Liquid Culture Systems for in vitro Plant Propagation

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Chapter 41 Optimisation of Panax ginseng liquid cell cultures for biomass accumulation and ginsenoside production C. Kevers 1* , G. Bare 2 , T. Gaspar 3 , P. Thonart 4 & J. Dommes 5 1, 3, 5 Plant Molecular Biology and Hormonology, Institute of Botany B 22, University of Liège, Sart Tilman, BE-4000 Liège, Belgium. *Corresponding author: Fax: 32-4-366 38 72; E-mail: c.kevers@ulg.ac.be; 2, 4 CWBI, B 40, University of Liège, Sart Tilman, BE-4000 Liège, Belgium Abstract: Solid calli and derived liquid cell cultures were initiated from one-year-old roots of Panax ginseng CA Meyer. Half-strength Murashige and Skoog medium supplemented classically with an auxin and a cytokinin did not appear favourable for biomass accumulation nor for a high ginsenoside content. Changes in the levels of mineral nutrients, sucrose and growth regulators were preliminary investigated here to improve growth and ginsenoside production in liquid cultures. The hypothesis that ginseng cells released growth inhibitors in the medium was not supported by the results obtained in experiments involving frequent transfers to fresh growth medium. Key words: auxin, benzo[b]selenienyl acetic acid, biomass, cytokinin, ginseng, inoculum size, medium composition Abbreviations: BSAA – benzo[b]selenienyl acetic acid; 2,4-D – 2,4-dichlorophenoxyacetic acid; DW – dry weight; HPTLC – high performance thin layer chromatography; Kin – kinetin; MS – Murashige and Skoog medium (1962); ZR - zeatin riboside 1. Introduction Panax root has been used in Oriental medicine since ancient times. The crude root extract is known to have tonic, stimulatory and adaptogenic properties (Hu, 1976) due to the presence of a wide range of saponins and sapogenins (Li, 1995). Recently, ginseng has become a popular tonic and health food complement in Western countries. Therefore, the demand for the plant has increased dramatically worldwide. Ginseng is expensive because of its long-term conventional (5-7 years) and troublesome production cycle. As 547 A.K. Hvoslef-Eide and W. Preil (eds.), Liquid Culture Systems for in vitro Plant Propagation, 547–555. © 2005 Springer. Printed in the Netherlands.
548 C. Kevers et al. a result, propagation methods of ginseng by plant tissue culture and particularly by somatic embryogenesis have been investigated. Somatic embryogenesis has been successfully induced on solid media, from derived calli of root (Chang and Hsing, 1980, Asaka et al., 1992, 1993a, Tirajoh et al., 1998), leaf (Tirajoh et al., 1998) or flower bud (Shoyama et al., 1997), or directly from zygotic embryos (Choi and Soh, 1996) or cotyledons (Choi and Soh, 1994). Culture of ginseng tissues in bioreactors was developed in order to produce fresh material containing ginsenoside saponins (Asaka et al., 1993b). With the same purposes, we already have developed techniques for plantlet multiplication through somatic embryogenesis (Kevers et al., 2000, Monteiro et al., 2002) and for biomass production through in vitro hairy root cultures (Kevers et al., 1999). In the present work, we investigated liquid cell cultures as a tool for biomass accumulation and ginsenoside production. 2. Material and methods One-year-old roots of Panax ginseng CA Meyer (from a Belgian field culture; provided by ORTIS Laboratories, Elsenborn, Belgium) were surface sterilised by successive incubations in ethanol (70%) for 3 min and in sodium hypochlorite (3%) for 20 min, and rinsed three times with sterile distilled water. Calluses were initiated from 3 mm long root sections that were cultured on a solid MS (Murashige and Skoog, 1962) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D 0.3 mg l -1 ) and kinetin (Kin 0.1 mg l -1 ). The cultures were incubated in darkness at 25 � 2 °C in 9-cm diameter Petri dishes for 6 weeks. The callus was subcultured every four weeks on a solid MS basal medium, supplemented with 2,4-D (1 mg l -1 ) and Kin (0.1 mg l -1 ) in darkness. Finely minced callus (0.3 to 10 g) was transferred to liquid medium in 100 ml conical flasks containing 50 ml of half strength MS (MS/2) medium supplemented with auxin 3-benzo(b)selenienyl acetic acid (BSAA) or 2,4-D, 0.3 or 1 mg l -1 and cytokinin (kinetin or zeatin riboside, ZR, 0.2 mg l -1 ) for one month with shaking (80 rpm) at 25 � 2 °C in darkness. To evaluate the effect of varying concentration of some mineral nutrients (KNO3, KH2PO4, NH4NO3, FeNaEDTA) and sucrose in the medium on the growth and the ginsenoside content of the callus, an experiment based on the statistical design of Plackett-Burman (Plackett and Burman, 1946) was performed. The method allows to screen a large number of factors to identify those that may be important, with the least number of observations. One way to design such
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LIQUID CULTURE SYSTEMS FOR IN VITRO
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A C.I.P. Catalogue record for this
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Contents Contributing Authors xiii
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Liquid culture systems for in vitro
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Contributing Authors Adelberg, J. 4
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Preface Plant cell, tissue and orga
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Chapter 1 General introduction: a p
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General introduction 3 2. Cell susp
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General introduction 5 rooted in gr
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General introduction 7 (Winkelmann
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General introduction 9 the bioreact
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General introduction 11 (TIS), resu
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General introduction 13 Barz W, Rei
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General introduction 15 Hohe A, Win
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General introduction 17 Shimazu T &
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I. Bioreactors
