Botanical Studies (2010) 51: 357-362.
                   REPRODUCTIVE BIOLOGY
In vitro flowering and mating system of Eulophia
graminea Lindl.
Chen CHANG1*, Wei-Hsin HU2,3, Ying-Chun CHEN4, Yu-Ling SU1, and Yi Tien CHIU1
1Department of Horticulture, National Chung Hsing University, 250, Kuo Kuang Rd., Taichung 402, Taiwan
2Department of Botany, National Museum of Natural Science, Taichung 404, Taiwan
3Department of Life Science, National Chung Hsing University, Taichung 402, Taiwan
4Taichung District Agricultural Research and Extension Station, Council of Agriculture, Changhua 515, Taiwan
(Received September 28, 2009; Accepted December 11, 2009)
ABSTRACT. The capsules of Eulophia graminea Lindl. were collected from a seashore and a city of subtropical Taiwan. The seeds from the capsules sown in vitro and germinated, hence developed into rhizomes where they either sprouted to form flower stems or developed leaf shoots with roots. The subculture rhizomes that produced a large amount of flower stems generated flowering. The plantlets with flower stems were then cultured in a medium for fruition. Some capsules formed through the autogamous mating system were harvested and their seeds were sown in vitro. A total of 4 generations were cultured over a 4 year period. There are three explanations that support the findings of the E. graminea and its strong colonization ability: the unique storage structure of the rhizome, the brief juvenile stage and the autogamous mating system.
Keywords: Autogamy; Eulophia graminea; Flowering; In vitro; Mating system; Rhizome.
introduction
The terrestrial orchid, Eulophia graminea Lindl., native only to subtropical and tropical Asia, has naturalized in Miami-Dade County in southeastern Florida; and found in woodchip mulched garden beds in the Northern Territories, Australia (Pemberton et al., 2008).
Within its native range, the E. graminea occurs in diverse habitats. In Singapore, it is reported to be widespread on wasteland, sandy beaches, lawns, roadsides and other exposed areas, as well as in secondary forests and parks (Tan and Sin, 1993). In Taiwan, the orchid grows among lowland shrubs, sandy beaches and coastal grassland (Su, 2000).
In 1997, experiments were done which suggested that E. graminea is a self-compatible but pollinator-dependent out-crossing species (Sun, 1997). Pemberton et al. (2008) performed pollination treatments which determined that the flowers of E. graminea are self compatible, but less fruit were set in self pollinated flowers, than in out-crossed flower. No fruit set occurred while plants were in isolation from pollinators; indicating that E. graminea is not autogamous. Wu (2004) tested the mating system of E. graminea in the wild of Tainan, Taiwan. There was 3.4% (2/69) fruition occurred in isolation from pollinators in the bagging plants.
*Corresponding author: E-mail: chenchang@dragon.
nchu.edu.tw; Tel: 886-4-22840340 ext 803; Fax:
886-4-22860574.
Williamson (1984) observed a mechanism in live plants by which self-pollination may be affected in eight species of the Eulophia from South Central Africa. At a certain level of maturity in the flowers, the pollinium becomes active and outgrowths begin to appear from the lower border of the pollinia to the stigma.
Wild capsules from the E. graminea were collected from a seashore and a city in Taiwan and brought to the laboratory where the seeds were harvested from the capsules and then propagated seeds in vitro.
Within this culture system, we could clearly observe the conversion morphology, flowering, and mating system in the medium. This significant data facilitate the understanding of the strong colonization capability of the E. graminea in a feral environment.
materials and methods
Plant material and seed germination
Capsules of E. graminea were collected from two locales in Taiwan: a seashore in Wuchi, (Taichung County July, 2002), and a roadside in Nankang (Taipei City, Taiwan, June, 2004). The capsules were sterilized with 2% NaOCl supplemented by 1 drop of Tween 20 for 15 min. The seed generation medium (GM) consisted of 1/4 strength MS salts (Murashige and Skoog, 1962), supplemented with (mg l-1): niacin (0.5), pyridoxine HCl (0.5), thiamine HCl (0.1), myo-inositol (100), activated charcoal (1,000), peptone (1,000), potato powder (6,000), coconut milk (150 ml/l), sucrose (20,000) and
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gelrite (4,000). Medium pH was adjusted to 5.2 before autoclaving for 15 min at 121°C. The seeds were sown in 20x150 mm Pyrex tubes each with 9 ml GM medium.
