Fourth Edition M E T H O D S I N M O L E C U L a R B I O Lo G Y

Methods in Molecular Biology 1815

Victor M. Loyola-Vargas Neftalí Ochoa-Alejo Editors Plant Cell Culture Protocols Fourth Edition M e t h o d s i n M o l e c u l a r B i o lo g y

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651 Plant Cell Culture Protocols

Fourth Edition

Edited by Víctor M. Loyola-Vargas

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Neftalí Ochoa-Alejo

Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico Editors Víctor M. Loyola-Vargas Neftalí Ochoa-Alejo Unidad de Bioquímica y Biología Departamento de Ingeniería Genética Molecular de Plantas Unidad Irapuato Centro de Investigación Científica de Yucatán Centro de Investigación y de Estudios Avanzados Mérida, Yucatán, Mexico del Instituto Politécnico Nacional Irapuato, Guanajuato, Mexico

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-8593-7 ISBN 978-1-4939-8594-4 (eBook) https://doi.org/10.1007/978-1-4939-8594-4

Library of Congress Control Number: 2018945533

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This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Preface

Plant cell, tissue, and organ culture techniques have been utilized for a long time and surely will continue to be important biological systems for a series of basic studies and also as biotechnological tools for clonal propagation of plants, for crop improvement programs, and for genetic manipulation of important crop species through genetic engineering or by genomic editing approaches. New avenues and possibilities for plant cell, tissue, and organ culture have been incorporated to enrich this fourth edition of Plant Cell Culture Protocols composed of 34 chapters dealing with a series of basic auxiliary protocols for tissue culture (confocal microscopy for immunolocalization of auxins, histological techniques and photographic analysis to follow morphogenetic events, and cytometry applied to the analysis of regenerated plants). A micropropagation chapter in the twenty-first century describing its importance, limitations, challenges, and possible solutions provides the reader with new horizons and perspectives, and also a collection of protocols for the micropropagation and embryo rescue of Agave spp., the conditions for the clonal propagation of Yucca spp., and the somatic embryogenesis-mediated plant regeneration systems for Cocos nucifera, Phaseolus vulgaris, Musa spp., Theobroma cacao, Quercus, and Jatropha curcas form part of the content of this volume. One of the most frequently faced problems in tissue culture is microbial contamination, and for many years it was thought that only those microorganisms present in the surface of the explants were important; however, endophytic bacteria very often can affect the establishment and the responses of cell, tissue, or organ cultures; because of the importance of endophytes, a description and identification of some commonly found endophytic bacteria as well as some of the effects caused by them and how to control this problem is provided in the current edition. Somaclonal variation is still an interesting issue and a protocol for the selection of molecular markers to estimate somaclonal variation in cell and tissue cultures is now presented here. Elimination of plant viruses through meristem isolation and subsequent culture or the use of thermotherapy combined with meristem culture are the regular methods to get virus-free plant materials of high phytosanitary quality; however, a protocol using cryotherapy represents a new alternative for this purpose and is integrated here. The production of haploid and doubled haploid plant production of carrot using induced parthenogenesis and ovule excision can be used for both basic and applied crop improvement programs. Conservation of germplasm of important crops has been always an issue of primary interest due to the potential utilization of genetic variation for crop improvement programs; therefore, protocols for the cryopreservation of pollen grains from pineapple and other bromeliads were considered as a part of the strategies for the preservation of germplasm of these plant species.

v vi Preface

Plant cell, tissue, and organ culture are used as systems to study the potential of different plant species to produce secondary metabolites; this is the case of the chili pepper (Capsicum chinense) protocol for the establishment of cell suspensions and immobilized placenta tissues, which are used as models to investigate the production of capsaicinoids, compounds responsible for the hot taste. Moreover, genetic transformation is certainly another tool of great value for the genetic manipulation of agricultural crops, but also when the aim is to carry out metabolic engineering of secondary metabolite pathways, such as the protocol for the Agrobacterium tumefaciens-genetic transformation of the Mayan medicinal species Pentalinon andrieuxii, which produces pentacyclic triterpenes with potential application in the pharmaceutical industry. In this fourth edition, a special focus was paid to the inclusion of protocols regarding the omics (transcriptomics, proteomics, and metabolomics) applied to different aspects of plant cell, tissue, and organ cultures. For example, protocols for the analysis of secondary metabolites (terpenes, carotenoids, phytosterols) through NMR-based metabolomics of Catharanthus roseus or hairy root cultures from several medicinal plants. Of relevance in this volume are the protocols for the application of proteomics and transcriptomics to study somatic embryogenesis and morphogenesis processes. Moreover, the participation of microRNAs and transcription factors as important actors in somatic embryogenesis is also described. Epigenetic changes involving histone modifications and changes in chromatin organization during biological processes can be analyzed using the chromatin immunoprecipitation assay (Chip) protocol presented in the current edition. Perhaps the most spectacular current tool for genomic editing is undoubtedly the CRISPR/ Cas9 technology, and a review on its use in plant tissue culture is reported. Among the miscellaneous applications of cell culture, the readers can consult and follow a protocol for the use of cell suspensions to test heavy metal toxicity and accumulation for a possible phytoremediation alternative. As in the previous editions of Plant Cell Culture Protocols, an Appendix of the composition of the most commonly used plant cell, tissue, and organ culture media is included. We would like to thank all the authors for their enthusiasm and the time devoted to prepare their chapters in which they are sharing the most invaluable richness: their expertise. Finally, we should make a special mention of gratitude to David Casey and John Walker, who always supported and guided us during this editorial journey.

