Jpn. J. Trop. Agr. 49(1): 14•\20, 2005

Inflorescence and Flower Initiation and Development in alismatifolia Gagnep ()

Seiichi FUKAI and Waraporn UDOMDEE

Horticultural Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795 Japan

Abstract Morphological development of inflorescence and floral organs of Curcuma alismatifolia Gagnep (Zingiberaceae), native to northern Thailand, was determined using a scanning electron microscope. Inflorescence of C. alismatifolia was a mixed inflorescence named thyrse; the main axis was indeterminate and secondary axes were determinate. Apex of the main axis produced primary bracts continuously. On the other hand, the apex of each axil produced secondary bracts and terminated in the first flower. The secondary bract subtended the secondary axis that terminated in the second flower. Tertiary and higher order bracts and flowers appeared similarly, leading to the formation of a cincinnus. The flowers underwent a petaloid transformation of the stamen (petaloid staminode), which is one of the characteristics of Zingiberaceae. The flowers consisted of a gamosepalous calyx, three petals, a polliniferous stamen, a labellum developed from two petaloid staminodes, two lateral petaloid staminodes, and a pistil. Ovule primordia had a lobed appendix that developed into a large seed aril. Key Words: aril, cincinnus, petaloid staminode, thyrse

flower development may enable to achieve effective Introduction flowering control and cut flower production with The genus Curcuma (Zingiberaceae) consists higher quality. of more than 50 species distributed in tropical Some -order undergo a Asia. Some Curcuma species have been used as petaloid transformation of the stamen (petaloid medicinal and aromatic plants. Curcuma species staminode) with considerable morphological have also a great potential as ornamental plants, variation (Kress, 1990; Weberling, 1989). The since they display a wide range of colors and family Zingiberaceae is one of eight families in morphological variations in their inflorescence. Zingiberales. Developmental studies on floral Curcuma alismatifolia Gagnep, native to northern organs in Zingiberales have been conducted for Thailand, was introduced to the world flower Cannaceae (Kirchoff, 1983), Costaceae (Kirchoff, market as a cut flower in the 1990s. Since the 1988), Lowiaceae (Kirchoff and Kunze, 1995), development of efficient propagation of this Marantaceae (Kirchoff, 1983), and Zingiberaceae species through tissue culture (Wannakrairoj, (Kirchoff 1997, 1998). The family Zingiberaceae 1997), a large number of corms with reasonable is sub-divided into four tribes, Hedychieae, price has become available for cut flower Globbeae, Zingibereae, and Alpinae. Each tribe production. Although the growth and flowering differs in floral morphology in terms of composition physiology of this species have been studied of floral organs. The androecia of Hedychieae (Azuma and Takano 1994; Hagiladi et al. 1997b; (the genus Curcuma belongs to this tribe) Krasaechai 1993; Kuehny et al. 2002; Ruamrungsri consists of a pollieniferous stamen, two petaloid et al. 2001; Takano and Azuma 1994), morphological staminodes forming a labellum, two lateral observations of the inflorescence and flower petaloid staminodes free from the labellum, and development in Curcuma have not been reported, an absent stamen (Kress 1990). except for a hand sketch drawn by Poobuapueon The objective of the present study was to et al. (1996). Elucidation of the inflorescence elucidate the morphological development of the and flower development is important for the inflorescence and floral organ of Curcuma alismatifo- analysis of the flowering physiology, because lia using a scanning electron microscope (SEM). better understanding of the inflorescence and Materials amd Methods Received Jun. 30, 2004 Accepted Dec. 18, 2004 Corms of C. alismatifolia 'Sharome Pink' and Fukai et al.: Inflorescence and floral development of Curcuma 15

