Liquid Culture Systems for in vitro Plant Propagation

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Chapter 25 Potential of flow cytometry for monitoring genetic stability of banana embryogenic cell suspension cultures Nicolas Roux 1 , Hannelore Strosse 2 , Arsenio Toloza 1 , Bart Panis 2 & Jaroslav Doležel 3 1 Plant Breeding Unit, FAO/IAEA Agriculture and Biotechnology Laboratory, International Atomic Energy Agency Laboratories, A-2444 Seibersdorf, Austria. Now working for the International Network for the Improvement of banana and Plantain (INIBAP), Parc Scientifique Agropolis 2, 34397 Montpellier Cedex 5, France. E-mail: N.Roux@cgiar.org. 2 Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven, Kasteelpark Arenberg 13, B-3001 Heverlee, Belgium. 3 Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, CZ-77200 Olomouc, Czech Republic. Abstract: Cell suspensions are the material of choice for rapid multiplication and for genetic engineering strategies such as in vitro mutagenesis and genetic transformation. Effective use of cell suspension cultures relies on the knowledge of several key parameters, which include genetic stability, kinetics of the cell cycle and a mode of plant regeneration. Here we report on the use of DNA flow cytometry for quality monitoring of banana cell suspension cultures. The method facilitates detection of ploidy changes and the occurrence of aneuploidy, which result in somaclonal variation of cell-suspension-derived plants. Flow cytometry could also be used to analyse the cell cycle kinetics by calculating the ratio of cells in the G2 and G1 phase of the cell cycle. This is important to determine the most appropriate moment for mutagenic treatment or for genetic transformation but also as an indicator on the proportion of cycling cells. In addition, the unicellular origin of somatic embryos was verified by treating embryogenic cell suspensions with colchicine and by determining the ploidy of regenerated plants by flow cytometric analysis. None of the plants regenerated from colchicine-treated embryogenic cell suspensions were mixoploid (chimeric). The application of flow cytometry will be discussed in relation to (a) the monitoring of genetic instability in DNA content of cell suspensions (b) the analysis of cell cycle and (c) the origin of somatic embryos of bananas and plantains. Key words: cell cycle, chimerism, DNA content, Musa acuminata, somatic embryogenesis Abbreviations: ECS - embryogenic cell suspensions; ITC - INIBAP International Transit Center-International Network for the Improvement of Banana and Plantain 337 A.K. Hvoslef-Eide and W. Preil (eds.), Liquid Culture Systems for in vitro Plant Propagation, 337–344. © 2005 Springer. Printed in the Netherlands.
338 Nicolas Roux et al. 1. Introduction The use of shoot-tip culture in Musa has allowed great progress in mass propagation (micropropagation), conservation (medium-term conservation and cryopreservation), elimination of virus diseases and exchange of germplasm (Van den Houwe et al., 1995; Panis et al., 1996). Unfortunately, the use of banana shoot tips as target tissues for genetic engineering strategies such as in vitro mutagenesis and genetic transformation leads to chimeric plants (Roux et al., 2001). Somatic embryogenesis is usually a preferred mode of regeneration over organogenesis because of the presumed single cell origin of the regenerants (Thorpe, 1988; Peschke and Phillips, 1992). The occurrence of off-types among regenerants has delayed the widespread industry acceptance of micropropagated bananas (Smith and Hamill, 1993). Before adopting somatic embryogenesis as a new method to support genetic improvement and mass propagation of bananas and plantains, further studies need to be undertaken to better understand the phenomenon of somaclonal variation. The aim of this study was to verify the ploidy level stability of embryogenic cell suspensions prior to the formation of embryos or plants. 2. Material and methods Embryogenic cell suspensions (ECS) of four triploid (2n=3x=33) Musa cultivars were kindly provided by the Laboratory of Tropical Crop Improvement at the Katholieke University of Leuven, Belgium. The suspensions were maintained in 100 ml Erlenmeyer flasks containing 15 ml of liquid maintenance media (Dhed’a et al., 1991). Erlenmeyer flasks were placed on an orbital shaker at 70 rpm. The medium was renewed every two weeks. For monitoring the genetic stability of ECS, flow cytometric analysis was performed according to Dolezel et al. (1997). The flow cytometric assay involved the use of nuclei isolated from chicken red blood cells (CRBC), which served as internal reference standard. The gain of the instrument was adjusted so that the Go/G1 peak of CRBC nuclei was positioned approximately at channel 100. The relative DNA content of ECS was expressed as a ratio of DNA content of CRBC and Musa (DNA index). By flow cytometric analysis, it was also possible to measure the proportion of cells in the G1 and G2 phase of the cell cycle since the DNA content during G2 is doubled compared to G1 phase. Through a combination of mixoploidy induction using colchicine and flow cytometry detection, Roux et al. (2001) could monitor the effectiveness of three micropropagation techniques to
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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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Chapter 30 Mass propagation of coni
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Chapter 31 Potentials for cost redu
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Potentials for cost reduction 405 c
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Potentials for cost reduction 407 c
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Potentials for cost reduction 409 s
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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 421 T
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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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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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494 Han Bouman & Annemiek Tiekstra
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V. Biomass for Secondary Metabolite
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510 E. Gontier et al. 1. Introducti
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512 E. Gontier et al. 2. Materials
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514 E. Gontier et al. developed dra
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516 E. Gontier et al. 3.3 Establish
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518 E. Gontier et al. Fresh weight
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520 E. Gontier et al. 3.5 Effect of
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522 E. Gontier et al. 3.6 Scale up
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524 E. Gontier et al. Lorenzo JC, G
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526 Dirk Wilken et al. 1. Introduct
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528 Dirk Wilken et al. running at 6
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530 Dirk Wilken et al. described a
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532 Dirk Wilken et al. packed cell
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534 Dirk Wilken et al. bioactive co
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536 Dirk Wilken et al. Fowler MW (1
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Chapter 40 Cultivation of root cult
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Cultivation of root cultures of Pan
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Chapter 41 Optimisation of Panax gi
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Optimisation of Panax ginseng liqui
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558 S. M. Nalawade et al. performan
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560 S. M. Nalawade et al. Plantlets
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562 S. M. Nalawade et al. leaves on
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564 S. M. Nalawade et al. roots (Fi
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Chapter 43 Production of taxanes in
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Production of taxanes 569 2. Materi
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Production of taxanes 571 Figure 2:
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Production of taxanes 573 various T
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Acknowledgments The editors thank t
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578 Contents 130, 131, 133, 134, 13
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580 Contents poplar, see Populus Po
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582 Contents bubble aerated 165 bub
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584 Contents ginsenoside content, p
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586 Contents plant growth regulator
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588 Contents -free microcorms 71 -f
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