the slopes from sea level up to 3000 m (Li et segregation of two genera has been supported al. 2003). Initially, P. chinensis originated in by numerous studies (Haraldson 1978, Ronse Eastern, Southeast and South Asia (US Forest De Craene and Akeroyd 1988, Ronse De Craene Service 2010), but later it has been introduced et al. 2000, Kim and Donoghue 2008, Burke to other geographical areas. The distribution et al. 2010, Sanchez et al. 2011, Schuster et al. and diversity centers of P. chinensis are the East 2011). Within Persicaria, Haraldson (1978)

—218— August 2016 The Journal of Japanese Botany Vol. 91 No. 4 219 recognized four sections based on vegetative materials were then sectioned. Serial sections anatomy viz., Cephalophilon, Echinocaulon, of 4–6 µm thickness were cut using a Leica Persicaria and Tovara, while Hong (1993) RM2255 rotary microtome (Leica Microsystems later suggested to include Rubrivena as a new GmbH, Germany) with disposable blades, section which was maintained by Ronse De stuck onto a slide glass, and dried with an Craene et al. (2000) based on fruit morphology electric slide warmer for 12 h. Dried slides were and anatomy. Persicaria chinensis belongs to stained with 0.1% Toludine blue O for 60–90 the section Cephalophilon and closely allied s, rinsed with running water and again dried with P. capitata and P. wallichii (Li et al. 2003, with an electric slide warmer for more than 6 h Kantachot et al. 2010). The species can be to remove water. The stained slides were then distinguished from other species of the section mounted with Entellan (Merck Co., Germany) Cephalophilon by heterostylous flowers, the and compressed using metal blocks for 2 days to perianth becoming fleshy in fruit, apparently remove air bubbles. After 2 days, the permanent berry-like in appearance (Li et al. 2003). slides were examined under an AXIO Imager Fruit morphology and anatomy have been A1 light microscope (Carl Zeiss, Germany). described as useful taxonomic features and Photomicrographs were taken with an AxioCam appear to be constant within sections Persicaria MRc5 attached camera system and seed coat and Polygonum (Ronse De Craene et al. 2000). measurements were made using AxioVision Although Persicaria chinensis was included in software for Windows (release 4.7, 2008). their study, detailed ontogeny of pericarp and Multiple image alignment was done using seed coat were lacking. Our primary objective Photoshop CS4 for Windows 2007. None of the in this study was to demonstrate and document image-alteration facilities of Photoshop were the development of pericarp and seed coat in the used to change the original images captured by species. We attempted to record comprehensive the camera. anatomy and micro morphological features No pre-treatments were applied in of flower, fruit and seed of P. chinensis for preparation for scanning electron microscopy morphological comparison and taxonomic (SEM). Before SEM imaging the achenes relationship within the genus Persicaria. were sputtered with gold in a KIC-IA COXEM Ion-Coater (COXEM. Co., Ltd, Korea). SEM Material and Methods imaging was carried out with a COXEM CX- Mature flower, young fruit and mature 100S scanning electron microscope (COXEM) fruit were collected from Seogwipo-si, Jeju- at 20 kv, at the seed test laboratory of the Korea do, Korea during November 2014. The National Arboretum. Scale bars in the images collected materials were fixed in FAA were added manually. (formalin : glacial acetic acid : 50% ethanol, 5:5:90, by vol.) before being stored in 50% Results ethanol. For serial sectioning, a microtome Persicaria chinensis has small flowers with resin technique was used. Microtome sections five free white or pinkish persistent tepals, were prepared using the following procedure. eight stamens, and one pistil consisting of three Plant materials were dehydrated in an ethanol styles and an ovary including an orthotropous series (50, 70, 80, 90, 95 and 100%). After ovule (Fig. 1A). Stigmas are capitate at the complete dehydration, they were passed through distal ends of style. The ovary wall is clearly combinations of alcohol/Technovit 7100 resin differentiated into three zones during anthesis: (3:1, 1:1, 1:3 and 100% Technovit) and then outer epidermis, mesophyll, and inner epidermis. embedded in Technovit 7100 resin. Embedded The outer epidermis is uniseriate, and formed by 220 植物研究雑誌 第 91 巻 第 4 号 2016 年 8 月

