The Differentiation and the Development of Flower Buds during the Life Cycle of graminifolia

Shuichi Hamatani Yoshiyuki Niimi The Hiroshima Botanical Garden Department of Bioresource Development 495 Kurashige, Saeki School of Bioresources Hiroshima 731-5156 Hiroshima Prefectural University Japan Hiroshima 727-0023 Japan

Keywords: tuberous root, terrestrial orchid, wild , formation of

Abstract We observed the differentiation and development of the flower buds of the Ponerorchis graminifolia Rchb. f., a terrestrial orchid with tuberous roots. We used the strain of Ponerorchis graminifolia that blooms in late May. The differentiation of the flower buds of Ponerorchis graminifolia began between September and December. First, the formation of was observed. In some cases, differentiation of the flower buds started in the early period, and the existence of nodes on the inflorescences were confirmed between early to mid-October. The increase in the number of nodes stopped in January. The differentiation of florets and bracts began in each inflorescence node after April, and then the number of nodes increased once again. In the middle of May, the floret formed clinandrium.

INTRODUCTION Ponerorchis graminifolia Rechb. f. is one of the small terrestrial orchids with tuberous roots, that are distributed throughout Honshu, Shikoku and Kyushu in Japan and Korea. Due to its attractive style, it is very popular and cultivated by many people in Japan who love wild . Thus, the timing of bud elongation, bloom, and formation of tuberous roots is already known. However, while detailed observations about the external formation have been made, the differentiation and the early development of flower buds of Ponerorchis graminifolia has not been reported. In this study, we researched the time of starting to differentiate and the process of developing flower buds, and we observed the life cycle of Ponerorchis graminifolia.

MATERIALS AND METHODS A population of Ponerorchis graminifolia, which were grown at Hiroshima Pref. Univ. from seed, was used for this research. We will temporarily call them the ‘Niimi strain’; they are an early flowering strain (Table 1). Cultivation and research were conducted in a field in the Asa-Minami ward in Hiroshima city with a custom method; rooting tuberous roots into soil in pots, and placing them outdoors through the year (Ishida, 1986). Research started in September 2002. Five plants were dissected every two or three weeks, then the stages of development of flower buds and new tuberous roots were observed. The stage of development of flower buds were determined by the stage of the earliest developed region in the plants. The air temperature in the field of the Asa-Minami are shown in Fig. 1.

RESULTS The results from the observation of the differentiation and the development of flower buds of Ponerorchis graminifolia ‘Niimi strain’ are shown in Table 2. The differentiation of flower buds occur on the growing point found in the shoot apices of buds, which are at the top of the tuberous roots (Fig. 2). Firstly, leaves were differentiated on the growing points (Stage I, Fig. 3). After a particular time, growing points became flat and broad (Stage II, Fig. 4). Then, distended growing points made

Proc. IXth Intl. Symp. on Flower Bulbs 361 Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS 2005 nodes and changed into primordia of inflorescences (Stage III, Fig. 5). Stage II was the first moment of the differentiation of flower buds. The first moments varied between individual samples. In some cases, differentiating their flower buds started in the early period, in the middle of September, and the existence of nodes on the inflorescence were confirmed between the start of October or the middle of October. On the other hand, in some cases the differentiating of flower buds started in the late period, December, and the existence of nodes on the inflorescence were confirmed in January. The increasing mean number of nodes on inflorescences, which were counted on the plants once they attained Stage III, stopped around January. In March, the elongations of buds started (Fig. 6), and rootings started nearly simultaneously. In April, the elongation of flower stalks (inflorescences and their proximal areas) in the insides of buds became noticeable, and the bracts and the formation of the primordia of florets were confirmed (Stage IV). After that, the increasing mean number of nodes on inflorescences restarted. Between the middle of April and the middle of May, inflorescences appeared from among leaves. On the other hand, and columns were formed in order (Stage V). In the middle of May, the floret formed clinandrium (Stage VI). In the end of May, Ponerorchis graminifolia ‘Niimi strain’ bloomed (Stage VII, Fig. 7). In the middle of June, the prime period of blooming, the formation of new tuberous roots started. Until around in September, the corpulence of new tuberous roots and the increase of the number of leaves around the growth points of new tuberous roots were observed (Table 3). In September, browning or blanching becomes conspicuous.

