Botanical Studies (2009) 50: 459-466.
ECOLOGY
Growth responses and changes of active components as influenced by elevations and orchid mycorrhizae on Anoectochilus formosanus Hayata
Shu-Fen CHENG and Doris Chi-Ning CHANG*
Department of Horticulture, National Taiwan Univsrsity, Taipei, Tai^wan 10617, ROC (Received February 6, 2009; Accepted April 17, 2009)
ABSTRACT. Two micropropagated lines, B and R, of Anoectochilus formosanus Hayata, were separately in­oculated with orchid mycorrhizal fungi (OMF), Rhizoctonia sp. R02 [a bi-nucleated isolate of Rhizoctonia sp. (Ceratobasidium sp. AG-A)] and R04 [a multi-nuclei isolate of Rhizoctonia solani, AG-6], and grown by plas­tic bag cultivation method (PBCM) at three elevations above sea level, including National Taiwan University (NTU, 10 m elevation), Xindian (500 m elevation) and Wufong (1,000 m elevation) for seven months. Results showed that the survival rates for ex vitro growth were more than 80%, and plant production was significantly increased and there was no need to apply any agrichemical. If this orchid was not grown by PBCM, then after 3-4 months of cultivation, all plants would die if pesticide or fungicide were not sprayed for every one or two weeks in greenhouse, and the cultivation period was shortened to 1-2 months compared with traditional cultured method. Plants grew in Wufong achieved the best growth performance among three elevations, the fresh weight of mycorrhizal A. formosanus was significantly higher than the non-mycorrhizal (NM) control. In Wufong, R04 showed better growth-enhanced effect on line B, while R02 stimulated growth enhancement for line R. For both lines of A. formosanus cultivated at National Taiwan University (NTU), R02 inoculated plants contained higher level of phenolic compounds and hepatoprotective agent AFEE (A. formosanus ex­traction with ethyl acetate) compared with the non-mycorrhizal (NM) control. Analyses and measurements of antioxidant capacity by Trolox Equivalent Antioxidant Capacity (TEAC) showed that most of the antioxidant index of mycorrhizal plants were significantly higher than the non-mycorrhizal control. PBCM was proven to be a very labour saving, i.e. this cultural method can save all the human cost of irrigation and fertilization dur­ing the rest of cultivation period, and this is an effective method for mass production of agrichemical free A. formosanus plants. Inoculation of orchid mycorrhizal fungi such as R02 or R04 can significantly increase the production of this orchid with higher antioxidant capacity and hepatoprotective agent content for medicinal use.
Keywords: Anoectochilus formosanus Hayata; Antioxidation capacity; Hepatoprotective agent; Orchid mycorrhizal fungi (OMF); Phenolic compounds; Plastic bag cultivation method (PBCM).
Abbreviations: ACP, acid phosphatise; AFEE, A. formosanus extraction with ethyl acetate; CCI4, carbon tetrachloride; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FRAP, Ferric reducing antioxidant power; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; MDA, malondialdehyde; NM, non-mycorrhizal; NTU, National Taiwan University; OMF, orchid mycorrhizal fungi; PBCM, plastic bag cultivation method; PPF, photosynthetic photon flux; R02, Rhizoctonia sp. (Ceratobasidium sp. AG-A)R04, Rhizoctonia solani AG-6; AG-6, Anastomosis group 6.; SOD, superoxide dismutase; TEAC, Trolox Equivalent Antioxidant Capacity.
INTRODUCTION
Anoectochilus formosanus Hayata is a terrestrial orchid species from wild forests in Taiwan. It is an ornamental plant with medicinal value, and is widely used for treat­ing high blood pressure and hyperliposis, lowering blood
sugar level, and improving control of diabetes, liver dis­eases, heart diseases and lung diseases (Yen et al., 1996). It also displays anticancer and antivirus effects (Lin, 2007; Wu, 2007). Due to its multitudinous medicinal effects, the number of A. formosanus in the wild has been greatly reduced by intentional picking and uprooting. In recent years, with the advance of tissue culture techniques, large-scale propagation can be achieved by using seedlings and micropropagated plantlets. Future prospects for the
*Corresponding authors: E-mail: chengsf@ntu.edu.tw; Tel: +886-2-33662463 ext. 102; Fax: +886-2-83695732.
