USE OF TISSUE CULTURE FOR THE MASS PROPAGATION OF PATHOGEN-FREE PLANTS Hsin-Sheng Tsay Department of Applied Chemistry, Chaoyang University of Technology 168 Gifeng E. Rd., Wufeng, Taichung 41301, Taiwan

ABSTRACT Different in vitro culture techniques have been used for rapid plant propagation. Pathogenfree plants have been produced using techniques such as seed culture, meristem culture, micropropagation using axillary or adventitious shoot buds, and somatic embryogenesis. This Bulletin discusses the importance of in vitro culture techniques for the production of pathogen-free medicinal plants such as Anoectochilus formosanus, Limonium wrightii and Corydalis yanhusuo.

INTRODUCTION Plant tissue culture technology has been successfully used for the commercial production of pathogen-free plants (Debergh and Maene 1981), and to conserve the germplasm of rare and endangered species (Fay 1992). Techniques such as meristem culture (Hu and Wang 1983) and hot-water treatment of explants before in vitro culture (Hol and van der Linde 1992, Langens-Gerrits et al. 1998) have been used to produce plants free from pathogens. In Taiwan, plant tissue culture is being used for the mass production of medical plants that in the wild are fairly rare, and which are becoming scarcer as time goes by. Medicinal plants are important to the health of many people in developing countries. Approximately 80% of people in developing countries still rely on traditional medicine for their primary health care needs. This usually involves the use of plant extracts (Vieira and Skorupa 1993). Many medicinal plant species are

disappearing at an alarming rate, as a result of rapid agricultural and urban development, deforestation and indiscriminate collection. Plant tissue culture technology may help to conserve rare and endangered medicinal plants. Many important Chinese medicinal herbs have been successfully propagated in vitro, either by organogenesis (Erdei et al. 1981, Shoyama et al. 1983, Hatano et al. 1986, Matsumoto et al. 1986, Hiraoka and Oyanagi 1988, Nishioka 1988, Tsay et al. 1989, Huang et al. 2000, Chen et al. 2001, Chueh et al. 2001) or by somatic embryogenesis (Hiraoka et al. 1986, Kitamura et al. 1989, Tsay and Huang 1998, Sagare et al. 2000). In vitro propagated plants of many important medicinal species were found to be uniform, showing less variation in their content of secondary metabolites than their wild/cultivated counterparts (Yamada et al. 1991). This paper describes the propagation of Anoectochilus formosanus using nodal explants, Limonium wrightii through adventitious shoot buds, and Corydalis yanhusuo by means of somatic embryogenesis.

Keywords: Anoectochilus formosanaus, Corydalis yanhusuo, germplasm conservation, Limonium wrightii, medicinal plants, micropropagation, pathogen-free plants, plant tissue culture, shoot morphogenesis, somatic embryogenesis

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TISSUE CULTURE OF THE ORCHID ANOECTOCHILUS FORMOSANUS Micropropagation using nodal explants Anoectochilus formosanus Hayata (Orchidaceae), is an important medicinal herb. It belongs to a group of terrestrial orchids commonly known as “Jewel Orchids”, because of their attractive foliage (Teuscher 1978). A. formosanus is an endemic species in Taiwan. It grows to a height of 20 – 30 cm, and has velvety-textured leaves, the upper surface of which is dark green with intricate silver veins. The lower surface of the leaves is pale purple in color. A. formosanus is found at an altitude of 800 – 1500 m in the central mountain range of Taiwan and the offshore island of Lanyu. It is also found in the Japanese Ryukyu Islands, and Fujian province in Mainland China (Anon 1999). In Taiwanese folk remedies, the whole plant of A. formosanus, fresh or dried, is boiled in water and taken internally to treat chest and abdominal pains (Hu 1971), diabetes, nephritis (Chiu and Chang 1995), fever, hypertension, impotence, liver and spleen disorders and pleurodynia (Kan 1986). The fresh herb is applied externally as a treatment for snake-bite (Kan 1986). The current market price of the fresh herb collected from its natural habitat is around US$320 per kilogram, while the dried herb sells for US$3200 per kilogram. A. formosanus is a slow-growing perennial herb. It reproduces from seed, but seedlings take 2 – 3 years to reach maturity and bear seed. It flowers only once a year, in winter (October to December). The indiscriminate collection of plants, often before they are old enough to bloom, has decimated the species so that its survival is in question. A reduction in the population size of A. formosanus may lead to a reduced gene flow, inbreeding depression and reduced fitness. The development of a rapid in vitro propagation system for A. formosanus would not only help in conserving the germplasm and provide new opportunities for commercial cultivation, but would also help to take some of the pressure off the natural populations

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(Shiau et al. 2002). In Taiwan, A. formosanus is cultivated on a limited scale by a few farmers. The species is difficult to cultivate, as it is delicate and highly susceptible to stem and rhizome rot caused by Fusarium oxysporum (Hsieh et al. 1994). This problem could be overcome by artificial cross-pollination, using elite individuals of A. formosanus to produce robust seedlings which are less likely to get infected by the disease. Another possibility is the in vitro propagation of elite plants. Methods for the in vitro mass propagation of A. formosanus have already been developed, using shoot tip and/or nodal explants (Chow et al. 1982, Liu et al. 1987, Ho et al. 1987, Tai 1987, Tsay 1999) and seeds from capsules collected from wild (Ho et al. 1987, Lee et al. 1992) or handpollinated plants (Shiau et al. in press). We have developed a method of using nodal explants for the rapid production of diseasefree plants of A. formosanus (Fig. 1).

