Liquid Culture Systems for in vitro Plant Propagation

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Chapter 14 Somatic embryo germination of Psidium guajava L. in the Rita� temporary immersion system and on semisolid medium Rafael Gómez Kosky 1 , J. Vilchez Perozo 2 , N. Albany Valero 2 & D. Agramonte Peñalver 1 1 Instituto de Biotecnología de las Plantas. Universidad Central “Marta Abreu” de Las Villas (UCLV). Carretera a Camajuní Km 5 ½ Santa Clara, Villa Clara, Cuba Fax: 53-42-281329, E-mail: koskyrg@yahoo.es 2 Fundación Gran Mariscal de Ayacucho. Caracas, República Bolivariana de Venezuela Abstract: Germination of somatic embryos and development to plants is recognized as one of the most critical stages in the process of plant propagation via somatic embryogenesis. The objective of the study was to investigate the germination of somatic embryos of Psidium guajava cv. Cuban Red Dwarf EEA 18-40 in the RITA� system and on semisolid medium. Somatic embryos were obtained from immature zygotic embryos which were cultured on the major salts of MS medium at half strength, supplemented with 400 mg l -1 L-glutamine, 100 mg l -1 ascorbic acid, 60 g l -1 sucrose and 1 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos at the heart and torpedo stages were transferred for germination into RITA� vessels containing liquid half strength MS medium of the major salts supplemented with 0.25 mg l -1 6- benzylaminopurine (6-BAP), 10 µg l -1 Biobras-6 (brassinosteroid analogue) and 20 g l -1 sucrose or to semiliquid medium of the same composition (solidified with 2.5 g l -1 Gellum Gum, Spectrum�) in 250 ml glass vessels. The germination percentage, fresh weight and number of somatic embryos with complete germination were determined. After 10 weeks of culture the highest germination percentage (91%) and fresh weight (1.22 g) were achieved in the temporary immersion system, being statistically superior to those obtained from semisolid culture medium (81.79% and 1.03 g respectively). Key words: Guava, liquid medium, somatic embryogenesis 1. Introduction Among 150 Psidium species guava (Psidium guajava L.) presents the greatest potential from an economic point of view. Difficulties in the 225 A.K. Hvoslef-Eide and W. Preil (eds.), Liquid Culture Systems for in vitro Plant Propagation, 225–229. © 2005 Springer. Printed in the Netherlands.
226 Rafael Gómez Kosky et al. application of conventional propagation techniques have generated considerations of other possible forms of vegetative propagation (Pontikis, 1996). Especially somatic embryogenesis, which has been investigated as a tool for genetic improvement and for the propagation of elite trees (Vilchez, 2001). The germination of somatic embryos has proved to be one of the most critical stages in somatic embryogenesis (Merkle et al., 1995). Temporary Immersion Systems (TIS) have been successfully used in several protocols for the germination of somatic embryos of some species (Etienne-Barry et al., 1999; Gómez et al., 2000). In this research, germination of guava somatic embryos in TIS was studied and compared to cultures on semisolid medium. 2. Materials and methods Somatic embryos of Psidium guajava L. cv. ‘Cuban Red Dwarf EEA 18- 40’, were obtained from immature zygotic embryos at the torpedo and cotyledonary stages. These were cultured on the major salts of MS medium (Murashige and Skoog, 1962) at half strength, supplemented with 400 mg l -1 L-glutamine, 100 mg l -1 ascorbic acid, 60 g l -1 sucrose and 1 mg l -1 2,4dichlorophenoxyacetic acid (2,4-D). Subculturing was performed at threeweek intervals using the same culture medium. The operations and characteristics of the RITA�system (CIRAD, France) have been described by Etienne-Barry et al., (1999). For embryo germination each RITA ® unit contained 200 ml of liquid MS culture medium with 50% of the major salts supplemented with 0.25 mg l -1 6benzylaminopurine (6-BAP), 10 �g l -1 Biobras-6 (brassinosteriod analogue from Nature Product Lab., Havana University) and 20 g l -1 sucrose. The inoculum density per RITA vessel was 800 mg fresh weight of somatic embryos at the heart or torpedo stages. The immersion frequency was one minute every 12 hours. The experiments were duplicated. For control cultures 250 ml glass vessels were used with 30 ml of semisolid culture medium (solidified with 2.5 g l -1 Gellan gum, Spectrum ® ) consisting of the same composition as used for the RITA ® units. Ten culture vessels were inoculated with the 800 mg fresh weight of somatic embryos as described for RITA ® cultures. The experiment was carried out in a growth chamber with the natural photoperiod in Cuba according to the time of the year (November-January) and a photosynthetic photon flow superior to 60 µmol m -2 s -1 . The temperature regime was 25 ± 2.0 ºC. After ten weeks of culture, the percentage of germinated embryos and fresh weight were determined. The
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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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Page 206 and 207: 198 Elio Jiménez González 1. Intr
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Page 218 and 219: 210 Elio Jiménez González Escalan
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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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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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