460
Botanical Studies, Vol. 50, 2009
development and application of these techniques to A. formosanus are therefore very bright. However, traditional intensive culture has several drawbacks. Survival rates of transplanted tissue-cultured plantlets are low, and plants tend to grow slowly and cultivation period is long. Addi­tionally, the occurrence of diseases such as root and stem rots caused by Fusarium oxysporum and Pythium ultimum, bacterial soft rot disease, and pest infections by red spider mites and scale insects often result in the continuous usage of pesticides and fungicides during cultivation (Yen et al., 1996; Chang, 1999). How to raise the survival rate of the seedlings or plantlets for ex vitro growth and control cul­tivating environment for shortening growth period as well as reducing the chances of harmful infection are the major concern for the cultivation of A. formosanus.
In this study, we applied the plastic bag cultivation method (PBCM), which was developed in our laboratory (Chang et al., 2007), and combined with the inoculation of Rhizoctonia spp. of orchid mycorrhizal fungi (OMF) (R02 and R04) for the cultivation of A. formosanus. The PBCM offered several advantages such as timesaving, labour saving, and pesticide or fungicide free. Appropriate ecological conditions are important determinants for the successful artificial culture of A. formosanus. Low temperature has been known to favor the growth of A. formosanus and lower pathogen occurrence (Lee, 2001; Chang and Chou, 2007). However, many people tried to grow this orchid in low elevations for practical reasons. Therefore, we decided to conduct the experiments in three locations with different elevations and growth temperature including NTU campus (10 m elevation above sea level), Shihzihtou Mountain in Xindian (500 m elevation) and Wufong (1,000 m elevation) to determine the most suitable site for the cultivation of A. formosanus.
The tissue cultured A. formosanus plants are considered to be less medicinally effective than the wild plants, which generally have 3-5 fold higher prices on the market. Previ­ous studies demonstrated that A. formosanus inoculated with OMF exhibits higher level of superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP) activities, and higher ascorbic acid, phenolic compounds, flavonoids, polysaccharides and phosphoric acid contents, and as a result they are more effective as medicinal source (Chou, 2004; Chang and Chou, 2007). In this study, we would like to achieve three goals. (1) To evaluate the possibility for using the PBCM for the mass production of A. formosanus. (2) To understand the inter­actions between the cultivating lines and the environments such as elevation and temperature. (3) To analyze and determine the antioxidant capacity and hepatoprotective activity among the mycorrhizal and non-mycorrhizal A. formosanus.
MATERIALS AND METHODS
Mycorrhizal inocula
In this experiment, two strains of Rhizoctonia spp.
including R02 (Rhizoctonia sp.; Ceratobasidium sp.; AG-A) and R04 (Rhizoctonia solani; AG-6) were isolated and obtained stable research results in our laboratory for more than ten years. Their pathogenicity was tested in our laboratory for many times and was indicated by Chang (2008), and all proved to be nonpathogenic. They were cultured on the medium which is prepared by mixing peat with 20% V8 juice to reach field capacity (Chang and Chou, 2007). All A. formosanus lines in this experiment were inoculated with single fungal strain. Each isolate is evenly inoculated under the roots of per plant in an amount of 0.1-0.2 g of inoculum. R02 was identified as a bi-nucleated isolate of Rhizoctonia sp. (Ceratobasidium sp.; AG-A, accession NO. DQ102413. 99% match, unpublished data), while R04 was a multi-nuclei isolate of Rhizoctonia solani; AG-6 (Lee, 2001).