Method Healthy, two-year-old plants of A. formosanus were used as a source of nodal explants (Fig. 1). Nodal explants 2-4 cm in length were cultured in liquid Murashige and Skoog’s (MS) medium (Murashige and Skoog 1962) supplemented with 2.0 mg/LBA (N 6 benzyladenine) and 0.5 mg/1NAA ( α naphthaleneacetic acid) in a 500-mL Erlenmeyer flask. After two months of culture, axillary shoot buds began to emerge from the explants (see arrows in Fig. 2). Axillary shoots elongated and rooted when subcultured on agar-gelled half-strength MS medium with 2.0 mg/1 BA + 0.5 mg/1 NAA and 0.2% (w/v) activated charcoal and incubated for 2 months (Fig 3). Plants with well-developed rhizomes were washed with tap water, transferred to plastic trays containing a peat moss:vermiculite mixture and acclimatized in a growth chamber (Fig. 4). Over 90% of the plants survived after being transplanted to the potting mixture. This method is being used to produce diseasefree elite plants of A. formosanus on a commercial scale in Taiwan.

Fig. 1

Fig. 3

Fig. 2

Fig. 4

Fig. 1.

Propagation of disease-free plants of Anoectochilus formosanus Hayata, using stem node explants from elite plants. Healthy, 2-year-old plant of A. formosanus grown in a growth chamber.

Fig. 2.

Emergence of axillary shoot buds (arrows) from nodal explants of A. formosanus after 2 months of culture in liquid MS medium with 2.0 mg/1 BA and 0.5 mg/1 NAA in 500-ml Erlenmeyer flask and incubation on an orbital shaker with a rotary motion of 100 rev/min.

Fig. 3.

Elongation of axillary shoots of A. formosanus after culture on agar-gelled half-strength MS medium supplemented with 2.0 mg/1 BA + 0.5 mg/1 NAA and 0.2% (w/v) activated charcoal in 500-ml Erlenmeyer flasks.

Fig. 4.

A. formosanus plants with well-developed rhizomes which have been transferred to 42x31x11 cm plastic trays containing peat moss:vermiculite (1:1 v/v) and incubated at 25 oC/20oC day/night temperature and light at 44 µE m-2 s-1 in a growth chamber.

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CULTIVATION OF A PERENNIAL HERB Propagation of Limonium wrightii (Hance) Ktze. (Pumbaginaceae), an important traditional medical plant, using adventitious shoot buds Limonium wrightii is a herbaceous perennial plant. It is found on the Bonin Islands and the Ryukyu Islands of Japan, in the southern part of Taiwan, and on the Taiwanese islands of Lanyu and Lutao (Li 1978). It grows among rocks along the seashore. In Taiwan, L. wrightii is cultivated from seeds on a limited scale by a few farmers on Lutao Island. However, it is not possible to produce a large number of elite plants within a short period using seeds, because it flowers only in the autumn, and seed set and germination are very poor. The dried plants with the leaves removed are used to make traditional Chinese medicinal preparations. These are mainly used for the treatment of asthma, tuberculosis, colds, hypertension and backache and also as a fortifying agent (Kan 1978). Because of the increasing demand and high price, L. wrightii plants have been collected indiscriminately from their natural habitat, and now are very rare in the wild. To conserve the germplasm and encourage commercial cultivation of L. wrightii, we have recently developed a method of rapid in vitro plant propagation using the shoot tip, leaf base and inflorescence node explants (Huang et al. 2000). Fig. 5 shows the propagation of L. wrightii using leaf base explants. Healthy plants of L. wrightii were collected from farmers on Lutao Island and raised in a growth chamber. Leaves were taken from the plants in the growth chamber. Their surface was disinfected, and the leaves were cultured on MS medium supplemented with 2.0 mg/1 BA and 0.2 mg/L NAA for two months. Well-developed adventitious shoots formed on the leaf base explants (Fig. 6). Adventitious shoots were multiplied by subculturing on 2x-strength MS medium supplemented with 2.0 mg/LBA and 0.2 mg/L NAA (Fig. 7). When shoot multiplication medium was used, an average of eight shoot buds could be produced per shoot. Shoots were easily rooted on MS basal medium with 1.0 mg/Lindole-3-butyric acid (IBA) (Fig. 8).

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Using this protocol, it is possible to produce a large number of healthy, uniform plants of this endangered species.