Mérida, Yucatán, Mexico Víctor M. Loyola-Vargas Irapuato, Guanajuato, Mexico Neftalí Ochoa-Alejo Contents

Preface...... v Contributors ...... xi

Part I I ntroduction

1 An Introduction to Plant Tissue Culture: Advances and Perspectives �� 3 Victor M. Loyola-Vargas and Neftalí Ochoa-Alejo

Part II cell Culture the Fundaments

2 Micropropagation in the Twenty-First Century �� 17 Jean Carlos Cardoso, Lee Tseng Sheng Gerald, and Jaime A. Teixeira da Silva 3 Cellular and Morpho-histological Foundations of In Vitro Plant Regeneration �� 47 Diego Ismael Rocha, Lorena Melo Vieira, Andréa Dias Koehler, and Wagner Campos Otoni 4 Bacterial Endophytes in Plant Tissue Culture: Mode of Action, Detection, and Control �� 69 Mona Quambusch and Traud Winkelmann 5 Digital Photography as a Tool of Research and Documentation in Plant Tissue Culture �� 89 Victor Gaba, Yehudit Tam, Danny Shavit, and Benjamin Steinitz 6 Selection of Molecular Markers for the Estimation of Somaclonal Variation �� 103 Octavio Martínez 7 Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9 �� 131 Víctor M. Loyola-Vargas and Randy N. Avilez-Montalvo

Part III Protocols

8 Micropropagation of Agave Species �� 151 Benjamín Rodríguez-Garay and José Manuel Rodríguez-Domínguez 9 Protocol for the Micropropagation of Coconut from Plumule Explants �� 161 Luis Sáenz, José Luis Chan, María Narvaez, and Carlos Oropeza 10 Micropropagation of Yucca Species �� 171 Yessica López-Ramírez, Alejandra Palomeque-Carlín, Lucía Isabel Chávez Ortiz, Ma. de Lourdes de la Rosa-Carrillo, and Eugenio Pérez-Molphe-Balch

vii viii Contents

11 Auxin Immunolocalization in Coffea canephora Tissues �� 179 Ruth E. Márquez-López, Ángela Ku-González, Hugo A. Méndez-Hernández, Rosa M. Galaz-Ávalos, and Víctor M. Loyola-Vargas 12 Somatic Embryogenesis in Common Bean Phaseolus vulgaris L. �� 189 José Luis Cabrera-Ponce, Itzel Anayetzi González-Gómez, Claudia G. León-Ramírez, José A. Sánchez-Arreguín, and Alba E. Jofre y Garfias 13 Induction of Somatic Embryogenesis in Jatropha curcas �� 207 Rosa M. Galaz-Ávalos, Heydi G. Martínez-Sánchez, and Víctor M. Loyola-Vargas 14 In Vitro Proliferation of Female Buds for Induction of Somatic Embryogenesis from False Horn Plantain (AAB, cv. Curraré) �� 215 Rosa Maria Escobedo-Gracia-Medrano, Carlos Iván Cruz-Cárdenas, Lucila Aurelia Sánchez-Cach, José Roberto Ku-Cauich, and Wilma Aracely González-Kantún 15 Somatic Embryogenesis in Theobroma cacao L. �� 227 Claudia Garcia, Jean-Philippe Marelli, Juan Carlos Motamayor, and Cristiano Villela 16 Somatic Embryogenesis of Quercus suber L. From Immature Zygotic Embryos �� 247 Pilar S. Testillano, Aránzazu Gómez-Garay, Beatriz Pintos, and María C. Risueño 17 Cryotherapy: A Novel Method for Virus Eradication in Economically Important Plant Species �� 257 Min-Rui Wang, Long Chen, Zhibo Zhang, Dag-Ragnar Blystad, and Qiao-Chun Wang 18 Cryopreservation of Pineapple Shoot Tips by the Droplet Vitrification Technique �� 269 Fernanda Vidigal Duarte Souza, Everton Hilo de Souza, Ergun Kaya, Lívia de Jesus Vieira, and Ronilze Leite da Silva 19 Cryopreservation of Pollen Grains of Pineapple and Other Bromeliads �� 279 Fernanda Vidigal Duarte Souza, Everton Hilo de Souza, and Ronilze Leite da Silva 20 Application of in Casa Pollination and Embryo Rescue Techniques for Breeding of Agave Species �� 289 Benjamín Rodríguez-Garay, Sigifredo López-Díaz, José Manuel Rodríguez-Domínguez, Antonia Gutiérrez-Mora, and Ernesto Tapia-Campos 21 Haploid and Doubled Haploid Plant Production in Carrot Using Induced Parthenogenesis and Ovule Excision In Vitro �� 301 Agnieszka Kiełkowska, Adela Adamus, and Rafal Baranski 22 Using Flow Cytometry Analysis in Plant Tissue Culture Derived Plants �� 317 Rosa María Escobedo-Gracia-Medrano, Martha Josefa Burgos-Tan, José Roberto Ku-Cauich, and Adriana Quiroz-Moreno Contents ix