Protocorms were subcultured in the same medium for rhizome development. The rhizomes were subcultured for a period between 4-6 months. All rhizome cultures were exposed to artificial light of 1000 lux (daylight fluorescent tubes FL-30D/29, 40 w, China Electric Co, Taipei, Taiwan) with an average light/dark cycle of 16/8 h at 25 1°C.
In vitro fruit set.
Plantlets with a 1-cm pseudobulb of E. graminea were transferred to the induction medium for fruit setting (FM). The medium formula was WPM basal salts (Lloyd and McCown, 1981) with (mg l-l): myo-inositol (100), activated charcoal (1,000), BA (1.0), NAA (0.6), sucrose (20,000) and solidified with agar (7,000). The medium pH was adjusted to 5.2. The in vitro capsules were collected and seeds were sown on the GM medium.
All statistical analyses were carried out using One-way ANOVA Duncan's multiple range test at a 95% confidence level with the COSTAT statistical software.
Scanning electron microscopy
Samples for scanning electron microscopy were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer for 4 h, then dehydrated using an ethanol series, dried in a critical-point dryer (HCP-2, Hitachi, Japan), and finally coated with gold using an ion coater (E1010, Hitachi, Japan)
before viewing with a Hitachi S-3000N scanning electron microscope (Chang et al., 2005).
In vivo pollen tube growth
The style of in vitro self-pollinated flowers were collected and fixed in FAA (50% ethanol : acetic acid : formaldehyde = 18:1:1) for 24 h, rinsed in demineralized water, and softened in 3 M NaOH for 2 h at 60°C. Afterwards, the style were rinsed in demineralized water and placed in a drop of aniline blue solution with 2% glycerol, squashed under a cover slip. After staining, it was observed by fluorescence microscopy (Peter et al., 2004).
results
Seed germination and plantlet establishment
Seeds of E. graminea (Figure 1a) were sown in vitro. After 2-3 months, the embryos swelled and broke out of the testa, and then formed protocorms (Figure 1b). The protocorm enlarged, and produced rhizomes with multiple buds (Figure 1c). The six month old rhizomes were subcultured in the same medium and then subcultured again for 4 months. The apex of the rhizome formed either vegetative buds (Figure 1d) or flower stems (Figure 1e). After the vegetative buds grew into a complete plantlet with expanded leaves, and were transplanted in a greenhouse where they survived and were healthy (Figure 1f). Subsequently, several plants were blooming the following year.
Figure 1. The seed germination, rhizomes development, plantlet establishment and flowering in vitro of Eulophia graminea. (a) Seed (bar = 70 (im); (b) Protocroms (bar = 100 (im); (c) Rhizomes (bar = 0.60 mm); (d) Shoots derived from rhizomes (bar = 1.3 cm); (e) Flower stems derived from rhizomes (bar = 6 mm); (f) Healthy seedling grown in greenhouse (bar = 1.9 cm).
CHANG et al. ― In vitro flowering of Eulophia graminea
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The ordinary phenomenon of in vitro flowering
12 months after being sown, the flower stems protrude and elongate from rhizomes derived from seeds collected from different locales (Wuchi, Taichung County and Nankang, Taipei City), showing 5.3% and 6.8% flowering of all the samples collected and cultivated.
The morphology of in vitro flowers
Upon the investigation the third subcultures, 44-61% rhizomes spontaneously produced flower stems from the apex. Most Eulophia graminea rhizomes formed flower stems only, while other rhizomes formed flower stems and shoots. The rhizomes that formed flower stems or only had fewer and smaller flowers than those of the plants grown in vivo (the greenhouses) (Table 1). A comparison of the in vitro and in vivo flower stem indicates that 10 cm in vitro flower stems produced up to 2.4 flower buds, where as plants grown in vivo (the greenhouse) produced 11 flowers for each 30 cm flower stems. There were 23.4% in vitro flower buds that did not bloom.