DISCUSSION There are many reports about flower bud formation of native orchid in Japan, that is Bletilla striata Rchb. f. (Nakagawa and Sawa, 1977), Calanthe discolor Lindl. (Komiya and Kosugi, 1971), Calanthe siebolidii Decne. (Nakagawa and Sawa, 1977), Calanthe triplicata (Willem.) Ames (Kosugi and Yokobori, 1972), Cymbidium goeringii Rchb. f. (Nakagawa and Sawa, 1977; Guo et al., 1990), Dendrobium moniliforme Sw. (Nakagawa and Sawa, 1977; Sawa and Nakagawa, 1978a), Habenaria radiata Spreng. (Kosugi and Yokoi, 1973; Tsuji et al., 1990), Neofinetia falcata (Thunb.) Hu (Nakagawa and Sawa, 1977; Sawa and Nakagawa, 1978b) and Sediera japonica Garay et Sweet (Syn. Aerides japonica) (Hasegawa and Yamauchi, 1995). However, there is no report about flower bud formation of Ponerorchis graminifolia. In this study, it became evident that the differentiation of the flower buds of Ponerorchis graminifolia began between September and December, when browning of the plants became remarkable above-ground. Habenaria radiata makes organs underground like Ponerorchis graminifolia does. The flower bud formation starts after sprouting in Habenaria radiata (Tsuji et al., 1990). However, in this strain of Ponerorchis graminifolia it started before sprouting. Firstly, it was confirmed that the growing points on the shoot apices formed leaves became flat and broad. Secondly, the growing points formed nodes and became primordia of inflorescences. Then, the number of nodes on the inflorescences increased until around January. Since around March, the elongation of the flower stalks and the starting of formation of the bracts and primordia of florets occurred, and the increasing number of nodes on the inflorescences restarted. In this study, we could not clarify the reason whether the increasing number of nodes on the inflorescences stopped in January was due to cold temperature, dormancy or another reason. Hereafter, the optimum temperature of growth of inflorescences and the style of dormancy must be clarified, if we want to regulate flowering. It is known that fertilization in the period after blooming is important for obtaining large tuberous roots. The result that the formation of new tuberous roots started from the middle of June of blooming period to about September of browning period, corroborates this. It is also known that the size of the buds on the tops of tuberous root are important for whether the bud takes flower or not. The correlation

362 between the size of tuberous root and the size of the bud on the top of the tuberous root must be researched.

Literature Cited Guo, Z., Goi, M. and Hasegawa, A. 1990. Flower formation in Cymbidium goeringii Reichb. fil. (in Japanese). J. Japan. Soc. Hort. Sci. 59(Suppl.2):829. Hasegawa, A. and Yamaguchi, M. 1995. Effect of temperature and day length on growth and flower formation of Aerides japonica (in Japanese). J. Japan. Soc. Hort. Sci. 64(Suppl.1):546-547. Ishida, G. 1986. How to grow and increase for wild orchids (in Japanese). Seibundo-Shinkosya, Japan. p.18-21. Komiya, K. and Kosugi, K. 1971. Studies on flower bud formation of orchids (3) Flower bud formation in Calanthe discolor (in Japanese). Japan. Soc. Hort. Sci. (Suppl.1):236-237. Kosugi, K. and Yokohori, H. 1972. Studies on flower bud formation of orchids (7) Flower bud formation in Calanthe triplicata (in Japanese). Japan. Soc. Hort. Sci. (Suppl.1):344-345. Kosugi, K. and Yokoi, M. 1973. Studies on flower bud formation of orchids (9) Flower bud formation in Habenaria radiata (in Japanese). Japan. Soc. Hort. Sci. (Suppl.1):314-315. Nakagawa, T. and Sawa, Y. 1977. Studies about flowering of native orchids in Japan (1) Effects of day length on the flowering in the five of native orchids (in Japanese). Abstr. Japan. Soc. Hort. Sci. (Suppl.1):536. Sawa, Y. and Nakagawa, T. 1978a. Studies on flowering of native orchids in Japan (2) Effects of temperatures on the flowering in Dendrobium moniliforme (in Japanese). Abstr. Japan. Soc. Hort. Sci. (Suppl.2):306-307. Sawa, Y. and Nakagawa, T. 1978b. Studies on flowering of native orchids in Japan (3) Effects of temperatures on the flowering in Neofinetia falcata (in Japanese). Abstr. Japan. Soc. Hort. Sci. (Suppl.2):308-309. Tsuji, T., Amaki, W. and Higuchi, H. 1990. The Histological observation of tuber formation and flower bud differentiation in Habenaria radiata (in Japanese). J. Japan. Soc. Hort. Sci. 59(Suppl.1):570-571.