23 Procedure for Estimating the Tolerance and Accumulation of Heavy Metals Using Plant Cell Cultures �� 333 Antonio Bernabé-Antonio, Amalia Maldonado-Magaña, María Elena Estrada-Zúñiga, Leticia Buendía-González, and Francisco Cruz-Sosa 24 Proteomics as a Tool to Study Molecular Changes During Plant Morphogenesis In Vitro �� 339 André Luis Wendt dos Santos, Ricardo Souza Reis, Angelo Schuabb Heringer, Eny Iochevet Segal Floh, Claudete Santa-Catarina, and Vanildo Silveira 25 Proteomic Analysis of Non-model Plant Tissues Using Phenol Extraction, Two-Dimensional Electrophoresis, and MALDI Mass Spectrometry �� 351 Petra Peharec Štefanić, Mario Cindrić, and Biljana Balen 26 Chromatin Immunoprecipitation (ChiP) Protocol for the Analysis of Gene Regulation by Histone Modifications in Agave angustifolia Haw �� 371 Rosa Us-Camas and Clelia De-la-Peña 27 Transcription Factors: Their Role in the Regulation of Somatic Embryogenesis in Theobroma cacao L. and Other Species �� 385 Claudia Garcia, Dahyana Britto, and Jean-Philippe Marelli 28 MicroRNA Expression and Regulation During Maize Somatic Embryogenesis �� 397 Brenda Anabel López-Ruiz, Vasti Thamara Juárez-González, Elva Carolina Chávez-Hernández, and Tzvetanka D. Dinkova 29 Elaboration of Transcriptome During the Induction of Somatic Embryogenesis �� 411 Elsa Góngora-Castillo, Geovanny I. Nic-Can, Rosa M. Galaz-Ávalos, and Víctor M. Loyola-Vargas 30 Induction of Specialized Metabolism in In Vitro Cultures of Capsicum chinense Jacq �� 429 Felipe A. Vázquez-Flota and María de Lourdes Miranda-Ham 31 Analysis of Terpenoid Indole Alkaloids, Carotenoids, Phytosterols, and NMR-Based Metabolomics for Catharanthus roseus Cell Suspension Cultures �� 437 Mohd Zuwairi Saiman, Natali Rianika Mustafa, and Robert Verpoorte 32 Transformed Root Culture: From Genetic Transformation to NMR-Based Metabolomics �� 457 Andrey S. Marchev, Zhenya P. Yordanova, and Milen I. Georgiev 33 Genetic Transformation of Pentalinon andrieuxii Tissue Cultures �� 475 Yeseña Burgos-May, Elidé Avilés-Berzunza, Luis Manuel Peña-Rodríguez, and Gregorio Godoy-Hernández

Appendix A: The Components of the Culture Media �� 493

Index �� 503 Contributors

Adela Adamus • Faculty of Biotechnology and Horticulture, Institute of Plant Biology and Biotechnology, University of Agriculture, Krakow, Poland Elidé Avilés-Berzunza • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Randy N. Avilez-Montalvo • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Biljana Balen • Faculty of Science, Division of Molecular Biology, Department of Biology, University of Zagreb, Zagreb, Croatia Rafal Baranski • Faculty of Biotechnology and Horticulture, Institute of Plant Biology and Biotechnology, University of Agriculture, Krakow, Poland Antonio Bernabé-Antonio • Departamento de Madera, Celulosa y Papel, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico Dag-Ragnar Blystad • Division of Biotechnology and Plant Health, Norwegian Institute of Bioeconomy Research, Ås, Norway Dahyana Britto • Mars Center for Cocoa Science, Itajuípe, BA, Brazil Leticia Buendía-González • Facultad de Ciencias, Universidad Autónoma del Estado de México, Toluca, Estado de Mexico, Mexico Yeseña Burgos-May • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Martha Josefa Burgos-Tan • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A C. ,. Mérida, Yucatán, Mexico José Luis Cabrera-Ponce • Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, CP, Guanajuato, Mexico Jean Carlos Cardoso • Laboratory of Plant Physiology and Tissue Culture, Department of Biotechnology, Plant and Animal Production, Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP, Brazil José Luis Chan • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Lucía Isabel Chávez Ortiz • Unidad de Biotecnología Vegetal, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico Elva Carolina Chávez-Hernández • Instituto de Ecología, Universidad Nacional Autónoma de México, Ciudad de México, México Long Chen • State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China Mario Cindrić • Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia Carlos Iván Cruz-Cárdenas • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A .C ., Mérida, Yucatán, Mexico; Centro Nacional de Recursos Genéticos, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Tepatitlán de Morelos, Jalisco, Mexico