' Kimono Pink', derived from clonal propagation, thyrse: the main axis was indeterminate and the were planted in plastic boxes (20 x 60 x 15cm) secondary axes were determinate, cymose. containing a mixture of sandy soil: manure (3:1) Inflorescence with the same structure can be in March. They were grown in a greenhouse observed in Hedichieae species; Hedycium

maintained at a minimum temperature of 15•Ž (Krirchof 1997) and Scaphochlamys (Krirchof under natural photoperiods, dug up in Decem- 1998). ber, and stored under dry conditions at room The primary bracts grew upward and covered temperature until next March. New shoots of the thyrse (Figure 1-E). The shoot apex of the ' Sharome Pink' at various developmental stages thyrse produced primary bracts continuously

were collected during the period from April to (Figure 1-F) until the late stage of thyrse June in both 2002 and 2003 for morphological development. About 20 primary bracts devel- observations. oped at the flowering time. Primary bracts at Pollen of C. alismatifolia 'Kimono Pink' was lower positions on the thyrse (7.7 •} 0.12 primary mounted on the stigmas of 'Sharome Pink' bracts from the bottom, n=30) remained green because C. alismatifolia is self incompatible and the subsequent 11.5 •} 0.27 (n=30) primary (Fukai unpublised). The stigmas were harvested bracts became thin and displayed a pink color at 1, 2, 3, 5, and 7 days after pollination. referred to as 'coma bract'. Samples were fixed immediately with FAA

(formalin: acetic acid: 70% ethanol; 5:5:90). The Development of cincinnus and flower specimens for SEM observation were dissected The secondary inflorescence developed in under a binocular microscope, dehydrated in an the axil of the primary bracts was a cincinnus

ethanol-acetone series, dried at the critical point, (scopioid cyme): a cylindrical inflorescence, with coated with Pt, and observed with a SEM (5- axes on different planes, branching alternately 2150; Hitachi, Ltd.). Some other specimens for to one side and the other. fluorescence microscope observation were stained The swollen apex of the axil became rounded

with 0.1% aniline blue for 24 h before observation. (Figure 2-A) and terminated in the first flower. The secondary bract appeared below the first Results and Discussion flower of the cincinnus (Figure 2-B). The direction Inflorescence initiation and development of the second bract development varried depending New shoots developed from corms produced on the position on the thyrse (Fig. 2-A and -B). 4 to 5 sheath leaves followed by 3 to 4 green The secondary bract was initiated in the opposite leaves before inflorescence initiation. The vegeta- direction of the raising of the phyllotactic helix tive shoot apex was flat and showed a triangular- in the first primary bract on the thyrse. However, shaped leaf primordium which surrounded more in higher primary bracts, the secondary bract than half of the apex with distichous phyllotaxy tended to be initiated in the direction of the (Figure 1-A). The first sign of inflorescence raising of the phyllotactic helix. The secondary initiation, dome formation (Figure 1-B), occurred bract subtended the secondary axis that produced when the shoot grew to a length of about 10 to a tertiary bract and terminated in the second 15 cm with considerable variation. Inflorescence flower of the cincinnus (Figure 2-C). The tertiary initiation occurred earlier in thicker stems, bract subtended the tertiary axis and the same which emerged from corms with larger storage sequence continued for higher order bracts and roots, as described by Hagiladi et al. (1997a). flowers, leading to the formation of the cincinnus

The reproductive shoot apex produced (Figure 3). Cincinni of this genotype produced crescent-shaped leaves (Figure 1-C) (primary about seven to eight flowers. Nevertheless, only bracts) with spiral (2/5) phyllotaxy. An even ratio the first three to four flowers developed and of clockwise and counter-clockwise phyllotactic bloomed at lower positions on the thyrse. The helix was observed in the arrangement of the number of developed flowers in a cincinnus primary bracts. Each primary bract subtended decreased at the upper positions on the thyrse. an axillary bud that developed into a secondary The flower primordia produced three sepal inflorescence, leading to the formation of a primordia sequentially (Fig. 2-C): the first one mixed inflorescence (Figure 1-D). The structure appeared in the farthest corner of the flower

of such a mixed inflorescence was referred to as primordia from the second flower, the second 16 Jpn. J. Trop. Agr. 49 (1) 2005

Fig. 1 Inflorescence initiation and development of C. alismatifolia 'Sharome Pink'.

A; Vegetative shoot apex. Bar= 200ƒÊm.

B; Dome formation and initiation of the first primary bract (arrow). Bar= 200ƒÊm.