There was 50% flowers of the E. graminea in vitro integrity flower parts (Figure 2a), 40% of the flowers lacked both the left petal and the right petal (Figure 2b),
and 10% lacked the right flower petal only. The in vitro plantlets had similar flower lengths as those of the plants grown in vivo but the labellum and ovaries were shorter (Table 2). Moreover, not many of the in vitro flowers fully opened and their petals also deteriorated. All the in vitro flowers had a normal column, and functional anther and stigma (Figure 2c, d).
In vitro fruiting
The life-spin of in vitro flowers was 2-3 weeks. The column apex shown- with the anther cap lifted to expose the globular pollinia following the blooming flower (Figure 2c, d). The pollinia shown below the main portion
Table 1. Characteristics of in vivo and in vitro panicle and flower of Eulophia graminea.
Panicle*
Flower*
Length (cm) Flower buds.
Length (cm) Width (cm)
In vivo 32.6 a 11.2 a
2.2 a 2.5 a
In vitro 10.4 b 2.4 b
1.7 b 2.0 b
*The means with 10-20 replicates with the same letters were not significantly different at p<0.05.

Figure 2. Flowering, self-pollination and fruition of Eulophia graminea in vitro. (a) In vitro flowering (bar = 4 mm); (b) In vitro flowering lacking two petals (bar = 2.5 mm); (c) Apex of column with anther cap lifted (bar = 2.5 mm); (d) Apex of column with anther cap lifted to exposed the pollinia (bar = 1 mm); (e) The pollinia position below the main portion of the androclinium grown touching the stigma in an early fruition stage (bar = 1 mm); (f) Fruit set (bar = 1 cm).
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Table 2. A comparison of in vivo and in vitro flower organ of Eulophia graminea.
Flower organ (cm)*
Dorsal sepal
Left sepal
Right sepal Left petal
Right petal
Lip
Column
Ovary
In vivo
1.34 a
1.42 a
1.42 a 1.10 a
1.12 a
1.56 a
0.62 a
1.66 a
In vitro
1.22 a
1.08 a
1.16 a 1.04 a
1.06 a
1.24 b
0.56 a
1.14 a
*The means with 5 replicates with the same letters were not significantly different at p<0.05.
of the androclinium where it comes into contact with the stigma and where the ovary subsequently grew and became enlarged (Figure 2e, f). The pollen tubes were observed penetration into the style (Figure 3). Finally, there was 18% flowering and automatic fruition without artificial pollination in the isolation culture condition.
The in vitro cultured fruit required more than two months to mature and produced seeds with in vitro embryos (Figure 4a). Several hundred seeds in one in vitro fruit were undersized (Figure 4b). The various germination rates of in vitro seeds sown on the GM medium were evaluated through constant observation under a microscope after a period of 4 months (Figure 4c). The average germination percentage from three independent flask was 59.6% (16/25, 19/34, 30/51). In vitro flower stems protruded after 11 months of culturing in 2 subcultures, and the flowers were developed with a labellum and expanded petals and sepals (Figure 4d). After two months, the in vitro fruit turned yellowish and had normal seeds. Formation of second generation
Figure 3. The pollen tubes growing in the style of Eulophia graminea. The style was examined at the time of the pollinia touched the stigma in an early fruition stage as figure 2f. (bar = 10 μM).
Figure 4. Fruition, seed germination and the secondary generation flowering of Eulophia graminea in vitro. (a) Mature fruit in vitro (bar = 1 cm); (b) Seed derived from in vitro fruit (bar = 100 (m); (c) Seed germination and rhizome formation (bar = 2 mm); (d) In vitro flowering of secondary generation (bar = 5 mm).
CHANG et al. ― In vitro flowering of Eulophia graminea
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plantlets starting from when in vitro seeds were sown to in vitro fruit maturation took 14 months. The third generation in vitro plantlets took 12 months from seed to fruit maturation. Subsequently, the fourth-generation seeds were harvested and sown in vitro. Figure 5 shows the process of the in vitro sexual reproduction cycle of E. graminea.
discussion
Eulophia graminea has strong colonization abilities and is widespread throughout subtropical and tropical Asia. Eulophia graminea has also been detected in Florida and in Australia (Pemberton et al., 2008). Eulophia graminea is often found in man-made habitats; such as recently built roadside slopes, abandoned agricultural fields, secondary grassland or lawns, and open fields near construction sites (Sun, 1997). What is the reason for the widespread occurrence of this particular species?
