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, 2002-07-01

Different in vitro culture techniques have been used for rapid plant propagation. Pathogen-free 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.

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 (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 hand-pollinated plants (Shiau et al. in press). We have developed a method of using nodal explants for the rapid production of disease-free 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 (N⁶-benzyladenine) and 0.5 mg/1NAA (a- 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 disease-free elite plants of A. formosanus on a commercial scale in Taiwan.

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). 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. 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 embryo-derived 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,L-tetrahydropalmatine and D-corydaline (Lee et al. 2001).

Conclusions

Tissue culture technology makes it possible to produce a large number of disease-free 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.

References

Anon. 1999. Anoectochilus formosanus Hayata. In: Chung-Hua-Ben-Tsao, Vol. 8. Shanghai Science Technology Press, Shanghai, pp. 672-673. (In Chinese).
Chen, C.C., S.J. Chen, A.P. Sagare and H.S. Tsay. 2001. Adventitious shoot regeneration from stem internode explants of Adenophora triphylla (Thunb.) A.DC. (Campanulaceae) _ an important medicinal herb. Botanical Bulletin of Academia Sinica 42:1-7.
Chiu, N.Y. and K.H. Chang. 1995. Anoectochilus formosanus Hayata. In: The Illustrated Medicinal Plants of Taiwan, Vol. 4 SMC Publishing Inc., Taipei, Taiwan, pp. 282-283. (In Chinese).
Chow, H.T., W.C. Hsieh and C.S. Chang. 1982. In vitro propagation of Anoectochilus formosanus. Journal of Science and Engineering 19: 155-165. (In Chinese).
Chueh, F.S., C.C. Chen, A.P. Sagare and H.S. Tsay. 2001. Quantitatiave determination of secoiridoid glucosides in in vitro propagated plants of G. davidii var. formosana by high performance liquid chromatography. Planta Medica 67: 70-73.
Debergh, P.C. and L.J. Maene. 1981. A scheme for commercial propagation of ornamental plants by tissue culture. Scientia Horticulturae 14: 335-345.
Erdei, I., Z. Kiss and P. Maliga. 1981. Rapid clonal multiplication of Digitalis lanata in tissue culture. Plant Cell reports 1: 34-35.
Fay, M.F. 1992. Conservation of rare and endangered plants using in vitro methods. In: Vitro Cellular and Developmental Biology. pp. 1-4. 28pp.
Hatano, K., Y. Shoyama and I. Nishioka. 1986. Multiplication of Pinellia ternate by callus culture of leaf segment. Shoyakugaku Zasshi 40: 188-192.
Hiraoka, N., T. Kodama, M. Oyanagi, S. Nakano, Y. Tomita, N. Yamada, N. Yamada, O. Iida and M. Satake. 1986. Characteristics of Bupleurum falcatum plants propagated through somatic embryogenesis of callus cultures. Plant Cell Reports 5: 319-321.
Hiraoka, N. and M. Oyanagi. 1988. In vitro propagation of Glehnia littoralis from shoot-tips. Plant Cell Reports 7: 39-42.
Ho, C.K., S.H. Chang and Z.Z. Chen. 1987. Tissue culture and acclimatization in Anoectochilus formosanus Hay. Bulletin of Taiwan Forestry Research Institute 2: 83-105. (In Chinese).
Hol, G.M.G.M. and P.C.G. van der Linde. 1992. Reduction of contamination in bulb-explant cultures of Narcissus by a hot-water treatment of parent bulbs. Plant Cell, Tissue and Organ Culture 31: 75-79.
Hsieh, S.P.Y., W.R. Chuang, S.F. Huang. 1994. Stem rot of Taiwan Anoectochilus and its causal organism. Plant Pathology Bulletin 3: 140-146. (In Chinese).
Hu, S.Y. 1971. The Orchidaceae of China. Quarterly Journal of the Taiwan Museum 24: 67-103.
Hu, C.Y. and P.J. Wang. 1983. Meristem, shoot tip, and bud cultures. In: Evans, D.A., Sharp, W.R., Ammirato, P.V. and Y. Yamada (Eds.). Handbook of Plant Cell Culture: Techniques for Propagation and Breeding. Vol. 1. Macmillan Publishing Co., New York, pp. 177-227.
Huang, C.L., M.T. Hsieh, W.C. Hsieh, A.P. Sagare and H.S. Tsay. 2000. In vitro propagation of Limonium wrightii (Hance) Ktze. (Plumbaginaceae), an ethno-medicinal plant, from shoot-tip, leaf- and inflorescence-node explants. In vitro Cellular and Developmental Biology _ Plant 36: 220-224.
Kamigauchi, M. and K. Iwasa. 1994. VI Corydalis spp.: In vitro culture and the biotransformation of protoberberines. In: Biotechnology in Agriculture and Forestry, Y.P.S. Bajaj (Ed.). Vol. 26. Springer-Verlag, Berlin Heidelberg, pp. 93-105.
Kan, W.S. 1978. Manual of Vegetable Drugs in Taiwan. Part 2. Medicine Publishing Inc., Taipei, Taiwan, pp. 43-44.
Kan, W. S. 1986. Anoectochilus formosanus Hay. In: Pharmaceutical Botany. National Research Institute of Chinese Medicine, Taipei, Taiwan, pp. 647. (In Chinese).
Kitamura, Y., H. Miura and M. Sugii. 1989. Plant regeneration from callus cultures of Swertia pseudochinensis. Shoyakugaku Zasshi 43: 256-258.