C; Reproductive shoot apex with crescent-shaped primary bracts. Bar = 200ƒÊm. D; Each bract subtended an axillary bud. Bar = 500ƒÊm.

E; Primary bracts grew upward and covered the inflorescence. Bar = 500/ƒÊm.

F; Shoot apex (arrow) of thyrse produced primary bracts continuously. Most primary bracts were removed in the picture. Bar =500ƒÊm. Fukai et al.: Inflorescence and floral development of Curcuma 17

Fig. 2 Development of cincinnus and flower of C. alismatifolia 'Sharome Pink'. A; Swollen apex of the axil. Bar = 100 ƒÊm. B; Secondary bract appeared below the first flower. Bar = 100/1 ƒÊm. C; Flower primordia producing sepal primordia. Arrows with numbers indicate the order of sepal primordium initiation. Bar = 1001ƒÊm. D; Three common primordia (petal-stamen primordia) appeared inside the sepal primordia. Bar= 200ƒÊm. E; Adaxial common primordium produced the polliniferous stamen and the upper petal. Other two abaxial common

primordia produced two lower petals and two petaloid staminodes (arrows) that fused and developed into a lip. Bar = 200ƒÊm. F; Top part of polliniferous stamen developed into thecae. Arrow indicates primordia of lateral petaloid stami ode. Bar = 100ƒÊm. G; Pistil primordia appeared. Bar = 200ƒÊm. H; Mature ovules. Arrows indicate lobe-shaped appendix (funiculus). Bar= 5001ƒÊm. I; Blooming flower. Abbreviations: cp, common primordia (petal-stamen primordia); ff, first flower; l, lip; Lp, lower petal; p, pistil; pb,

primary bract; ps, polliniferous stamen; sb, secondary bract; sf, second flower; sp, sepal; t, theca. one on the adaxial side near the second flower, primordia) appeared inside the sepal primordia and the third one on the abaxial side of the flower, (Figure 2-D). These primordia displayed a as described by Kirchoff (1998) in Scaphochlamys. triangular shape surrounding a central depression. Then, the sepal grew upward and covered the The adaxial common primordium was larger flower and developed into a gamosepalous calyx than the others. Each common primordium at the later stage of floral development. developed into a petal to the exterior and a Three common primordia (petal-stamen stamen to the interior. The adaxial common 18 Jpn. J. Trop. Agr. 49 (1) 2005

Fig. 3 Structure of cincinnus of C. alismatifolia ' Sharome Pink'. The axil of the primary bract terminated in the first flower. Secondary bract was born on the axis Fig. 4 Structure of flower of C. alismatifolia 'Sharome that terminated in the first flower. The second Pink'. bracts subtended an axis that bore the tertiary bract and terminated in the second flower of the ovules exhibited a unique lobe-shaped appendix cincinnus. This numbering sequence was continued (funiculus) on the placenta (Figure 2-H). The to the fourth and higher order bracts and flowers of structure developed into a jelly-like seed aril the cincinnus. Abbreviations: FS, flower stalk (main axis of (Figure 5-E). Most of the Zingiberaceae plants inflorescence); PB, primary bract; F, flower; SB, have developed arils (Liao and Wu, 2000). secondary bract; TB, tertiary bract; FB, fourth bract. Although the developed aril of Curcuma may function as an elaiosome, only a few reports primordium produced a polliniferous stamen have dealt with the myrmecochory of Zingiber- and an upper petal. Two other abaxial common aceae species (Pfeiffer et al. in press). Further primordia produced two lower petals and two studies should be carried out elucidate the petaloid staminodes that fused and developed function of seed arils in this species. into the labellum (lip) later (Fig. E). The same Flowers of each cincinnus opened sequen- sequence of events in which one common tially in a primary bract (Figure 2-I). Cup-shaped primordium developed into two different func- stigma was filled with stigmatic exudate on top tional organs, petal and stamen, have also been of the stigmas two days after flowering, indicating observed in Allium (Kamenetsky, 1997) and that they were receptive. Compatible pollen Freesia (Fukai and Goi,1998) . germinated on stigmas within a few hours after The top part of the polliniferous stamen pollination (Figures 5-A and B). The pollen became enlarged and divided into two parts, tubes grew vigorously in a style and reached the which developed into thecae (Figure 2-F). Two ovary within 2 days following pollination. Once other primordia appeared between the upper fertilization occurred, the flowers wilted and and lower petal primordia (Figure 2-F). They part of the corolla tube coiled on the ovary subsequently developed into lateral petaloid (Figure 5-C). Pollen tubes grew in the mucilage staminodes. Pistil primordium appeared from on the surface of the ovary inner wall (Figure 5- the central depression and grew upward later D); they then reached a micropyle of the ovule, (Figure 2-G). Finally the flower consisted of a which was also covered with mucilage. The gamosepalous calyx, three petals, a polliniferous ovary cracked and mature seeds appeared about stamen, a labellum developed from two petaloid 40 to 50 days after fertilization (Figure 5-F). staminodes, two lateral petaloid staminodes, and Micro-propagation of C. alismatiflolia has already a pistil (Fig. 4). been established and clonal corms of selected Ovary and ovules developed in a trilocular genotypes are available commercially. On the style with axial placentation. Mature round other hand, seed propagation of Curcuma species Fukai et al.: Inflorescence and floral development of Curcuma 19

