Liquid Culture Systems for in vitro Plant Propagation

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278 Ch. Wawrosch et al. 3. Results and discussion As previously reported the main factor, which triggers the formation of adventitious shoots on nodules of Charybdis sp. is light (Wawrosch et al., 2001). The highest regeneration frequency (208.7�23.5 per gram of initial nodule explants) was observed in the AM system with semisolid medium. However, about 7 % of the regenerants were small buds, which were not suitable for further processing. For this reason the number of regenerated shoots is presented separately (Table 1), which was 194.3�23.0 for the AM system. Shoots regenerated on semisolid medium were typically of healthy appearance and did not display any symptoms of hyperhydration (Figure 1B). In contrast, the results obtained in submerged liquid culture were significantly different. In the LM system 59.7�3.6 shoots were formed which is about 29 % of the regeneration frequency in the AM system. Not only were the values per container lower than those achieved with the AM system, but practically all shoots were severely hyperhydrated, too (Figure 1C). It is well known that one of the advantages of temporary immersion culture systems lies in the higher multiplication rates when compared to semisolid medium. However, this could not be verified for our Charybdis nodule micropropagation system. In the TI setup (Figure 1D) an immersion of 5 min every 24 hours (TI 1) resulted in 25.4�3.2 shoots per g which was only about half of the regeneration frequency under LM conditions and was significantly lower than on semisolid medium. However, only a minor portion of the shoots showed symptoms of hyperhydration (Figure 1E). A comparison of the total number of shoots obtained from one container or culture unit (Table 1) reveals that the use of the TI 1 system resulted in significantly higher amounts of regenerants. Although the inoculum was approximately 5-fold that of the AM system it should be taken into consideration that the TI system is more labour saving. Indeed the nodules are just loaded into the bottle (using e.g. a sterile funnel) whereas for the AM systems containers have to be opened, nodules have to be placed firmly onto the medium, and the containers have to be closed again. In order to increase the regeneration rate the immersion frequency was doubled in the TI 2 system. Although a significant increase in the number of shoots per container was observed it was noted that just a doubling of the time of immersion resulted in completely hyperhydrated shoots (Figure 1F). It seems evident that shoots of Charybdis numidica are very sensitive to contact with liquid medium, on the other hand the more frequent immersion did not significantly increase the number of regenerated shoots per gram of nodules (Table 1).
Shoot regeneration from nodules of Charybdis 279 Table 1: Regeneration rates (average�SE) from nodules of Charybdis numidica clone UN26 and hyperhydration on semisolid (AM), submerged in liquid medium (LM), and in temporary immersion (TI) systems Culture method Total regenerants per g inoculum* Shoots per g inoculum Shoots per container** Hyperhydration (score 0-3) AM 208.7�23.5 a 194.2�23.0 a 194.2�23.0 ab 0 LM 59.7�3.6 b 55.0�3.8 b 130.4�7.5 a 3 TI1 (5 min every 24h) TI2 (5 min every 12h) 38.3�1.6 b 25.4�3.2 b 270.5�34.5 b 1 48.0�2.4 b 36.6�1.2 b 437.0�1.0 c 3 Mean separation within columns by Duncan´s multiple range test at p=0.05 * shoots + buds ** AM: baby food jar/c. 1 g inoculum; LM: 250ml Erlenmeyer flask/c. 2 g inoculum; TI1 and TI2: 1-litre flask/c. 10 g inoculum/500 ml medium Micropropagation of Charybdis sp. through nodule culture offers a convenient alternative to other in vitro methods. Growth of the nodules (without regeneration) can be achieved in liquid MS medium supplemented with 3 % sucrose and 20 µmol BA in the dark, with an up to 8-fold increase in biomass depending on the clone (Wawrosch et al., 2001). Two different procedures can be proposed for the regeneration of shoots. Recently a multiplication protocol described for a Phalaenopsis hybrid suggested the multiplication of protocorm-like bodies in liquid culture systems followed by conversion into plantlets on semisolid medium (Park et al., 2000). Similarly, nodules of Charybdis sp. can be multiplied in liquid medium and subsequently transferred to semisolid medium for shoot regeneration. In order to achieve higher shoot numbers per unit, larger containers and/or higher inoculum density could be tested. As an alternative, using a temporary immersion system with low immersion frequency and duration, large amounts of shoots per container can be produced with less labour input. Because the shoots can be rooted ex vitro during acclimatization (unpublished results) the procedure is labour and time saving. Studies are currently in progress on the suitability of a TI system for nodule growth. Using TI culture for both nodule growth and shoot regeneration would allow for a production system with least expenditure of labour: at the end of the growth period only nutrient medium and light conditions would have to be changed for shoot regeneration.
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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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Page 232 and 233: Chapter 14 Somatic embryo germinati
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Page 250 and 251: 244 C. Damiano et al. Over the last
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Page 268 and 269: 264 Katerina Grigoriadou et al. cul
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Page 306 and 307: 304 Christopher Hunter & Lionel Lev
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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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Potentials for cost reduction 407 c
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Potentials for cost reduction 409 s
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Potentials for cost reduction 411 N
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Potentials for cost reduction 413 G
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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 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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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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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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