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22 Wayne R. Curtis 1. Introduction
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24 Wayne R. Curtis headspace double
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26 Wayne R. Curtis that is required
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28 Wayne R. Curtis C L y � P O2
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30 Wayne R. Curtis since there is a
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32 Wayne R. Curtis 5 cm 30 cm 30 o
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34 Wayne R. Curtis Pˆ N � � me
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36 Wayne R. Curtis Radial Flow Impe
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38 Wayne R. Curtis CS = Medium diss
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40 Wayne R. Curtis Murashige T & Sk
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42 Anne Kathrine Hvoslef-Eide et al
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Chapter 4 Practical aspects of bior
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Practical aspects of bioreactor app
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Chapter 5 Simple bioreactors for ma
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Simple bioreactors for mass propaga
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96 K. Y. Paek et al. opportunity fo
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98 K. Y. Paek et al. disadvantages
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100 K. Y. Paek et al. 3.1 Dissolved
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102 K. Y. Paek et al. optimizing bi
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104 K. Y. Paek et al. cosmetics, wh
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106 K. Y. Paek et al. protocol, mor
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108 K. Y. Paek et al. use. In order
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110 K. Y. Paek et al. a b c d e f F
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112 K. Y. Paek et al. a b c d e f F
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114 K. Y. Paek et al. Escalona M, L
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116 K. Y. Paek et al. Son SH & Paek
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118 Seppo Sorvari et al. economical
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120 Seppo Sorvari et al. membrane w
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122 Seppo Sorvari et al. 3. Results
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124 Seppo Sorvari et al. D.O. 160 1
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Chapter 8 Cost-effective mass cloni
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Cost-effective mass cloning of plan
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144 Eun-Joo Hahn & Kee-Yoeup Paek 1
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146 Eun-Joo Hahn & Kee-Yoeup Paek F
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148 Eun-Joo Hahn & Kee-Yoeup Paek T
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150 Eun-Joo Hahn & Kee-Yoeup Paek 3
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152 Eun-Joo Hahn & Kee-Yoeup Paek E
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Chapter 10 Control of growth and di
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Control of growth and differentiati
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Chapter 11 Temporary immersion syst
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Temporary immersion system: a new c
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198 Elio Jiménez González 1. Intr
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200 Elio Jiménez González Several
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202 Elio Jiménez González Figure
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204 Elio Jiménez González exploit
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206 Elio Jiménez González weeks i
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208 Elio Jiménez González germina
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210 Elio Jiménez González Escalan
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Chapter 13 Use of growth retardants
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Use of growth retardants for banana
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Chapter 14 Somatic embryo germinati
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Somatic embryo germination of Psidi
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Somatic embryo germination of Psidi
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232 Tino Hempfling & Walter Preil N
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234 Tino Hempfling & Walter Preil 3
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236 Tino Hempfling & Walter Preil F
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238 Tino Hempfling & Walter Preil F
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240 Tino Hempfling & Walter Preil 4
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242 Tino Hempfling & Walter Preil R
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244 C. Damiano et al. Over the last
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246 C. Damiano et al. 2.2 Temporary
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248 C. Damiano et al. Table 3: Wate
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250 C. Damiano et al. hyperhydricit
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Chapter 17 Optimisation of growing
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Optimisation of growing conditions
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264 Katerina Grigoriadou et al. cul
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266 Katerina Grigoriadou et al. 2.4
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268 Katerina Grigoriadou et al. mic
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270 Katerina Grigoriadou et al. of
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272 Katerina Grigoriadou et al. 4.