Fig. 5 Fertilization and seeds of C. alismatifolia 'Sharome Pink'. A; Top of stigma. * abaxial side of pistil. Bar = 500ƒÊm. B; Germinated pollen on stigma.

C; Coiled corolla tube (arrow). D; Growing pollen tubes (arrow heads) in mucilage on the inner wall surface of the ovary. iw, inner wall. Bar = 200ƒÊm. E; A pollen tube (arrow heads) penetrated into a micropyle (arrow). Bar = 100ƒÊm.

F; Mature seeds. Bar=2mm. is poorly documented and the seed germination Hortic. 430: 755-761. physiology of this species remains to be elucidated. Kamenetsky, R. 1997 Inflorescence of Allium speces (subgenus Melamocrommyum): structure and devel- Acknowlegements opment. Acta Hortic. 430: 141-146. The authors thank Atsushi Hasegawa, Kirchoff, B. K. 1983 Floral organogenesis in five genera of the Marantaceae and in canna (Cannaceae). Amer. University farm of Kagawa University and Hiroshi Muraguchi, Kagawa Agricultural Experimental J. Bot. 70: 508-523. Kirchoff, B. K. 1988 Inflorescence and flower development Station, for providing materials. We also in Costus scaber (Costaceae). Can. J. Bot. 66: 339-345. thank Chalermsri Nontaswatsri for her assistance Kirchoff, B. K. 1997 Inflorescence and flower develop- in collecting literature on Curcuma. ment in the Hedychieae (Zingiberaceae): Hedychium. References Can. J. Bot. 75: 581-94. Kirchoff, B. K 1998 Inflorescence and flower development Azuma, A. and K. Takano 1994 Studies on the flowering in the hedychieae (Zingiberaceae): Scaphochlamys control of Curcuma alismatifolia.II. On rest of corm, kunstleri (Baker) Holttum. Int. J. Plant Sci. 159: 261-274. and its breaking of rest. The storage condition of Kirchoff, B. K. and H. Kunze 1995 Inflorescence and corm. Bull. Kochi Agric. Res. Cent. 3: 37-45*. floral development in Orchidantha maxillarioides Fukai, S. and M. Goi 1998 Floral initiation and development (Lowiaceae). Int. J. Plant Sci. 156: 159-171. in freesia. Tech. Bull. Fac. Agric. Kagawa Univ. 50: Krasaechai, A. 1993 Daylength effect on the flowering of 69-72. Curcuma sparganifolia. Proc. 31th Kasetsart Univ. Hagiladi, A., N. Umiel, Z. Gilad and X. -H. Yang 1997a An. Con.: Plant: 32-36. Curcuma alismatifolia I. Plant morphology and the Kress, W. J. 1990 The phylogeny and classification of the effect of tuberous root number on flowering date Zingiberales. An. Missouri Bot. Gar. 77: 698-721. and yield of inflorescence. Acta Hortic. 430: 747-753. Kuehny, J. S., M. J. Sarmiento and P. C. Branch 2002 Hagiladi, A., N. Umiel, and X. -H. Yang 1997b Curcuma Cultural studies in ornamental ginger. In: Trends in alismatifolia II. Effects of temperature and daylength new crops and new uses. (Janick, J. and E. Whipkey on the development of flowers and propagules. Acta eds.) ASHA Press (Alexandria) 477-482. 20 Jpn. J. Trop. Agr. 49 (1) 2005