Plant materials
The micropropagated plantlets of A. formosanus were purchased from a tissue culture company Puli, Taiwan. Two lines of A. formosanus, line B (wild type) and line R (hybrid of the wild type and red stem type), were used in the experiment. The parental plants for both lines were collected from the wild-grown A. formosanus. The cutting segments of A. formosanus were cultivated on the agar medium for eight to ten months until root growth and bud burst, and then the plantlets over 6 cm height were se­lected. The tissue culture vessels were placed in the green­house for a week before transplanting for acclimation.
Culture medium and method for the cultivation
The culture medium for A. formosanus used is a compost of peat and coconut fiber blended in a ratio of 7:4 (v:v). The plants for each line including non-mycorrhizal (NM) control and mycorrhizal treatment and the ex vitro
plants were cultivated by PBCM (Chang et al., 2007), to
culture 10 plants of A. formosanus in 5 inch plastic pot, and every two of the 5 in. pots were covered with an OPP transparency plastic bags (35.5x55 cm). In preliminary experiments, all cultivars would die if pesticide or fungicide were not sprayed every one or two weeks after three or four months of cultivation, thus PBCM was applied in this experiment. One gram Hyponex No. 5 re-suspended in one liter water was added into the growth medium and field capacity of water was attained. Totally three experimental sites were included: 1) Greenhouse in the campus of National Taiwan University [10 m eleva­tion above sea level, an average photosynthetic photon flux (PPF) at 58.3 [imol+m-2+s-1 (maximum 63.5 [imol+m-2 •s-1 at noon)], 2) Shihzihtou Mountain in Xindian [500 m elevation, an average PPF at 53.2 imol+m-2+s-1 (maximum 57.7 imol+m-2+s-1 at noon)], and 3) Wufong [(1,000 m el­evation, an average PPF at 55.4 imol+m-2+s-1 (maximum 60.9 imol+m-2+s-1 at noon)]. Each treatment contained 400 plants for evaluating the practical use of mass production, and treatments were arranged by a completely randomized design (CRD). The plants cultured by PBCM were
CHENG and CHANG ― Mycorrhizal Anoectochilus formosanus 461
transported to the Xindian and Wufong on April 14-15, 2005 respectively. All plants were shipped back to NTU, than random sampling (5 plants per pot, total of 40 pots, i.e. 200 plants per treatment) for statistical analyses on
Nov. 14-16, 2005.
Analysis of the antioxidant capacity
Two methods were used to examine the antioxidant capacity. The Folin-Ciocalteu method was used to examine the total phenolic contents (Kujala et al., 2000). And Trolox Equivalent Antioxidant Capacity (TEAC) was measured according to the method reported by Arnao et al. (1996). Spectrophotometer was used to determine the absorbance at 734 nm. Regression equation of standard curve was obtained based on the relation of absorbance and concentration of trolox. The absorbance was substituted into the equation to attain the TEAC value of the sample.
Analysis of the hepatoprotective agent
Analysis of the concentration of hepatoprotective agent was performed according to the method reported
by Wu et al. (Wu et al., 2007). The weight of a dry A.
formosanus plant was precisely measured and suitable quantity of water was added. The sample was extracted for three times using ultrasonic treatment and then proc­essed by gravity filtration. The filtrate was diluted, the target ingredient AFEE was determined quantitatively by high-performance liquid chromatography (HPLC). Test samples and reference standard were processed following the procedures described above and then examined by HPLC. After obtaining the standard calibration curve, the values of test samples were substituted into the regression equation of the standard reference to calculate their concentrations. The analyses were performed by Shih Hwa Biotech Co.