xi xii Contributors

Francisco Cruz-Sosa • Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de Mexico, Mexico Ma. de Lourdes de la Rosa-Carrillo • Unidad de Biotecnología Vegetal, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico Clelia De-la-Peña • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Tzvetanka D. Dinkova • Facultad de Química, Departamento Bioquímica, Universidad Nacional Autónoma de México, Ciudad de México, México Rosa Maria Escobedo-Gracia-Medrano • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A .C ., Mérida, Yucatán, Mexico María Elena Estrada-Zúñiga • Centro de Investigación en Recursos Bióticos-Facultad de Ciencias, Universidad Autónoma del Estado de México, Toluca, Estado de Mexico, Mexico Victor Gaba • Department of Plant Pathology and Weed Science, Agricultural Research Organization—The Volcani Center, Rishon LeZion, Israel Rosa M. Galaz-Ávalos • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Claudia Garcia • Mars Center for Cocoa Science, Fazenda Almirante, Itajuípe, BA, Brazil Milen I. Georgiev • Group of Plant Cell Biotechnology and Metabolomics, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria Gregorio Godoy-Hernández • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, México Aránzazu Gómez-Garay • Faculty of Biology, Department of Plant Biology I, UCM, Madrid, Spain Elsa Góngora-Castillo • CONACYT Research Fellow-Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Itzel Anayetzi González-Gómez • Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, CP, Guanajuato, Mexico Wilma Aracely González-Kantún • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A .C ., Mérida, Yucatán, Mexico Antonia Gutiérrez-Mora • Unidad de Biotecnología Vegetal, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Zapopan, Jalisco, Mexico Alba E. Jofre y Garfias • Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, CP, Guanajuato, Mexico Vasti Thamara Juárez-González • Facultad de Química, Departamento Bioquímica, Universidad Nacional Autónoma de México, Ciudad de México, México Ergun Kaya • Department of Molecular Biology and Genetics, Faculty of Science Mugla Sitki Kocman University Koteki, Koteki, Mugla, Turkey Agnieszka Kiełkowska • Faculty of Biotechnology and Horticulture, Institute of Plant Biology and Biotechnology, University of Agriculture, Krakow, Poland Andréa Dias Koehler • Laboratório de Cultura de Tecidos—LCT, Instituto de Biotecnologia Aplicada à Agropecuária-BIOAGRO/Departamento de Biologia Vegetal, Campus Universitário, Universidade Federal de Viçosa, Viçosa, MG, Brazil José Roberto Ku-Cauich • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A C. ,. Mérida, Yucatán, Mexico Contributors xiii

Ángela Ku-González • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Claudia G. León-Ramírez • Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, CP, Guanajuato, Mexico Sigifredo López-Díaz • Departamento de Investigacion, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad, Michoacán-Instituto Politécnico Nacional, Jiquilpan, Michoacán, Mexico Yessica López-Ramírez • Unidad de Biotecnología Vegetal, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico Brenda Anabel López-Ruiz • Facultad de Química, Departamento Bioquímica, Universidad Nacional Autónoma de México, Ciudad de México, México Victor M. Loyola-Vargas • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Amalia Maldonado-Magaña • Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Estado de Morelos, Mexico Andrey S. Marchev • Group of Plant Cell Biotechnology and Metabolomics, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria Jean-Philippe Marelli • Mars Center for Cocoa Science, Fazenda Almirante, Itajuípe, BA, Brazil Ruth E. Márquez-López • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Octavio Martínez • Unidad de Genómica Avanzada (Langebio), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Guanajuato, México Heydi G. Martínez-Sánchez • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Hugo A. Méndez-Hernández • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico María de Lourdes Miranda-Ham • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Juan Carlos Motamayor • Mars Center for Cocoa Science, Fazenda Almirante, Itajuípe, BA, Brazil Natali Rianika Mustafa • Natural Products Laboratory, Institute of Biology, Leiden University, Leiden, The Netherlands María Narvaez • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Geovanny I. Nic-Can • CONACYT Research Fellow-Campus de Ciencias Exactas e Ingeniería, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico Neftalí Ochoa-Alejo • Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, Guanajuato, Mexico Carlos Oropeza • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Wagner Campos Otoni • Laboratório de Cultura de Tecidos—LCT, Instituto de Biotecnologia Aplicada à Agropecuária-BIOAGRO/Departamento de Biologia Vegetal, Campus Universitário, Universidade Federal de Viçosa, Viçosa, MG, Brazil Alejandra Palomeque-Carlín • Unidad de Biotecnología Vegetal, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico xiv Contributors

