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Embryology of a Himalayan Glasshouse ， nobile Hook.f. & Thoms. ()

a Y 吋i OMORl and Hideaki OHBA b

aYokosuka aYokosuka City Museum ，Fukadadai 95 ，Yokosuka ， 238-0016 ; bDep 釘 tment of Botany ，University Museum ，University of ， Hongo Hongo 7-3-1 ，Bunkyo ，Tokyo ， 113-0033 JAPAN (Received (Received on January 11 ， 2003)

The embryologic a1 process of a large perenni a1 Himalayan “glasshouse plan t" Rheum nobile was anatomically an a1 yzed. It s megag 卸 netophyte development was classi- fied fied as PO かgonum type ， embryogenic type as Polygonad type and endosperm formation as as the Nuclear type. These embryological features of R. nobile were almost the same as those those of R. palmatum and some species of Polygonum. The embryological evidences sug- gest gest that this species steadily carries out normal amphimixis and produces a great number

of of seeds at the final ye 紅 of its life under severe environmental conditions such as low temperature temperature with much rain ，strong window and intense UV radiation.

Key words: alpine plant ，embryology ，glasshouse plant ，Polygonaceae ，Rheum nobile.

Rheum nobile Hook .f. & Thoms. is the et al. 1999 ，Omori et al. 2000). l紅 gest perennial herb reaching 1- 1. 5 m in The reproductive morphology or ecology height ，which grows at subtropical alpine of the adapted to cool and wet envi- zone of the East ， at 4000 -4 500 m ronment as in the East Himalayas has not above the sea leve l. It is called as been performed. Regardless of low tempera- 'glasshouse' 'glasshouse' plant (Ohba 1988) because of ture during growing season and poor insect having having pale yellow translucent cover- fauna as pollinators in the Himalayan alpine ing ing the 1紅 ge compound raceme. In this 紅 ea ， regions ， R. nobile produced many matured we cannot find any large plant except R. fruits. This suggests autogamy and apomixis nobile ， as short growing season ，low tem- might occur in this species. Embryological perature ， strong wind and UV radiation make data are not enough in the alpine plants. But shrubs and herbs dwarfish. Under such se- alpine species in general have higher ploidy vere vere conditions ， R. nobile grows in 1訂 ge ro- levels than lowland species (Bliss 1971 in sette sette for seven to eight years and produces k りmer 1999) ，which are much correlated to many in fruits large infructescence at the autogamy and apomixis. For making sure of final final year (Masuzawa et al. 1993). the reproduction of this species ，we exam- In In a series of the ecological and morpho- ined the embryology next to the pollen de- logical studies studies logical about R. nobile ，its main habi- velopment (Omori and Ohba 1996). tats ， the photosynthetic rate of the rosulate The embryological studies conceming of leaves ， the anatomical features of the bracts ， Polygonaceae have not done sufficiently and so on have been analyzed (Terashima (Sou とges 1948 ，Comer 1976). Wang et al. et et al. 1993 ，Omori and Ohba 1996 ，Ki kuchi (1992) investigated developmentally only the

-145- 146 146 植物研究雑誌第78 巻第3号 平成15 年6月 ovule ovule and macrosporangium of R. palmatum. cut in 7-10 凹n by rot 訂 y microtome. For For clarifying the role of the translucent Counterstaining was by Heidenhain's Hema- bracts bracts of R. nobile and a series of studies we toxylin ，Safranin and Fast green FCF. have have here investigated microclimatic condi-

tions tions (Ki kuchi et al. 1999) ， the mor 田 Results phology phology (Omori et al. 2000 ，Omori 2002) of We examined 60 ovules of six individuals this this species. As ap 紅 t of these investigations at various stages from egg cell to he 紅 t- we present here the results conceming the shaped embryo. We did not find any abnor- development development of ovule ，embryo and seed coa t. mal embryo sac and embryo in all samples from the anatomical standpoin t. Materials Materials and Methods Megagametophyte and nucellus - The The in f1 0rescence and infructescence of ovule of Rheum nobile is attached to basal Rheum nobile Hook.f. & Thoms. were col- placenta and orthotropous ， and bitegmic lected lected near Banduke ， at 4300 m above sea (Fig. 1: U ， o i). The outer in 旬gument is two- level ， in Ja りale Himal in the east or three-cells thick and the inner one-two (Ohba 1992) and were stocked in FAA (five cells thic k. The micropyle is formed only by p紅 ts stock form a1 in; five p紅白 glacial acetic the inner integument and the nucellus is acid; acid; 90 parts of 50 % ethanol) solution. crassinucellate (Fig. 3). After After development a1 stages from f1 0ral bud Megaspore mother cell (Fig. 3.: mc) di- to to young fruit were dehydrated by n-butanol- vides transversely at the first meiotic divi- ethanol ethanol series ， they were embedded in sion ， forming a dyad. The embryo sac (Fig. Palaplast Palaplast or Histosec (56-58 OC) and were 4) finally consists of one egg cell (ec) ，two