RESULTS AND DISCUSSION
Plastic bag cultivation method (PBCM)
PBCM has been applied for the cultivation of A. formosanus (Figure 1 ). This method offers several advantages. No more watering or fertilization is needed during the whole cultivation period, and the potential thread of pest attack and labor need for the in cultivation management are greatly reduced (Chang and Chou, 2007). In this study, PBCM was applied to large-scale cultivation of A. formosanus in three sites with different elevations, and more than 80% survival rate was reached. Cultivation of A. formosanus in greenhouse, chemical spray program is intensively needed and regularly performed. Most of the plants would be attacked, if pesticide, fungicide and mites-killing chemical are not regularly sprayed. By PBCM, survival rate of the ex vitro growth of plantlets and thus plant production are greatly increased without applying any pesticide, fungicide or any other agrichemicals and the growth period is also shortened. As a result of our
Figure 1. Plastic bag cultivation method (PBCM) for Anoectochilus formosanus. The metal wire (MW) was to prevent the collapse of plastic bag in the wildness.
studies, PBCM has now been promoted in the Wufong area and applied to large-scale and year-round commercial cultivation for more than 100,000 plants, and the survival rates were more than 90%.
Inoculation of OMF for the cultivation of A. formosanus
PBCM was adopted for the vessel cultivation of line B and line R of A. formosanus for both mycorrhizal and non-mycorrhizal control plants. After seven months of growth in three locations, the best plant growth was achieved in Wufong (1,000 m elevation). The number of leaves, fresh weight and chlorophyll meter reading of the plants in this location were all higher than those placed in other sites (Figures 2, 3 and 4). In addition, among the plants in
Wufong, those inoculated with OMF (either R02 or R04)
attained better growth than the non- mycorrhizal control. Based on the fresh weight, R04 showed better growth-promoting effect on line B, while R02 stimulated better growth for line R (Figures 2 and 3). Therefore, different lines of A. formosanus plants appeared to react differently to different OMF, and OMF inoculation does influence the growth of plants. Compared to the non-mycorrhizal control, OMF inoculation obviously promoted the plant growth and development. The fresh weight of mycorrhizal plants was consistently higher in all three elevations (Fig­ures 2 and 3). We also found that fresh weight of plants in Wufong was the heaviest in all three treatments even though taller plants were observed in two lower elevations such as NTU and Xindian (Figures 2 and 3). Further exam-
462 Botanical Studies, Vol. 50, 2009
2 0^64^
Jeqiclsl P8 tooLISVM i_d
^7^i Plant height Eaaaaa Leaf number i^^i Fresh weight
6} 1L-6{3AA L-saJ
Jeqolnu n pu& ud
s H-6est qs^lj
Inoculum &, Location
Figure 3. Growth of orchid mycorrhizal fungi (Rhizoctonia sp. R02 and R04) inoculated and non-mycorrhizal (NM) plants of Anoectochilus formosanus (line R) with plastic bag cultivation
method (PBCM) at National Taiwan University (NTU, 10 m of
elevation), and in Xindian (500 m) and Wufong (1,000 m) for seven months.
Figure 2. Growth of orchid mycorrhizal fungi (Rhizoctonia sp. R02 and R04) inoculated and non-mycorrhizal (NM) plants of Anoectochilus formosanus (line B) with plastic bag cultivation
method (PBCM) at National Taiwan University (NTU, 10 m of
elevation), and in Xindian (500 m) and Wufong (1,000 m) for seven months.
ination found that the plants in Wufong displayed shorter internodes but thicker stems, while the plants at NTU gen­erally showed the opposite features with longer internodes and thinner stems. We speculate that temperature probably was the cause of heavier fresh weight for the Wufong's plants. The average temperatures of the NTU and Xindian were about 30-23°C and 27-18°C respectively, while the Wufong site was around 25-15°C. When comparing A. formosanus plants cultivated in phytotron, those cultivated plants under controlled temperature at 20/15°C (day/ night) had shorter internodes and thicker stem; samples cultivated in phytotron at 30/25°C (day/night) grew taller and had thinner stem (unpublished data). Based on our studies, we conclude that temperature plays a critical role on the growth of A. formosanus, and is the main reason for the higher fresh weight of the plants grown in Wufong. Therefore, the ideal growing location for A. formosanus is at high elevation which has lower temperature and greater day/night temperature difference. Previous studies reported that when line C and line T of A. formosanus was inoculated either with Rhizoctonia sp R02 or R04, growth-promoting effects are obviously observed (Chang et al., 2007). In this experiment, two other different lines, line B and line R, were chosen and also inoculated with R02 or R04, and similar growth-enhancing effects were also observed. The results indicated that OMF inoculation can promote the growth of A. formosanus both in the cultiva­tion vessel and after transplantation. The absorbing areas of roots are increased as a result of OMF inoculation and therefore the plantlets can efficiently absorb nutrients and water. It was also reported that the quantity of chlorophyll as well as the efficiency of photosynthesis are increased due to OMF inoculation (Chu, 2000). In our experiments, the chlorophyll meter readings among mycorrhizal plants were generally higher than the controls (Figure 4). How­ever, when fresh weight and chlorophyll meter readings were cross-referenced, positive correlation was not found.