PRODUCTION OF MEDICINAL TUBERS Propagation of disease-free tubers from somatic embryo-derived plants of Corydalis yanhusuo W.T. Wang (Fumariaceae) The dried and pulverized tubers of Corydalis yanhusuo are used in traditional herbal remedies in China, Japan, and Korea. C. yanhusuo is also known as Rhizoma corydalis (Tang and Eisenbrand 1992). The tubers are used in traditional Chinese medicine to treat gastric and duodenal ulcers, cardiac arrhythmia (Kamigauchi and Iwasa 1994), rheumatism and dysmenorrhea (Tang and Eisenbrand 1992). C. yanhusuo is cultivated as an annual crop, using the tubers. However, the plants are susceptible to fungal diseases, especially downy mildew caused by Peronospora corydalis de Bary. This infects the tubers, and may cause losses of 30 – 50% when conditions are suitable for fungal growth (Gao et al. 1991). To improve the yield and quality of the tubers, it is necessary to have pathogen-free planting material. Pathogen-free plants of C. yanhusuo can be obtained from seed, but the seeds have a low germination rate (Hu and Liang 1996). They also take a long time to germinate, because of the extremely immature state of the zygotic embryo at the time of dispersal. Both warm and cold stratification treatments are required to induce the germination of seeds. Furthermore, the growth of the seedling is very slow. During the first year, the plant forms only a small, immature tuber. Mature tubers can be obtained only in the following year, after a period of dormancy (Hu and Liang 1996). Recently, we have reported an efficient method of plant regeneration by means of somatic embryogenesis from tuber-derived callus (Sagare et al. 2000). Using this protocol, it is possible to produce a large number of disease-free plants and tubers of C. yanhusuo in a short time (Fig. 9). Mature, healthy tubers were used to establish embryogenic callus cultures of C.

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 5.

Rapid in vitro propagation of Limonium wrightii (Hance) Ktze. mature plant of L. wrightii in 14-cm diameter pot

Healthy,

Fig. 6.

Direct regeneration of shoots from the basal part of a leaf explant cultured on MS medium, supplemented with 2.0 mg/LBA and 0.2 mg/1 NAA after two months of incubation.

Fig. 7.

Rapid multiplication of adventitious shoots in a 500 ml Erlenmeyer flask with 2x-strength MS medium supplemented with 2.0 mg/LBA and 0.2 mg/ LNAA.

Fig. 8.

Rooted axillary shoots after two months of culture in MS basal medium, supplemented with 1.0 mg/1 IBA.

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Fig. 11

Fig. 9

+ Fig. 10 Fig. 9.

Fig. 12

Production of disease-free plants and tubers of Corydalis yanhusuo through somatic embryogenesis. Embryogenic callus derived from mature, healthy tubers after three months of culture on MS medium supplemented with 2.0 mg/LBA and 0.5 mg/LNAA and incubation in darkness.

Fig. 10. Tuber-derived embryogenic callus showing the formation of a cotyledonarystage somatic embryo (C) and converted somatic embryo (arrows) after five weeks of culture on medium supplemented with 1.0 mg/L zeatin riboside. Fig. 11. Converted somatic embryos with well-developed shoot and root after culture in half-strength liquid MS medium which contains 1.0 mg/L zeatin riboside and has been on a rotary shaker for two weeks. Fig. 12. Somatic embryo-derived tubers (arrows) formed after 9 months of culture on half-strength MS medium supplemented with 3% sucrose.

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yanhusuo. Embryogenic callus was induced by culturing mature tuber pieces on MS medium, supplemented with 2.0 mg/LBA and 0.5 mg/1 NAA, and incubating them in darkness for three months. Somatic embryos were induced by subculturing the primary callus on MS medium supplemented with 1.0 mg/L zeatin, with two weeks of culture in light. Fig. 10 shows a cotyledonary-stage somatic embryo (c) and a converted somatic embryo (arrow) formed on primary callus. Somatic embryos converted into plantlets when they were transferred into liquid MS medium supplemented with 1.0 mg/L zeatin riboside (Fig. 11). Converted somatic embryos were cultured on half-strength MS medium, supplemented with 3% sucrose to promote the further development of plantlets and tubers. Well-developed, healthy tubers were obtained after six months of culturing somatic embryoderived plants (Fig. 12). These tubers can be used as disease-free planting materials. Tubers derived from somatic embryos of C. yanhusuo have recently been shown to contain pharmaceutically important compounds, such as D,Ltetrahydropalmatine and D-corydaline (Lee et al. 2001).

CONCLUSIONS Tissue culture technology makes it possible to produce a large number of diseasefree and uniform plants of medicinally important species. Such plants can be used for the extraction of medicinally important compounds, or for pharmacological studies. They can also help in germplasm conservation, and can be reintroduced into protected habitats such as National Parks. Pathogen-free plants maintained under in vitro conditions can also be used for the safe exchange of germplasm across national borders.

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