Figs. Figs. 1-2. Gynoecium (L S: longitudinal section) and fruit (LS) of Rheum nobile. nobile. Fig. 1. Gynoecium: ovary wall (ow) ， outer integument (oi) ， inner integument integument (i i) ，nucellus (nu). Scale bar= 100μm. Fig. 2. Fruit ，pericarp (p e) ， seed coat (sc) ，epidermis of nucellus (ep). Scale bar=50μm. June June 2003 Journal of Japanese Botany Vo l. 78 No. 3 147 synergids ，two pol 紅 nuclei (p n) and three thin inner layer. The endotesta withers and antipodal antipodal cells (α c) ，therefore the type of degenerates as the seed ripens. megagametophyte development of R. nobile is is classified as Polygonum type (Willemse Discussion and van Went 1984). Rheum nobile has an orthotropous ， Early Early embryogenesis and endosperm- bitegmic and crassinucellate ovule on the The zygote is located at the micropylar end basal placenta ，and the megaspore mother of of the embryo sac (Fig. 5: zy). The first divi- cell divides transversely at first division. sion sion is transverse ，dividing the zygote into an These morphological features 紅 e almost in apical apical cell ca and a basal cell cb (Figs. 6，16- accordance with the previous description of 1). 1). The apical cell ca divides longitudinally P. palm αtum (Wang et al. 1992) and two times into the quadrant cells q， whereas Polygonaceae (Corner 1976). Corner (1 976) the the basal cell divides transversely ， forming did not decide whether the seed coat type of the the middle cell m and the proximal cell ci. this family was truly exotestal seed or not ， The proximal cell ci divides transversely into because the seed coat formation of the two superposed cells n and n' ， and the cell Polygonaceae had not been studied suffi- n' n' becomes 0 and p cells by transverse divi- ciently. The seed coat type of R. nobile is sion sion (Figs. 7 ，16-2). The quadrant cells q di- supposed to be exotestal seed coat because vide vide transversely into two cell layers 1 and the outer epidermis of the outer integument l' l' and the middle cell m divides longitudi- remains as mechanical layer. The seed coat nally nally (Fig. 16-3). The celllayers 1 and l' di- of R. nobile is morphologically similar to vide vide periclinally and 紅 e formed protoderms that of R. palm αtum (Marek 1958) in having

(Figs. (Figs. 16 四 3-5). firm testa with tannin. In the case of The middle cell layer m further divides Polygonum pensylvanicum ， the outer layer of

transversely transversely into two to three cell layers nucellus and two integuments s勘ink to be 四 (Figs. (Figs. 16-5-6: m) ，its proximal layer contri- come thin cover of the seed (Neubauer butes butes to the formation of the initials of the 1971). root cortex (Figs. 16-7-8: iec). The celllayer Rheum nobile agrees with Polygonum n divides transversely into two or three cell persicaria (Sou とges 1948) and P. layers layers (Figs. 16-5-7: n) to form the root cap pensyl να nicum (Neubauer 1971) in its basic (Figs. (Figs. 14 ， 15: ra). Based on the differentia- embryological characteristics and in the de- tion tion of the initials of the root cortex iec ， the viation of the initial cell of root cortex iec. embryogenic embryogenic type of R. nobile is classified as These common embryological characters Polygonad Polygonad type (Yamazaki 1982). The draw a conclusion that R. nobile is also in- procambium is recognized by subsequent cluded in Polygonum variation of Asterad cell cell divisions (Figs. 14 ， 15: pr). type by Johansen (1 950) and Polygonad type After After fertilization ， the primary endosperm by Yamazaki (1 982). nucleus nucleus becomes directly free nuclear (Fig. The fruits of R. nobile 紅 e fully ripened in 8:fn). 8:fn). As the embryo develops ， the cell walls spite of so harsh condition of alpine zone of 紅 e formed from the periphery. Therefore the the Himalayas that it was expected at first endosperm formation of R. nobile is the that the agamospermy rather than normal Nuclear Nuclear type (Vijayaraghavan and Prabhakar sexual reproduction occurred. However ， the 1984). 1984). megaspore formation and embryogeny Seed coat- The seed coat consists of two (embryogenesis) examined here 紅 e normal layers layers of exotesta; a sclerenchymatous and and co 町 espond to the previous reports in the tanniferous tanniferous outer layer (Fig. 2: sc) and very genus (Soueges 1948 ，Wang et al. 1992). 148 148 植物研究雑誌第78 巻第3号 平成15 年6月 June June 2003 Journal of Japanese Botany Vo l. 78 No. 3 149