Tables

Table 1. Flowering day of Ponerorchis graminifolia ‘Niimi strain’ and other .

Species Cultivars or strains Flowering day Ponerorchis graminifolia Niimi strain 27 May Suisin 24 May Kou-itten 2 June Ginryu 4 June Muten Kei 6 June Daihakuchou 10 June Heiwa 13 June Iwashimizu 19 June Ebisu-no-hikari 21 June Ponerorchis graminifolia var. kurokamiana Toubun 23 May Utage 28 May Ponerorchis graminifolia var. micropunctata Usugesyou 12 June Ohchou 20 June

363

Table 2. The differentiation and the development of the flower buds and the growth of new buds of Ponerorchis graminifolia ‘Niimi strain’.

Number of nodes on 0.0 0.0 1.8 1.5 3.8 4.6 5.8 4.8 5.4 4.8 8.2 9.8 9.0 3.0 5.7 5.3 the inflorescence1 Stage of Stage VIII ***** ***** ***** growing Stage VII **** ***** point or Stage VI ***** * flower Stage V *** 2 bud Stage IV ** Stage III **** *** **** **** ***** ***** ***** Stage II *** Stage I ***** ** * ** * * 2 19 5 21 7 16 4 15 29 19 13 25 14 6 25 14 Sep. Sep. Oct. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May May Jun. Jul. Jul. Aug. 2001 2002 1 We count 0 to the plants which are in Stage I or Stage II. 2 The stage of development of growing points or flower buds decided the stage of earliest developed region of the plants. Stage I: the stage when leaves are differentiated on the growing point. Stage II: the stage when growing point becomes flat and broad. Stage III: the stage when growing point makes nodes and changes into primordium of inflorescence. Stage IV: the stage when inflorescence forms bracts and florets. Stage V: the stage when floret forms tepals and columns. Stage VI: the stage when floret forms clinandrium. Stage VII: the blooming stage. Stage VIII: finished blooming. One asterisk (*) represents one individual.

364

Table. 3. The differentiation and the development of the flower buds and the development of the buds on new tuberous roots of Ponerorchis graminifolia ‘Niimi strain’.

Stage of growing point Stage III *** ** **** or flower bud Stage II ** * ** Stage I ***** ***** ***** ***** *** * * * Number of leaves 4.2 5.0 5.3 7.2 7.8 6.4 7.0 7.2 Length of new tuberous root (mm) 9.8 15.0 12.8 17.4 19.6 17.8 16.8 16.0 Thickness of new tuberous root (mm) 6.6 7.5 7.5 9.4 9.8 11.4 8.4 8.0 14 Jun. 6 Jul. 25 Jul. 14 Aug. 6 Sep. 26 Sep. 10 Oct. 1 Nov. 2002 One asterisk (*) represents one individual.

365 Figures

(°C)

40

35 Max 30 Min 25

20 15 10 5 0 -5 1- 11- 21- 1- 11- 21- 1- 11- 21- 1- 11- Sep Oct Nov Jan Feb Mar May Jun Jul Sep Oct 2001 2002

Fig. 1. Air temperature (°C) of the cultivating field (Asa-Minami ward in Hiroshima).

Fig. 2. Bud on top of the tuberous Fig. 3. Shoot apex of bud in Stage I. root. Bar indicates 2 mm. A: Leaf. B: Growing point. Bar indicates 0.5 mm.

Fig. 4. Growing point in the shoot apex becomes flat and broad (Stage II). Arrow shows growing point. Bar indicates 0.5 mm.

366

Fig. 5. Shoot apex of bud changes into primordium of inflorescence (Stage III). Arrows show nodes. Bar indicates 0.5 mm.

180 160 Length of bud on the to p of tuberous root 140 Length of flow er stalk 120 100

(mm) 80 60 40 20 0 1-Jan 1-Feb 1-M ar 1-Apr 1-M ay 1-Jun

2002

Fig. 6. Length of the bud on the top of tuberous root and length of flower stalk.

Fig. 7. Flower of Ponerorchis graminifolia ‘Niimi strain’. Bar indicates 1.0 cm.

367