Petra Peharec Štefanić • Faculty of Science, Division of Molecular Biology, Department of Biology, University of Zagreb, Zagreb, Croatia Luis Manuel Peña-Rodríguez • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Eugenio Pérez-Molphe-Balch • Unidad de Biotecnología Vegetal, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Mexico Beatriz Pintos • Pollen Biotechnology of Crop Plants, Biological Research Centre, Madrid, Spain Mona Quambusch • Abteilung Waldgenressourcen, Nordwestdeutsche Forstliche Versuchsanstalt, Hann . Münden, Germany Adriana Quiroz-Moreno • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán A C. ., Mérida, Yucatán, Mexico María C. Risueño • Pollen Biotechnology of Crop Plants, Biological Research Centre, Madrid, Spain Diego Ismael Rocha • Instituto de Biociências, Universidade Federal de Goiás, Jataí, GO, Brazil José Manuel Rodríguez-Domínguez • Unidad de Biotecnología Vegetal, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Zapopan, Jalisco, Mexico Benjamín Rodríguez-Garay • Unidad de Biotecnología Vegetal, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Zapopan, Jalisco, Mexico Luis Sáenz • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Mohd Zuwairi Saiman • Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia; Centre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala Lumpur, Malaysia; Natural Products Laboratory, Institute of Biology, Leiden University, Leiden, The Netherlands José A. Sánchez-Arreguín • Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, CP, Guanajuato, Mexico Lucila Aurelia Sánchez-Cach • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán A .C ., Mérida, Yucatán, Mexico Claudete Santa-Catarina • Laboratório de Biologia Celular e Tecidual, CBB, UENF, Campos dos Goytacazes, RJ, Brazil Angelo Schuabb Heringer • Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil; Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENF, Campos dos Goytacazes, RJ, Brazil Eny Iochevet Segal Floh • Laboratory of Plant Cell Biology, Department of Botany, Institute of Biosciences, University of São Paulo, São Paulo, Brazil Danny Shavit • Shavit Professional Scientific Photography, Kfar Saba, Israel Lee Tseng Sheng Gerald • Laboratory of Plant Physiology and Tissue Culture, Department of Biotechnology, Plant and Animal Production, Centro de Ciências Agrárias, Universidade Federal de São Carlos, Araras, SP, Brazil Ronilze Leite da Silva • State University of Feira de Santana (UEFS), Feira de Santana, Bahia, Brazil Vanildo Silveira • Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil; Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENF, Campos dos Goytacazes, RJ, Brazil Contributors xv

Ricardo Souza Reis • Laboratório de Biotecnologia, Centro de Biociências e Biotecnologia (CBB), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ, Brazil; Unidade de Biologia Integrativa, Setor de Genômica e Proteômica, UENF, Campos dos Goytacazes, RJ, Brazil Everton Hilo de Souza • Federal University of Recôncavo da Bahia (UFRB), Cruz das Almas, Bahia, Brazil; Scholarship of Coordination for the Improvement of Higher Education Personnel (CAPES) at CAPES-EMBRAPA Program in Embrapa Cassava and Fruits (CNPMF), Cruz das Almas, Bahia, Brazil Fernanda Vidigal Duarte Souza • Embrapa Cassava and Fruits, Cruz das Almas, Bahia, Brazil; Federal University of Recôncavo da Bahia (UFRB), Cruz das Almas, Bahia, Brazil Benjamin Steinitz • Institute of Plant Sciences, Agricultural Research Organization— The Volcani Center, Rishon LeZion, Israel Yehudit Tam • Department of Plant Pathology and Weed Science, Agricultural Research Organization—The Volcani Center, Rishon LeZion, Israel Ernesto Tapia-Campos • Unidad de Biotecnología Vegetal, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Zapopan, Jalisco, Mexico Jaime A. Teixeira da Silva • Kagawa, Japan Pilar S. Testillano • Pollen Biotechnology of Crop Plants, Biological Research Centre, Madrid, Spain Rosa Us-Camas • Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Felipe A. Vázquez-Flota • Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Mérida, Yucatán, Mexico Robert Verpoorte • Natural Products Laboratory, Institute of Biology, Leiden University, Leiden, The Netherlands Lívia de Jesus Vieira • Scholarship of Coordination for the Improvement of Higher Education Personnel (CAPES) at CAPES-EMBRAPA Program in Embrapa Cassava and Fruits (CNPMF), Cruz das Almas, Bahia, Brazil Lorena Melo Vieira • Laboratório de Cultura de Tecidos—LCT, Instituto de Biotecnologia Aplicada à Agropecuária-BIOAGRO/Departamento de Biologia Vegetal, Campus Universitário, Universidade Federal de Viçosa, Viçosa, MG, Brazil Cristiano Villela • Mars Center for Cocoa Science, Fazenda Almirante, Itajuípe, BA, Brazil Min-Rui Wang • State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China; Division of Biotechnology and Plant Health, Norwegian Institute of Bioeconomy Research, Ås, Norway Qiao-Chun Wang • State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China André Luis Wendt dos Santos • Laboratory of Plant Cell Biology, Department of Botany, Institute of Biosciences, University of São Paulo, São Paulo, Brazil Traud Winkelmann • Institut für Gartenbauliche Produktionssysteme, Leibniz Universität Hannover, Hannover, Germany Zhenya P. Yordanova • Faculty of Biology, Department of Plant Physiology, Sofia University “St . Kliment Ohridski”, Sofia, Bulgaria Zhibo Zhang • Division of Biotechnology and Plant Health, Norwegian Institute of Bioeconomy Research, Ås, Norway Part I