Fig. 1. Longitudinal section of flower and young fruit of Persicaria chinensis. A. Whole flower. B. Ovary. C. Part of ovary wall magnified. D. Young fruit. E. Young embryo, arrow heads indicate endosperm nuclei. F. Young fruit wall magnified. Abbreviations: em. Embryo. enc. Endocarp. es. Embryo sac. exc. Exocarp. hy. Hypostase. iep. Inner epidermis. ii. Inner integument. me. Mesophyll. msc. Mesocarp. oep. Outer epidermis. oi. Outer integument. sc. Seed coat. vb. Vascular bundle. large radially elongated cells with thin walls and The pericarp clearly differentiated into conspicuous nucleus (Fig. 1B, C). The mesophyll three histological zones: exocarp, mesocarp, is formed of 7–8 layers of isodiametric cells of and endocarp developing correspondingly varying sizes (Fig. 1C). The inner epidermis is from the outer epidermis, mesophyll, and inner mostly uniseriate, but periclinal division can be epidermis of the carpel (Fig. 1F). The exocarp seen in few places giving rise to two cell layers does not undergo many alterations in young (Fig. 1C). The early development of embryo sac fruit and remains single layered, and the cells is missing in this study; however, the available are considerably higher than wide in cross and data confirmed that the ovule is bitegmic and longitudinal section, with sinuate anticlinal and both integuments remain bilayered in mature periclinal walls and narrow unbranched lumina embryo sac which later developed into seed (Figs. 1F, 2A, B). During the fruit development coat (Fig. 1D, E). The hypostase is formed and the cells of the mesocarp lose protoplasts, endosperm development is of nuclear type (Fig. become irregular in shape, and then develop 1D, E). many intercellular spaces (Fig. 2A−C). Small August 2016 The Journal of Japanese Botany Vol. 91 No. 4 221

Fig. 2. Transverse and longitudinal section of fruit of Persicaria chinensis. A. TS of premature fruit. B, C. Premature fruit wall arrow heads indicate the innermost layer of tepal. D. Seed coat. E. LS of mature fruit. F. TS of mature fruit wall and seed coat. Abbreviations: hy. Hypostase. em. Embryo. enc. Endocarp. ent. Endotesta. exc. Exocarp. ext. Exotesta. msc. Mesocarp. mst. Mesotesta. sc. Seed coat. teg. Tegmen. tp. Tepal. vb. Vascular bundle. and inconspicuous vascular bundles developed that are persistent as a white papery layer even in the outer mesocarp region, attached with the until the fruit becomes complete dry (Figs. 2A− exocarp (Fig. 2B, C). The cells of the endocarp C, 3A−C). In mature fruit wall, the exocarp is also lose protoplasts and elongated tangentially composed of highly lignified cells, 100−120 (Fig. 2C). The endocarp may be uniseriate or µm thick and protective in nature comprising biseriate. narrow elongated-rectangular cells with slit like The young seed coat comprised of both testa lumen and undulating anticlinal walls (Figs. 2E, and tegmen. The testa composed of uniseriate F, 3G, H). The mesocarp is characterized by 2−3 exotesta representing by large thick walled layers of crushed cells; numerous small vascular cells, 2−3 cell layered mesotesta with small bundles are also present in this zone (Fig. 2F). thin walled cells, and single layered endotesta The endocarp is either completely collapsed characterized by thin walled elongated cell (Fig. or represented by crushed cells. Mature seed 2D). The tegmen is either single or bi-layered, coat is under-developed and comprised 3−4 and the cells are radially elongated and with full layers of discontinuous cells. The exotesta is of protoplast (Fig. 2D). single-layered with tangentially elongated and The fruits are surrounded by fleshy tepals rectangular cells, the mesotesta is represented 222 植物研究雑誌 第 91 巻 第 4 号 2016 年 8 月

Fig. 3. Light and scanning micrographs of mature fruit of Persicaria chinensis. A, B. Mature fruits covered with fleshy tepals under LM. C. Mature fruit without most of tepal under LM. D. Mature fruit under SEM. E, F. Surface details of fruit (middle portion between ridges) under SEM. G, H. TS of mature fruit under SEM arrow heads indicate the tubercles formed by undulation of wall. Abbreviations: exc. Exocarp. msc. Mesocarp. sc. Seed coat. tp. Tepal. by one or two layers of thin-walled parenchyma are developed from the undulating anticlinal cells and the endotesta is weakly represented by wall of the exocarp cells (Fig. 3G, H). a single cell layer (Fig. 2F). The mature fruit is black, trigonous with Discussions the average size of 2.5−3 × 1.6−2.1 mm (Fig. The morphology of the fruit is relatively 3C, D). The external fruit surface represents a simple and basically similar in most of the faintly reticulate pattern that consists of slight Polygonaceae (Graham and Wood 1965, Roth concavity in the periclinal walls of the exocarp 1977, Brandbyge 1993). The most conspicuous and bordered by small ridges (Fig. 3E). Both the feature of the pericarp in the family is the periclinal concavity and ridges are covered with exocarp or epidermis, which is sclerified in numerous tubercles (Fig. 3F). These tubercles most species (Ronse De Craene et al. 2000). In August 2016 The Journal of Japanese Botany Vol. 91 No. 4 223