Inoculum & Location
Figure 4. Chlorophyll meter reading value of orchid
mycorrhizal fungi (Rhizoctonia sp. R02 and R04) inoculated and non-mycorrhizal (NM) plants of Anoectochilus formosanus (line B and line R) with plastic bag cultivation method (PBCM) at National Taiwan University (10 m of elevation), and in Xindian (500 m) and Wufong (1,000 m) for seven months.
This is may be due to the fact that plant growth is indeed influenced by various factors.
Analysis of the antioxidant capacity
1. Comparison and analysis of the phenolic com­pounds. Phenolic compounds are widely distributed in the plant kingdom. They are plant secondary metabolites which are often used as an index for antioxidant capacity (Castelluccio et al., 1995). Our analyses revealed that A. formosanus line B grown in Wufong contained higher lev­el of phenolic compounds; however the levels of phenolic compounds of the NM control and mycorrhizal plants in this location were no significant difference (Figure 5A). The levels of phenolic compounds of mycorrhizal plants at
CHENG and CHANG ― Mycorrhizal Anoectochilus formosanus
463
(63S 5uj) s!IOUSLId leloh
B'MM
B-R02
mm
i i
R-NM
R-R02
R-R04
(lueld3d /3VE> _spo JOLSlJd leio
10 500 1000 10 500 1000
Elevation (m) Elevation (m)
Figure 5. Changes of phenolic compounds level of two lines of Anoectochilus formosanus inoculated with Rhizoctonia sp. R02 and R04 in the bag culture at National Taiwan University (10 m of elevation), and in Xindian (500 m) and Wufong (1,000 m) for seven months. A, level of phenolic compounds per gram; B, level of phenolic compounds per plant.
NTU and in Xindian were higher than the non mycorrhizal control (Figure 5A). The levels of phenolic compounds among line R plants inoculated with R04 in three locations were similar (Figure 5A). For the NM and R02-inoculated, the levels of phenolic compounds were higher at NTU and in Wufong; the R02-inoculated group had higher phenolic compounds level, with 5.06 mg/g in the NTU plants and 4.96 mg/g in the Wufong ones. Considering the dilution effect of fast-growing plants, we converted the level of phenolic compounds into the whole plant basis as shown in Figure 5B. Results showed that the level of phenolic compounds was the highest among the plants grown in Wufong both for line B and line R, and the phenolic com­pound levels found in the OMF inoculated plants were higher than the NM plants (Figure 5B). Previous studies indicated that the cultivation environment affects the types and quantity of phenolic compounds in a plant (Dumas et al., 2003; Roussous et al., 2007). Our results demonstrate that cooler area such as Wufong was more suitable for the growth of A. formosanus and the level of phenolic com­pounds found obtained from these plants was higher.
2. Test of Trolox Equivalent Antioxidant Capacity
(TEAC). In previous experiments, antioxidant capacity of A. formosanus was tested by TEAC, FRAP (Ferric reducing antioxidant power) and DPPH (1,1-diphenyl-2-picrylhydrazyl) method. In this study, TEAC was chosen to determinate of the antioxidant capacity by its comparative stability and significant differences in data reading between the mycorrhizal and non-mycorrhizal plants.
Our results showed that the TEAC readings of the my-corrhizal line B at NTU and in Xindian were higher than the NM plants. In line R plants, the R02-inoculated ones at NTU and in Wufong had higher TEAC readings (Figure 6).