Langens-Gerrits, M., M. Albers and G.J. De Klerk. 1998. Hot-water treatment before tissue culture reduces initial contamination in Lilium and Acer. Plant Cell, Tissue and Organ Culture 52: 75-77.
Lee, S.G., C.L. Huang and C.C. Lin. 1992. Development of natural crude drug resources from Taiwan (XI) _ study on economic cultural practices in Anoectochilus culture. Journal of Chinese Medicines 3: 53-60. (In Chinese).
Lee, Y.L., A.P. Sagare, C.Y. Lee, H.T. Feng, Y.C.Ko, J.F. Shaw and H.S. Tsay. 2001. Formation of protoberberine-type alkaloids by the tubers of somatic embryo-derived plants of Corydalis yanhusuo. Planta Medica 67:839-842..
Li, H.L. 1978. Plumbaginaceae. In: Flora of Taiwan, H.L. Li, T.S. Liu, T.C. Huang, T. Koyama and C.E. Devol (Eds.). Vol. 4. Epoch Publishing Co., Ltd. Taipei, Taiwan, Republic of China, pp. 90-93.
Liu, S.Y., H.S. Tsay, H.C. Huang, M.F. Hu and C.C. Yeh. 1987. Comparison of growth characteristics and nutrient composition between plants of Anoectochilus species from mass vegetative propagation by tissue culture techniques. Journal of Agricultural Research of China 36: 357-366. (In Chinese).
Matsumoto, M., M. Nagano, Y. Shoyama and I. Nishioka. 1986. New vegetative propagation method of Rehmannia glutinosa. Shoyakugaku Zasshi 40: 193-197. (In Japanese).
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.
Nishioka, I. 1988. Clonal multiplication of medicinal plants by tissue culture. Shoyakugaku Zasshi 42: 1-11.
Sagare, A.P., Y.L. Lee, T.C. Lin, C.C. Chen and H.S. Tsay. 2000. Cytokinin-induced somatic embryogenesis and plant regeneration in Corydalis yanhusuo (Fumariaceae) _ a medicinal plant. Plant Science 160: 139-147.
Shiau, Y.J., U.C. Chen, S.R. Yang, A.P. Sagare and H.S. Tsay. 2002. Conservation of Anoectochilus formosanus, a medicinally important terrestrial orchid, by synchronizing flowering, hand-pollination, and in vitro culture of seeds. Bot. Bull. Acad. Sin. 43: 123-130.
Shoyama, Y., K. Hatano and I. Nishioka. 1983. Clonal multiplication of Pinellia ternate by tissue culture. Planta Medica 49: 14-16.
Tai, K.S. 1987. In vitro propagation of Anoectochilus formosanus (Hayata). Journal of the Agricultural Association of China 137: 42-54. (In Chinese).
Tang, W. and G.Eisenbrand. 1992. Corydalis turtschaninovii Bess. F. yanhusuo Y.H. Chou et C.C. Hsü. In: Chinese Drugs of Plant Origin, Chemistry, Pharmacology, and Use in Traditional and Modern Medicine, W. Tang and G. Eisenbrand (Eds.). Springer- Verlag, Berlin Heidelberg, pp. 377-393.
Teuscher, H. 1978. Erythrodes, Goodyera, haemaria and Macodes, with Anoectochilus. American Orchid Society Bulletin 47: 121-129.
Tsay, H.S. 1999. Tissue culture of Anoectochilus formosanus. Paper presented at the 16th World Orchid Conference, Vancouvor, Canada.
Tsay, H.S., T.G. Gau and C.C. Chen. 1989. Rapid clonal propagation of Pinellia ternate by tissue culture. Plant Cell Reports 8: 450-454.
Tsay, H.S. and H.L. Huang. 1998. Somatic embryo formation and germination from immature embryo-derived suspension-cultured cells of Angelica sinensis (Oliv.) Diels. Plant Cell Reports 17: 670-674.
Vieira, R.F. and L.A. Skorupa. 1993. Brazilian medicinal plants gene bank. Acta Hortculturae 330: 51-58.
Yamada, Y., Y. Shoyama, I. Nishioka, H. Kohda, A. Namera and T. Okamoto. 1991. Clonal micropropagation of Gentiana scabra Bunge var. buergeri Maxim.and examination of the homogeneity concerning the gentiopicroside content. Chemical and Pharmaceutical Bulletin 39: 204-206.
Yonemitsu, H., M. Kusube and M. Noguchi. 1998. Studies on propagation by tissue culture of Platycodon grandiflorum A.DC. Natural Medicines 52: 368-371.

Index of Images

Figure 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.
Figure 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.
Figure 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.
Figure 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 25OC/20OC Day/Night Temperature and Light at 44 Me M-2 S-1 in a Growth Chamber.
Figure 5 Rapid in Vitro Propagation of Limonium Wrightii (Hance) Ktze. Healthy, Mature Plant of L. Wrightii in 14-CM Diameter Pot
Figure 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.
Figure 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.
Figure 8 Rooted Axillary Shoots after Two Months of Culture in MS Basal Medium, Supplemented with 1.0 MG/1 Iba.
Figure 9 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.
Figure 10 Tuber-Derived Embryogenic Callus Showing the Formation of a Cotyledonary-Stage Somatic Embryo (C) and Converted Somatic Embryo (Arrows) after Five Weeks of Culture on Medium Supplemented with 1.0 MG/L Zeatin Riboside.
Figure 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.
Figure 12 Somatic Embryo-Derived Tubers (Arrows) Formed after 9 Months of Culture on Half-Strength MS Medium Supplemented with 3% Sucrose.