Introduction Chapter 1

An Introduction to Plant Tissue Culture: Advances and Perspectives

Victor M. Loyola-Vargas and Neftalí Ochoa-Alejo

Abstract

Plant tissue culture techniques are the most frequently used biotechnological tools for basic and applied purposes ranging from investigation on plant developmental processes, functional gene studies, commercial plant micropropagation, generation of transgenic plants with specific industrial and agronomical traits, plant breeding and crop improvement, virus elimination from infected materials to render high-quality healthy plant material, preservation and conservation of germplasm of vegetative propagated plant crops, and rescue of threatened or endangered plant species. Additionally, plant cell and organ cultures are of interest for the production of secondary metabolites of industrial and pharmaceutical interest. New technologies, such as the genome editing ones combined with tissue culture and Agrobacterium tumefaciens infection, are currently promising alternatives for the highly specific genetic manipulation of interesting agronomical or industrial traits in crop plants. Application of omics (genomics, transcriptomics, and proteomics) to plant tissue culture will certainly help to unravel complex developmental processes such as organogenesis and somatic embryogenesis, which will probably enable to improve the efficiency of regeneration protocols for recalcitrant species. Additionally, metabolomics applied to tissue culture will facilitate the extraction and characterization of complex mixtures of natural plant products of industrial interest. General and specific aspects and applications of plant tissue culture and the advances and perspectives are described in this edition.

Key words Aseptic culture, Genetic modified organisms, Large-scale propagation, Metabolic engineering, Plant cell culture, Proteomics, Transcriptomics

1 Introduction

Plant tissue culture is a broad term that refers to the culture of any part of a plant (cells, tissues, or organs) in artificial media, in aseptic conditions, and under controlled environments. This set of techniques emerged as an experimental approach to demonstrate the cell theory, which establishes that all living organisms are constituted of cells, the basic units of structure and reproduction, and also the totipotency concept, which is defined as the genetic potential of a cell to generate an entire multicellular organism [1]. Different attempts were conducted by several researchers to investigate the

Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo (eds.), Plant Cell Culture Protocols, Methods in Molecular Biology, vol. 1815, https://doi.org/10.1007/978-1-4939-8594-4_1, © Springer Science+Business Media, LLC, part of Springer Nature 2018 3 4 Victor M. Loyola-Vargas and Neftalí Ochoa-Alejo

conditions to initially achieve the growth of organs [2] or tissues [3] in an artificial nutrient culture medium [4] rather than isolated cells because of the complex nutritional and hormonal requirements they need. Nutrient solutions alone or supplemented with natural extracts were used as starting culture media, and some important results were reported [5]; however, the discovery of plant growth regulators was determinant for the successful establishment of in vitro plant tissue cultures [6, 7]. A key advance in plant tissue culture was the control of morphogenesis by using different levels and combinations of growth regulators [8], because this allowed the regeneration of entire plants, opening the possibility of using in vitro systems to study fundamental aspects of cell differentiation and development, and also for the application of tissue culture for different purposes. Some other relevant advances in plant tissue culture were the culture of meristems as a tool for get- ting virus-free plants [9]; the demonstration of totipotency in haploid or gametophyte cells, which made possible the faster generation of isogenic lines important for plant breeding programs [10, 11]; the rescue of hybrid embryos to overcome sexual incompatibility between plant species [12]; the enzymatic degradation of cell walls of plant cells to produce protoplasts and the fusion of these naked cells to eliminate sexual barriers between different plant species to render intraspecific or interspecific somatic hybrids [13, 14]; and the production of secondary compounds using cell or organ cultures [15], and perhaps the most relevant advance in plant tissue culture was the development and establishment of genetic transformation systems by Agrobacterium tumefaciens infection and through particle bombardment to allow the genetic manipulation of plant species [16] (Fig. 1).