Persicaria chinensis three carpels form the ovary enlargement in outer palisade region occurs both that encloses only one orthotropous ovule as in radially and tangentially and thin, straight, radial other Polygonaceae (Short 2011). The young walls begin to form slight nodulation. Later on fruit consists of a well-developed exocarp, lignification of the thickened cell walls begins several layers of parenchymatous mesocarp to occur and the protoplast of the palisade cells and an endocarp. The mesocarp and endocarp is enclosed by the thick cell walls. Mechanically collapsed along with the development of fruit. it is perhaps the most significant part of the This process begins after fertilization and pericarp and helps to protect the seeds. only the exocarp increases in thickness which It is known that the pericarp has several becomes heavily sclerified like in most species functions; its main function is to protect the of Polygonaceae. embryo (Roth 1977). If the pericarp becomes In young fruit the mesocarp consists of hard during development and takes over two distinct histological zones as described the seed protection, the integuments remain in Persicaria pensylvanica (as Polygonum parenchymatous or may even be reduced pensylvanicum) (Neubauer 1971) and and partly disintegrated or disappear during Fagopyrum (Heo et al. 2001). Especially in P. development. This statement makes sense here pensylvanica the two zones have been described for Persicaria, well organized integuments as outer pigmented and inner non-pigmented remain parenchymatous and reduced gradually mesophyll and the vascular tissue branches during the seed maturation. During early stage of throughout the outer portion of this pigmented development the seed coat comprises 6–7 layers zone (Neubauer 1971). Although we could not of cells; single layer of exotesta, 2–3 layers of identify such distinct pigmented zones in the mesotesta, an endotesta, and a thick tegmen with mesophyll in P. chinensis, it was obvious that 1–2 layers of palisade cells. However, in fully the mesophyll cells adjacent to the exocarp are mature fruit seed coat is weakly characterized smaller in size than the inner part and several with fragmented layers of cells. vascular strands pass through this region. In P. There is remarkable similarity in such pensylvanica, Neubauer (1971) described that pericarp structure in the genus Persicaria, as the disintegration of the inner layers of fruit wall most of the species share thick exocarp of narrow begins from the inside out and the mature fruit rectangular cells with undulating anticlinal walls consists of a remnant of outer mesocarp only. We and narrow lumina with or without dendritic also found similar phenomena in P. chinensis, in branches (Ronse De Craene et al. 2000). On the mature fruit the crushed remnant of mesophyll basis of the mature exocarp P. chinensis closely cells includes vascular bundles, which represents resembled P. nepalensis of sect. Cephalophilon, the remnant derived from the outer zone of the and P. hastatosagittata of sect. Echinocaulon mesophyll. (Ronse De Craene et al. 2000). Within the The most noticeable event during the family Polygonaceae this pericarp type has been pericarp development in Persicaria chinensis considered the most evolved type, whereas the is the transformation of exocarp. The exocarp exocarp with trapezoidal to triangular lumina, is structurally the most elaborate part during relatively square cells and straight anticlinal wall the pericarp development like in most of the which is found in species of the tribe Rheae has Persicarieae (Neubauer 1971, Ronse De Craene been considered the most basal one (see Ronse et al. 2000). At the beginning, it is a single De Craene et al. 2000; fig. 81). The exocarp layer of palisade cells that are elongated at right thickness in P. chinensis is 100−120 µm that angles to the surface of ovary. As the ovary lies within the range of sect. Cephalophilon. It undergoes a rapid increase in size, a considerable has been already mentioned that the commonly 224 植物研究雑誌 第 91 巻 第 4 号 2016 年 8 月 occurring surface pattern in Persicaria consists genus. of a reticulate arrangement of ridges covered with tubercles running along the anticlinal walls This study was financially supported by of the cells (Ronse De Craene et al. 2000). 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B. Ghimire，G. E. Choi，M. J. Jeong，H. Lee，K. M. Lee， S. W. Son，C. H. Lee，G. U. Suh：ツルソバ（タデ科）の 果実の形態と内部構造 ツルソバ Persicaria chinensis (L.) H. Gross（タデ科） で部分的に崩壊するかあるいは消失した．種子の外表面 の果皮と種皮の発生を理解するために，果実の形態と内 には垂層壁の凹みがあり，多数の微小突起で被われたリ 部構造を調べた．果皮は外果皮，中果皮，内果皮に明瞭 ッジで区切られる．種子全体の表面観はわずかに網目 に分化していた．外皮の細胞には波打つような垂層壁と 状となる．ツルソバのこの網目状のパターンはイヌタ 細くかつ枝分かれのない細胞間隙があり，横断面，縦断 デ属の他の種，とくにタニソバ P. nepalensis (Meisn.) H. 面ともに幅よりもはるかに長さが大きかった．若い種皮 Gross やウナギツカミ P. sieboldii (Meisn.) Ohki に似てい は外種皮と内種皮から成っていたが，成熟した種皮は た． 3–4 層の不連続な細胞から成っていた．果皮は硬くなっ （韓国・Korea National Arboretum, て種子の保護を担い，一方，種皮は退化して発生の途中 Plant Conservation Division）