Previous studies showed the A. formosanus extract dis­plays free radical scavenging activity (Wang et al., 2002).
V/////. R-R02 B-R04
(,)3mv> GV31
10 500 1000
Elevation (m)
Figure 6. The analyses of trolox equivalent antioxidant capacity (TEAC) values for two lines of Anoectochilus formosanus inoculated with Rhizoctonia sp. R02 and R04 and cultivated at NTU (10 m of elevation), and in Xindian (500 m) and Wufong (1,000 m), for seven months.
Under the treatment of A. formosanus, HepG22 cell dem­onstrated stronger solvency over free radicals and hydro­gen peroxide and maintain effective antioxidant capacity (Hsieh, 2001). Anoectochilus formosanus also contains various enzymes including superoxide dismutase (SOD), catalase, peroxidase and ascorbate peroxidase (Wang, 1999). In 2004, analyses of the enzyme activities and the plant components demonstrated that stronger antioxidant capacity was found in the mycorrhizal A. formosanus plants. Our results showed that mycorrhizal line B plants at NTU and in Xindian exhibited higher level of TEAC than the NM control plants. R02 inoculated line R plants cultivated at NTU and in Wufong had higher TEAC read­ing as well. The results demonstrated that the mycorrhizal
464
Botanical Studies, Vol. 50, 2009
A. formosanus plants showed different antioxidant capac­ity under different environments.
Analysis of the hepatoprotective components
Previous studies revealed that A. formosanus contains hepatoprotective components and thus displays hepatopro-tective effect (Huang, 2000). Ethyl estate (EtOAc) extract from A. formosanus can lower the GOT and GPT concen­trations of liver cells, increase the quantity of glutathione, and decrease the quantity of malondialdehyde (MDA), a product of lipid peroxidation, and thus the hepatoprotec-tive effect is achieved (Lin et al., 1991). Our studies dem­onstrated that OMF inoculation can increase the quantity of hepatoprotective agent AFEE, an ethyl acetate-philic fraction of the extracts of A. formosanus. The levels of AFEE were higher in either R02 or R04 inoculated line B and line R plants than those of the NM control plants at NTU. The level of AFEE found in the mycorrhizal line B plants was more than two folds higher than that of the NM control plants (Table 1). Therefore, our results showed that OMF inoculation can effectively promote the growth of A. formosanus, and also increase the levels of their medicinal components, their antioxidant capacity and the quantity of
AFEE (Table 1).
Our studies demonstrated that PBCM is an effective mass production method for producing agrichemical free A. formosanus plants. Both NM and mycorrhizal plants grew well in Wufong, due to the higher elevation, lower temperature and greater temperature difference for day and night. Inoculation of OMF (R02 or R04) significantly pro­moted the growth of A. formosanus, but the effects might vary dependent upon the combination of OMF and orchid line and as well as the growth environment. Generally our results showed that the mycorrhizal plants contained higher amount of phenolic compounds, stronger antioxi-dant capacity, and more abundance of the hepatoprotective agent AFEE in comparing to NM plants of all locations, but varied with the different environments. We presume
Table 1. The analysis of hepatoprotective activity of kinsenoside (AEEE) derived from mycorrhizal and non-mycorrhizal Anoectochilus formosanus plants cultivated at
NTU campus for seven months.
that the wild plants of A. formosanus may be all infected with some kind of OMF fungi and thus result in better me­dicinal effects than the tissue-cultured NM plants in green­house. Based on the results, we highly recommend PBCM for mass production of agrichemical free A. formosanus plants. One thousand meter of elevation is an ideal place for cultivating A. formosanus plants in Taiwan. The inocu­lation of Rhizoctonia sp, either R02 or R04, can signifi­cantly increase the production of A. formosanus containing higher antioxidant capacity and hepatoprotective agent for medicinal use.