Figs. Figs. 12-15. Embryogeny of Rheum nobile (2). Fig. 12: E 紅 ly stage of organization of cotyledons. Fig. 13: Cotyledons Cotyledons (co) develop. Figs. 14 ， 15: Root apex (ra) and procambium (p r) differentiate. Scales= 50 J.l m.

Figs. Figs. 3-5. Megaspore formation and zygote of Rheum nobile. Fig. 3. Nuce l1 us (LS): megaspore mother ce l1

(mc). (mc). Fig. 4. Embryo sac (LS): egg ce l1 (ec) ，pol 訂 nu c1 eus (p n) ，antipodal ce l1 (ac). Fig. 5. Prim ぽ yen- dosperm nu c1 eus (en) 加 d zygote (zy). Scale bars= lO J.l m.

Figs.6-1 1. Embryogeny of Rheum nobile (1). Fig. 6. Zygote divides transversely two ce l1 s. Fig. 7: Eight ce l1 ed stage. stage. Fig. 8. Young fruit (LS): Free nu c1 eus endosperm (か)，間 ly globular embryo (em). Fig. 9: Protoderm differentiates. differentiates. Fig. 10: Globul 訂 embryo. Fig. 11. Early heart-shaped embryo. Sc a1 e bars=5μm in Figs. 6- 7，10μm in Figs. 9-11 ，50μm in Fig. 8. 150 150 植物研究雑誌第78 巻第3号 平成15 年6月

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Fig. Fig. 16. Ill ustrated embryology of Rheum nobile. 1: Zygote divides transversely an apical cell ca and a basal basal cell cb. 2: ca cell divides longitudinally twice to become quadrant cells q， whereas cb cell divides transversely transversely a middle cell m and ci cells ， ci cell divides transversely n and n' cells ， n' cell divides trans- versely versely 0 and p cells. 3 -4: q cell divides transversely 1 and l' cells ，m cell divides longitudinally. 5: Protoderm Protoderm differentiates. 6: m cells divide transversely to become multi-cell layers. 7: Initial cell of radicle radicle (i ec) differentiates from the proximal part of m cells. 8: Cotyledons (cot) and root apex differen- ate. t1 ate.