2 Basic Principles of Cell, Tissue, and Organ Culture

Anyone who wishes to start plant tissue cultures should have in mind the following basic principles: (1) select an appropriate explant from a healthy and vigorous plant, (2) eliminate microbial contamination from the surface of the explant, (3) inoculate the explant in an adequate culture medium, and (4) provide the explant in culture with the proper controlled environmental conditions. In the case of in vitro regenerated plants, they are subjected to an adaptation process (acclimatization) in the greenhouse before the transference to ex vitro conditions. Depending on the part of the plant that is cultured, we can refer them as cell culture (gametic cells, cell suspension, and protoplast culture), tissue culture (callus and differentiated tissues), and organ culture (any organ such as zygotic embryos, roots, shoots, and anthers, among others). Each type of culture is used for different basic and biotechnological applications. Plant Tissue Culture Introduction 5

Fig. 1 (a) Mixotrophic callus from Catharanthus roseus. (b) Heterotrophic callus from Catharanthus roseus. (c) Suspension culture from Catharanthus roseus. (d) Regeneration of Catharanthus roseus plants from callus. (e) Root culture from Catharanthus roseus. (f) Somatic embryogenesis in Coffea canephora. (g) Protoplast from Coffea canephora. (h) Micropropagation of Agave fourcroydes. Pictures a, b, c, d, e, f, and g are from the authors’ laboratories. Picture h is a gift from the laboratory of Dr. Clelia De la Peña, from Centro de Investigación Científica de Yucatán

3 Micropropagation

Undoubtedly, micropropagation or in vitro clonal propagation is one of the most current extended commercial applications of tissue culture (see Chapters 2, 8, and 10). Plant tissue culture is an excellent tool for the asexual multiplication of those species that are naturally reproduced asexually, but it is also used to overcome some problems of germination of seeds in different plant species; for example, recalcitrant species are particularly characterized for their short-seed viability (recalcitrant seeds), and therefore, asexual multiplication is a good alternative. Although tissue culture can be 6 Victor M. Loyola-Vargas and Neftalí Ochoa-Alejo

applied for the micropropagation of almost any plant species, it is recommended only for those that are economically profitable. Among the plant species that are currently micropropagated at the commercial level, the ornamentals occupy the first place. Micropropagation of plants may be carried out through three different ways: (1) by promoting the proliferation of apical or axillary buds and then rooting them, (2) by inducing adventitious bud formation and its further rooting (see Chapters 2 and 3), and (3) by somatic embryo formation, maturation, and germination (see Chapters 9, 12, 13, 14, 15, and 16). Each alternative can be applied to several plant species at different efficiencies depending on the genetic background or regeneration capacity, the culture media, and the incubation conditions.

4 Plant Breeding and Genetic Improvement

Plant tissue techniques can be certainly powerful auxiliary tools for plant breeding and genetic improvement programs. Genetic variability detected in callus tissue and cell cultures can be due to genetic or epigenetic changes and represents an important possibility for recovering somaclonal variants or mutants with specific agronomic or industrial characteristics that can be exhibited at the cell or plant level [17]. Thousands or millions of cells constitute a piece of callus or a cell suspension, and they can be subjected to a selective pressure of different kinds of stresses to isolate resistant cells under controlled conditions. The recovered resistant cells may regenerate the entire resistant plants when cultured in adequate media. In this way, it is possible to generate plants resistant to drought, salinity, and cold or to biotic stress that affects crop yield [18]. A novel protocol for the estimation of somaclonal variation using molecular markers is described in Chapter 6. Isogenic or homozygous plants are important materials for breeding programs since they are used as parental lines to generate hybrid seeds, which when they raise plants, they have high yields. However, the generation of isogenic or homozygous lines can take five to ten cycles of self-fertilization by the traditional breeding techniques. By using microspore or anther culture, the time to produce isogenic lines may be reduced dramatically, because haploid plants can be regenerated in just one cycle of culture and then they can be diploidized by a colchicine treatment to get double- haploid plants with fixed homozygous sets of chromosomes [19, 20] (see Chapter 21). Anther or microspore culture can be also used to fix the characteristics of hybrid plants generated by parental crosses and conventional techniques. Embryo rescue and culture allow the recovery of hybrid plants from partially sexual compatible species. After cross-pollination between two different species, the development of the hybrid embryo occurs, Plant Tissue Culture Introduction 7

but the endosperm not necessarily accompanies the whole process of seed development, and at certain step, the hybrid embryo aborts; it is in that moment that the embryo can be rescued and cultured for further development [21, 22] (see Chapter 20). Intra- or interspecific hybrid plants can be also generated in sexual incompatible plant species through somatic hybridization using protoplasts from two different sources, which are fused by physicochemical methods. The hybrid cells are cultured to regenerate hybrid plants. Different somatic hybrid plants have been generated and described in the literature [23–27].