Acknowledgments. This project was financially supported by the Forestry Bureau, Council of Agriculture, Executive Yuan, Taiwan (94AS-9.4.1-FB-el, 95AS-11.4.1-FB-el,
96AS-11.4.1-FB-e1). We thank Dr. Tsu-Hwie Liu in the
Development Center for Biotechnology, Xizhi, Taipei, Taiwan and Dr. Wei-Chiang Shen in the Department of Plant Pathology and Microbiology, National Taiwan University for improving the manuscript.
LITERATURE CITED
Arnao, M.B., A. Cano, J. Hernandez-Ruiz, F. Garcia-Ca-novas, and M. Acosta. 1996. Inhibition by L-ascorbic acid and other antioxidants of the 2.2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) oxidation catalyzed by peroxidase: a new approach for determining total antioxidant status of foods. Anal. Biochem. 236: 255-261.
Castelluccio, C., G. Paganga, N. Melikian, G. Bolwell, J. Pridham, J. Sampson, and C. A. Rice-Evans. 1995. Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Lett. 368: 188-192.
Chang, D.C.N. 2008. The Screening of Orchid Mycorrhizal
Fungi (OMF) and their Applications. In W. H. Chen and H. H. Chen (eds.), Orchid Biotechnology. World Scientific Publishing Co. Pte. Ltd. 5 Joh Tuck Link, Singapore, pp. 77-98.
Chang, D.C.N. and L.C. Chou. 2007. Growth responses, enzyme activities, and component changes as influenced by Rhizoctonia orchid mycorrhiza on Anoectochilus formosanus Hayata. Bot. Stud. 48: 445-451.
Chang, D.C.N., L.C. Chou, and G.C. Lee. 2007. New cultivation
methods for Anoectochilus formosanus Hayata. Orchid Sci.
Biotechnol. 1: 56-60.
Chang, S.F. 1999. Integrated control of basal stem rot and stem rot of Anoectochilus. Master thesis, Department of Plant Pathology, National Chung Hsing University, 100 pp.
Chou, L.C. 2004. The mycorrhizal physiology and cultivation of Anoectochilus formosanus Hayata, Haemaria discolor var. dawsoniana and their F1 hybrids. PhD dissertation, Graduate Institute of Horticulture, National Taiwan University, 169 pp.
Chu, J.N. 2000. Isolation and inoculation of orchid mycorrhizal fungi and their effects on the seedling growth of Oncidium Goldiana X Onc. Guiena Gold. Master thesis, Department
Cultivar Rhizoctonia inoculum AFEE (g/g)
B
NM
0.0174±0.0003 b
R02
0.0417±0.0011 a
R04
0.0400±0.0019 a
R
NM
0.0126±0.0004 c
R02
0.0242±0.0029 a
R04
0.0191±0.0025 b
NM: non-mycorrhizal control group. Three replicates were conducted for each treatment. Means in each column followed by different letters were significantly different (P=0.05) as determined by LSD test.
CHENG and CHANG ― Mycorrhizal Anoectochilus formosanus
465
of Tropical Agriculture, National Ping Tung University of Science and Technology, 101 pp.
Dumas, Y., M. Dadomo, G. D. Lucca, and P. Grolier. 2003.
Effects of environmental factors and agricultural techniques on antioxidant content of tomato. J. Sci. Food Agric. 83: 369-382.
Hsieh, J.S. 2001. Oxidative stress mediate the anti-proliferative effect of Anoectochilus formosanus on hepatoma cells. Master thesis, Graduate Institute of Medicinal Science, China Medical College, 69 pp.
Huang, D.F. 2000. The mechanism study of hepatoprotective control of Boehmeria species and Anoectochilus species. Master thesis, Graduate Institute of Natural Products, Kaohsiung Medical University, 90 pp.
Kujala, T.S., J. M. Loponen, K.D. Klika, and K. Pihlaja. 2000.
Phenolics and betacyanins in red beetroot (Beta vulgaris) root: distribution and effect of cold storage on the content of total phenolics and three individual compounds. J. Agric.