We found no abnormal embryo in 60 sam- case (Omori and Ohba 1999) and absorb al- ples ples of six individuals ， and it is considered most UV radiation in the' 320 to 400 nm that that R. nobile reproduces sexually ， that is ranges (Omori et al. 2000) ， while the bract normal normal amphimixis is conducted ， from the removal experiment showed that some flow- anatomical anatomical viewpoin t. Similarly ， most of the ers yield abnormal pollen grains (Omori and pollen pollen grains were observed to be normal Ohba 1996). Therefore it is supposed that (Omori (Omori and Ohba 1996). male sterility was induced by low air tem- Rheum nobile is' considered to have the perature and the bracts play an important mechanism to protect from bad influence of role to produce normal pollen grains (Omori low low temperature and s住ong UV radiation to and Ohba 1996). develop develop normally megaspore ， pollen grains and and embryo in the alpine region. The translu- This study was supported by a Grant-in- cent cent bracts enveloping the inflorescence in Aid for Scientific Research (A) from the the the inside raise temperature over 15 degrees Japanese Society for the Promotion of than than air temperature in the' most pronounced Science ， No. 14255005. We would like to June June 2003 Joumal of Japanese Botany Vo l. 78 No. 3 151 thank thank . Dr. M. Wakabayashi (Tokyo first Centuries ， pp. 147-15 1. The Society of Metropolitan Metropolitan University) for his valuable Himalayan Botany ， Tokyo. comments. comments. 一一- and Ohba H. 1996. Pollen development of Rheum nobile Hoo k. f. & Thomson (Polygonaceae) ， with with reference to its sterility induced by bract re- References References mova l. Bot. J. Linn. Soc. 122: 269-278. Comer E. J. H. 1976. The seeds of Dicotyledons. 一一一 and 一一一 1999. Thermal condition of the inflores- Cambridge Cambridge Univ. Press ， Cambridge. cence of a glasshouse plant ，Rheum nobile Hoo k. f. Johansen Johansen D. A. 1950. Plant Embryology. Chronica & Thoms. and the microclimatic features of its Botanica Botanica Co. ，Massachusetts. habitat in eastem Himalayas. Newsletter of Ki kuchi T. ， Subedi M. N. ，Omori Y. and Ohba H. Himalayan Botany (25): 5-11. 1999. 1999. Habitats of alpine plants in Jaljale Himal ， 一一一， Takayama H. and Ohba H. 2000. Selective light eastem eastem Nepal ， with special reference to Rheum transmittance of translucent bracts in the nobile nobile (Polygonaceae). J. Jpn. Bo t. 74: 96- 104. Himalayan giant glasshouse plant Rheum nobile Komer C. 1999. Alpine Plant Life. Springer-Verlag ， Hoo k.主& Thomson (Polygonaceae). Bo t. 1. Li nn. Berlin Berlin Heidelberg. Soc. 132: 19-27. Marek S. 1958. European genera of Polygonaceae in Sou とges R. 1948. Embryogenie et classification. Essai the the light of anatomical and morphological investi- d'um systeme embryogenique (Partie speciale: gations gations on their fruits and seeds. Monographiae Premiere periode du systeme). Hermann & Cie ， Botanicae Botanicae 6: 57-79 ，tables 1-7. P 紅 is. Masuzawa T. ， Terashima 1.， Ohba H. and Omori Y. Terashima 1.， Masuzawa T. and Ohba H. 1993. 1993. 1993. Ecological study on agiant alpine plant Photosynthetic characteristics of a giant alpine (Rheum nobile) population in the eastem plant ，Rheum nobile Hook.f. et Thoms. and of some Himalayas ， Nepal. XV Intemational Botanical other alpine species measured at 4300 m ， in the Congress. Congress. Abstracts. p. 273. Eastem Himalayas ，Nepa l. Oecologia 95: 19 4- 20 1. Neubauer Neubauer B. F. 197 1. The development of the achene Vijayaraghavan M. P. and Prabhakar K. 1984. The en- of of Polygonum pensylvanicum: embryo ，endosperm ， dosperm. In: Johri B. M. (ed よ Embryology of and and peric 田p. Amer. J. Bo t. 58 (7): 655 -6 64. Angiosperm ， pp. 319-376. Springer Verlag ，Berlin Ohba H. 1988. The alpine flora of the Nepal Heidelberg. Himalayas: anHimalayas: introductory note. In: Ohba H. and Wang Y. ，Chen C. and Ding H. 1992. Development of Malla Malla S. B. (eds) ，The Himalayan plants 1: 19 -4 6. ovule and female gametophyte in Rheum palmatum University University of Tokyo Press ， Tokyo. L. Phytomorphology 42 (1 & 2): 72-79. 一一 1992. Botanical survey in Jaljale Himal ， east Willemse M. T. M. and van Went 1. L. 1984. The fe- Nepal Nepal in 199 1. Newsletter of Himalayan Botany male gametophyte. In: Johri B. M. (ed よEmbryo- (10): (10): 1 -4. logy of Angiosperm ， pp. 159-196. Springer Verlag ， Omori Y. 2002. Anatomical peculi ぽ ity of the translu- Berlin Heidelberg. cent cent bracts of Rheum nobile (Polygonaceae) and Yam 沼北i T. 1982. Recognized types in the early de- the the thermal condition of its inflorescence. In: velopment of the embryo and the phylogenetic sig- Noshiro Noshiro S. and Rajbhandari K. R. (eds.) ， nificance in the Dicotyledons. Acta Phytotax. Himalayan Himalayan Botany in the Twentieth and Twenty- Geobo t. 33: 40 0-4 09.

大森雄治ヘ大場秀章b .ヒマラヤ高山帯の温室型 植物 Rheum nobile の匪発生 ヒマラヤ高山帯に生育する大型で多年生の温室 れた知見から，本種が低温・強紫外線・多雨といっ 型植物，Rheum nobile (タデ科)の腔発生を解析 たヒマラヤ東部の高山帯特有の厳しい環境条件下 した.その発生タイプはタデ型で，正常な融合生 にあっても一生に 1 回の生殖を確実に行い，多数 殖が行われていていた.また，種皮構造・大胞子 の種子を生産していることが示唆された. 形成・大配偶体発生を既知のダイオウ属 (Rheum) (a. 横須賀市自然人文博物館，

のそれと比較したところ 互いに類似していた. b東京大学総合研究博物館) 本種は 1 団結実性多年草である.脹発生から得ら