Liao, J. -P. and Q. -G. Wu 2000 A preliminary study of the compounds, carbohydrates and abscisic acid in seed anatomy of Zingiberaceae. Bot. J. Linn. Soc. Cuycuma alismatifolia Gagnep. during dormancy. J. 134: 287-300. Hortic. Sci. Biotech. 76: 48-51. Pfeiffer, M., J. Nais and E. Linsenmair Myrmecochory in Takano, K. and A. Azuma 1994 Studies on the flowering the Zingiberaceae: Seed dispersal of Globbafyanciscii control of Cuycuma alismatifolia. I. The storage and G.propinqua by ants (Hymenoptera/Formicidae) condition of corm. Bull. Kochi Agric. Res. Cent. 3: 31- in rain forests on Borneo. J. Trop. Ecol. (in press). 36*. Poobuapueon, J., C. Suwanthada, P. Apavatjrut and Wannakrairoj, S. 1997 Clonal micropropagation of patumma P. Voraurai 1996 Floral development of Cuycuma (Cuycuma alismatifolia Gagnep). Kasetsart J. (Natu. alismatifolia. Proc. 2nd Nation. Confer. Flowers and Sci.), 31: 353-536. Ornamental Plants: 78-85. Weberling, F 1989 Morphology of flowers and inflorescences. Ruamrungsri, S., N. Ohtake, S. Kuni, C. Suwanthada, P. Cambridge University Press (Cambridge). p. 405. Apavatjrut and T Ohyama 2001 Changes in nitrogenous (*: in Japanese with English summary)

クル ク マ ア リス マ テ ィフ ォ リア(Curcuma alismatifolia Gagnep)の 花 序 お よ び 花 芽 の 分 化 と発 達

深 井 誠 一 ・ワ ラ ポ ン ウ ド ン デ ィ ー

香川 大 学 農 学部 〒761-0795木 田郡 三 木 町 池戸

要 約 北 部 タ イ 原 産Curcuma alismatifolia Gagnep(シ ョウ ガ科)の 花 序 お よ び 花 芽 の 分 化 と発 達 を走 査 型 電 子 顕 微 鏡 を 用 い て 明 ら か に し た.ク ル ク マ の 花 序 は,主 花 序 軸 の 茎 頂 が 苞 を形 成 し な が ら無 限 生 長 を し,一 方,各 苞 の 腋 芽 が 有 限 生 長 型 の 花 序 に 発 達 す る 密 錐 花 序(thyrse)と 呼 ば れ る複 合 花 序 で あ っ た.主 花 序 軸 上 の 各 腋 芽 は,1枚 の 二 次 苞 葉 を形 成 し先 端 が 花 と な っ て 止 ま り,二 次 苞 葉 の 腋 芽 が 発 達 し て 再 び1枚 の 苞 葉 を 形 成 して 先 端 が花 に な っ た.三 次 以 降 の 花 も 同 様 の 方 法 で 分 化 し,さ そ り型 花 序(cincinnus)に 発 達 した.花 は シ ョ ウ ガ 科 特 有 の 雄 性 器 官 の 弁 化 が 見 られ た.花 は3枚 の ガ ク片 が 筒 状 に 癒 合 した ガ ク 筒,3枚 の 花 弁,葯 を持 つ 雄 ず い,2つ の 仮 雄 ず い が 癒 合 し た 唇 弁,さ ら に2つ の 弁 化 し た 仮 雄 ず い,雌 ず い か ら な っ て い た.胚 珠 は 特 異 な形 態 の 付 属 体 を も ち,そ れ は種 子 が 成 熟 す る と き に大 き な 仮 種 皮 に 発 達 した. キ ー ワ ー ド 花 芽 分 化,cincinnus,thyrse