Several characteristics were discovered in this experiment to support the pervasive nature of this unique orchid. First, the germinated seed of the orchid rapidly develop into a specific storage organ rhizome with abundant nutrient contents and multiple-buds. The seedlings established are like some terrestrial orchids of the same genus plant, the E. cucullata, E. streptopetala, E. petersii (McAlister and van Staden, 1998), E. hormusjii (Vij et al., 1989), and other genera orchids such as Cymbidium sinense (Chang, and Chang, 2000), Cymbidiium ensifolium (Chang and Chang, 2003) and Geodorum densiflorum (Sheelavantmath et al., 2000). We deduced that the rhizome structure would enhance seedling survival in wild habitats such as barren landscapes, drought ridden area or salty environment.
Second, the orchid has a brief juvenile stage that endures less than one year, which is a shorter span than other 82 species and cultivars orchids require. For example, the next known shortest juvenile stage is the Spathoglottis Penang Beauty which lasts 2 years 11 months
and 13 days while the longest stage is the Aranda Lucy Laycock which has a juvenile stage of 13 years 3 months (Goh et al., 1982). The diminutive juvenile stage of the E. graminea then moves quickly into the reproduction stage, with flowering and fruiting seeds phases.
Third, the orchid has several mating systems that can help it adapt to diverse habitats, such as the self-compatible but pollinator-dependent outcrossing species in Florida and Hong Kong (Sun, 1997; Pemberton et al., 2008), and our in vitro culture demonstrating autogamy. Four generations of the autogamous mechanism were observed. Particular attention was focused on how the pollinia automatic growth to stigma initiating pollination and subsequent fruition. Eulophia graminea develops an autogamous mating system that to a non-pollinator environment as other 8 species of the Eulophia from South
Central Africa (Williamson, 1984).
conclusion
The seeds of the orchid, E. graminea.- native to Taiwan- germinated in vitro and developed into rhizomes. They then develop shoots and root into a whole plantlet. We cultured the rhizome in vitro which spontaneously sprouted flower stems followed by flowering and subsequent fruition to complete a full life cycle. Overall, there were 4 years of cultures that spawned 4 generations. We deduce that there are three reasons for these findings which all support the premise that the E. graminea has potent colonization ability in the wild: the unique storage structure of the orchid rhizome, the brief juvenile stage of the orchid, and autogamous mating system.
Acknowledgements. The authors would like to thank the Council of Agriculture of the Republic of China, Taiwan, for financially supporting this research under Contract No. 96 AS-1.2.1-ST-a1(6). The authors thank Mr. Paul Davis for improving the English.
Figure 5. The processes of in vitro sexual reproduction 4 generations of Eulophia graminea.
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禾草芋蘭試管內開花與配育系統
 1胡維新2,3 陳盈君4 蘇昱綾1 邱翊恬1
1國立中興大學園藝學系
2國立自然科學博物館植物學組
3國立中興大學生命科學系
4行政院農業委員會台中區農業改良場
從臺灣的海岸及城巿收集禾草芋蘭的蒴果,無菌播種後,種子發育形成根莖,再從根莖頂芽形成花
莖,或形成莖葉發根後發育成為幼苗。將根莖繼代培養後,可形成更多的花莖及開花,試管內開花的禾
草芋蘭,未經人工授粉可自行自花授粉後結果實,採收試管內開花的果實再行無菌播種後,可在試管內
發芽、形成花莖,開花再結果而完成完整的生命週期,在培養的四年內共完成四個有性世代的培養歷
程。禾草芋蘭具有高度能力可在新的棲地生存下來,推測可能有以下三個原因,一為種子發芽後形成根
莖構造,可以在各種棲地具有較高的存活率,二為禾草芋蘭具有短幼年期,可在新的棲地中較快進行有
性繁殖,第三為在無授粉者的新棲地可能可以自花授粉,以繁衍後代。

關鍵詞:自花授粉;禾草芋蘭;開花;In vitro ;配育系統;根莖。