Food Chem. 48: 5338-5342.
Lee, G.C. 2001. Identification and improvement of production techniques for Anoectochilus formosanus Hayata and orchid mycorrhizal Fungi. PhD dissertation, Graduate Institute of Horticulture, National Taiwan University, 191 pp.
Lin, J.M., C.C. Lin, H.F. Chiu, and S.G. Lee. 1991. Evaluation
of the anti-inflammatory and liver-protective effects of
Anoectochilus formosanus, Ganoderma lucidum and Gynostemma pentaphyllum in rats. Paper presented at the seminar on the plants of medicinal and health, pp. 89-98.
Lin, W.C. 2007. Study of health keeping effects of Anoectochilus formosana Hayata. Agric. World 288: 8-13.
Roussous, P.A., A. Matsoukis, C.A. Pontikis, and A. Chronopou-lou-Sereli. 2007. Relations of environmental factors with the phenol content and oxidative enzyme activities of olive
explants. Sci. Hortic. 113: 100-102.
Wang, S.C. 1999. Studies on antioxidative enzymes in Anoectochilus formosanus Hayata. Master thesis, School of Pharmacy, National Taiwan University, 82 pp.
Wang, S.Y., Y.H. Kuo, H.N. Chang, P.L. Kang, H.S. Tsay, K. Lin, N.S. Yang, and L.F. Shyur. 2002. Profiling and
characterization antioxidant activities in Anoectochilus formosanus Hayata. J. Agric. Food Chem. 50: 1859-1865.
Wu, J.B., W.L. Lin, C.C. Hsieh, H.Y. Ho, H.S. Tsay, and W.C.
Lin. 2007. The hepatoprotective activity of kinsenoside from Anoectochilus formosanus. Phytother. Res. 21: 58-61.
Wu, K.B. 2007. The use and potential for Anoectochilus formosanus Hayata. Agric. World 288: 14-19.
Yen, D.M., S.C. Chen, and C.T. Liao. 1996. The art for propagation of Jin-Shiann Lian. Dong-Kung Farmer Association Published, 294 pp.
466
Botanical Studies, Vol. 50, 2009
台灣金線連於不同海拔地區接種蘭菌後其生長發育及
有效成分之變化
鄭淑芬張喜寧
國立台灣大學園藝學研究所
於台大(海拔10公尺)、新店(海拔500公尺)、五峰(海拔1000公尺)等三個不同海拔地區以
塑膠袋栽培法(PBCM),種植接種絲核菌屬蘭菌R02R04BR等兩種不同品系的台灣金線連,
栽培七個月,結果顯示以塑膠袋套袋栽培法栽培之所有臺灣金線連組培苗,於出瓶後其成活率均在
80%以上,且可顯著提高植株產量,及不需施用任何化學農藥,假使台灣金線連不是利用塑膠袋培法
(PBCM)栽種,則在溫室中栽培3-4個月後,每1-2個星期若不進行殺蟲劑與殺菌劑的噴施,則所有
植株將會死亡,和傳統栽培方法相比較,則可縮短栽培期1-2個月。其中種植於五峰,且接種蘭菌之
植株生長最佳,鮮重亦較對照組高,B品種則以接種蘭菌R04 R品種以接種蘭菌R02可顯著促進生
長。種植於台大之台灣金線連BR兩品系均以接種R02之菌根植株有較高的總酚類及保肝活性成分
AFEE之含量。以TEAC的方法分析測試其抗氧化能力,結果顯示大部分菌根植株較非菌根植株為高。
本研究結果證實塑膠袋栽培法為一非常省工(可省下栽培期間之所有灌溉與施肥之人力),且不需噴施
任何殺蟲劑及殺菌劑等化學藥劑即可大量生產臺灣金線連之有效方法,接種蘭菌R02R04可顯著增
加台灣金線連的產量,並提高抗氧化能力及保肝成分之含量。
關鍵詞:台灣金線連;抗氧化能力;保肝成分;蘭菌;總酚類含量;塑膠袋栽培法。