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274 Katerina Grigoriadou et al. Tei
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276 Ch. Wawrosch et al. possesses s
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278 Ch. Wawrosch et al. 3. Results
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280 Ch. Wawrosch et al. References
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Chapter 20 Propagation of Norway sp
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Propagation of Norway Spruce 285 2.
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Propagation of Norway Spruce 287 su
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Propagation of Norway Spruce 289 5.
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Propagation of Norway Spruce 291 bl
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Propagation of Norway Spruce 293 H
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296 M. Vágner et al. 1995). In liq
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298 M. Vágner et al. maturation pe
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300 M. Vágner et al. mature somati
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302 M. Vágner et al. Acknowledgeme
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304 Christopher Hunter & Lionel Lev
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314 Michel Petitprez et al. 1. Intr
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316 Michel Petitprez et al. 2.2 QTL
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318 Michel Petitprez et al. Figure
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320 Michel Petitprez et al. Table 1
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322 Michel Petitprez et al. Gentzbi
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324 F. Afreen et al. 1. Introductio
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326 F. Afreen et al. precotyledonar
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328 F. Afreen et al. Dry mass (mg/e
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330 F. Afreen et al. under photoaut
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332 F. Afreen et al. Figure 3: a) C
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334 F. Afreen et al. the plant qual
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Chapter 25 Potential of flow cytome
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Potential of flow cytometry 339 dis
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Potential of flow cytometry 341 Fig
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Potential of flow cytometry 343 tha
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Chapter 26 Somatic embryogenesis of
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Somatic embryogenesis of Gentiana 3
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Chapter 27 Induction and growth of
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Induction and growth of tulip 361 a
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Induction and growth of tulip 363 T
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Chapter 28 Micropropagation of Ixia
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Micropropagation of Ixia 367 for PA
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Micropropagation of Ixia 369 Biomas
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Micropropagation of Ixia 371 Figure
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Chapter 29 Micropropagation of Rosa
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Micropropagation of Rosa 375 The BA
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Micropropagation of Rosa 377 Platfo
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Micropropagation of Rosa 379 3. Res
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Micropropagation of Rosa 381 The ro
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Micropropagation of Rosa 383 4. Dis
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Micropropagation of Rosa 385 Murash
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Chapter 30 Mass propagation of coni
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Mass propagation of conifer trees 3
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Chapter 31 Potentials for cost redu
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Potentials for cost reduction 405 c
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Chapter 32 A new approach for autom
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A new approach for automation 417 (
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A new approach for automation 419 p
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A new approach for automation 423 D
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426 B. McAlister et al. 1. Introduc
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428 B. McAlister et al. 2.2 Multipl
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430 B. McAlister et al. Table 2: Mu
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432 B. McAlister et al. rest time -
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434 B. McAlister et al. Figure 3: M
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436 B. McAlister et al. Average mul
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438 B. McAlister et al. Table 4: Co
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440 B. McAlister et al. Semi-solid
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442 B. McAlister et al. Escalona M,
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444 Jeffrey Adelberg including Host
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446 Jeffrey Adelberg BioSystems (Ho
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448 Jeffrey Adelberg more prolific
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450 Jeffrey Adelberg vessel of liqu
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452 Jeffrey Adelberg Figure 4: Labo
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454 Jeffrey Adelberg constituted a
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456 Jeffrey Adelberg Acknowledgemen
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Chapter 35 Aeration stress in plant
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Aeration stress in plant tissue cul
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476 Hans R. Gislerød et al. 1. Int
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478 Hans R. Gislerød et al. biorea
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480 Hans R. Gislerød et al. A low
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482 Hans R. Gislerød et al. Table
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484 Hans R. Gislerød et al. common
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486 Hans R. Gislerød et al. can be
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488 Hans R. Gislerød et al. 4.1 Li
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490 Hans R. Gislerød et al. 4.3 Ca
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492 Hans R. Gislerød et al. Morten
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Page 532 and 533: Chapter 40 Cultivation of root cult
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Page 542 and 543: Optimisation of Panax ginseng liqui
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Page 552 and 553: 560 S. M. Nalawade et al. Plantlets
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Page 560 and 561: Production of taxanes 569 2. Materi
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Page 566 and 567: Acknowledgments The editors thank t
Page 568 and 569: 578 Contents 130, 131, 133, 134, 13
Page 570 and 571: 580 Contents poplar, see Populus Po
Page 572 and 573: 582 Contents bubble aerated 165 bub
Page 574 and 575: 584 Contents ginsenoside content, p
Page 576 and 577: 586 Contents plant growth regulator
Page 578: 588 Contents -free microcorms 71 -f
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