5 Genetic Engineering

Plant genetic engineering is possible thanks to the use of plant tissue culture systems combined with recombinant molecular biology techniques. The goal of plant genetic engineering is to manipulate genetic material from different organisms in such a way to have specific sequences coding for specific genes that confer particular characteristics when they are introduced and integrated into a plant genome. Once a gene of interest is isolated, a construct is prepared in an appropriate vector to carry out the genetic transformation using either biological (Agrobacterium tumefaciens-mediated infection) (see Chapter 33) or physical methods (usually microparticle bombardment). Genetic transformation has been achieved with important crops such as corn, wheat, cotton, rice and soybean, among others, and millions of hectares are currently planted with transgenic crops resistant to pests [28] or herbicides [29]. A reduction in the applications of toxic insecticides (organophosphorus insecticides) to control several pests is expected with the use of transgenic plants resistant to insects. Besides biotic factors, crop production and yield are much more frequently affected by abiotic factors (water stress, salinity, and cold, among others). Plants have evolved adaptation mechanisms to abiotic factors, but, in general, they are quite complex because they involve physiological, biochemical, and molecular processes. However, transgenic resistant plant crops to drought or salinity have been already generated [30–34], opening new opportunities of manipulation of complex abiotic resistant traits to cope with different environmental stresses. Genetic transformation has been also a powerful approach in basic science to carry out functional studies of plant genes (see Chapter 33).

6 Genome Editing

In the last decade, different genome editing techniques based on the use of sequence-specific nucleases have allowed precise manipulation of target genomic sequences opening the possibility of 8 Victor M. Loyola-Vargas and Neftalí Ochoa-Alejo

creating specific desirable mutations [35, 36]. Genome editing technology combined with plant tissue culture and genetic transformation has started to revolutionize the breeding and improvement programs of several crops. One of these technologies involves the CRISPR/Cas9 genome editing system [37–39]. Comparatively with genetic transformation, genomic editing technologies do not imply the use of foreign DNA to make a genetic change in the receptor plant, but the genetic change is carried out in the own genome of the plant species to be genetically modified [40]. A review of this novelty technology is described in Chapter 7. Examples of successful genetically edited modified plants using CRISPR/Cas9 include important crops such as rice [41, 42], wheat [43], corn [44], tomato [45], and potato [46], among others. Genome editing systems are also currently of high value for functional gene studies [47, 48].

7 Omics and Plant Tissue Culture

Genomics (the study of gene structure, function and regulation, and related techniques), transcriptomics (the study of the transcriptome or the set of genes that are transcribed in an organism), proteomics (the study of the set of proteins translated in an organism), and metabolomics (the study of all metabolites present in an organism) have become essential for the study of biological processes in plants. The knowledge on plant genomes, transcriptomes, proteomes, and metabolomes has impacted favorably in the com- prehension of complex developmental processes, such as in vitro organogenesis, embryogenesis, or dedifferentiation, and the genetic changes induced during in vitro conditions [49–51] (see Chapters 24, 25, and 29). Additionally, metabolomics can be very useful to investigate secondary metabolism not only during morphogenetic processes but mainly in cell, tissue, and organ cultures of plant species producing secondary metabolites of industrial and pharmaceutical interest [52, 53] (see Chapter 32).

8 Epigenetics in Plant Tissue Culture

Epigenetic changes (heritable changes in gene function that do not involve changes in the DNA sequence) affecting in vitro plant regeneration and also explaining the variation frequently observed in either cells or regenerated plants have been reported [54–61]. Due to the impact of these epigenetic changes on tissue cultures, it was considered convenient to include in this edition protocols regarding the analysis of histone modifications and gene regulation (see Chapter 26). Plant Tissue Culture Introduction 9

9 Preservation and Conservation of Plant Germplasm

Plant germplasms are the genetic resources that are collected and conserved for plant breeding and crop improvement programs, and they represent really true preserved treasures of genetic variability from which plant breeders start looking for specific desirable characteristics to be selected to increase the yield of crops. Plant germplasm of important crops such as corn and wheat are maintained as seed collections under low temperatures at the Centro Internacional de Mejoramiento de Maíz y Trigo (International Maize and Wheat Improvement Center; CIMMYT) in México, whereas rice germplasm is concentrated at the International Rice Research Institute (IRRI) in the Philippines. Plant germplasms of vegetatively propagated crops such as potato (Solanum tuberosum L.) and sweet potato (Ipomoea batatas L.) are preserved in the form of tubercles or under tissue culture conditions at the Centro Internacional de la Papa (International Potato Center; IPC) in Peru. Plant tissue culture offers excellent alternatives for the conservation of germplasm of those crops that are vegetatively propagated since thousands of plantlets may be conserved in small spaces under controlled conditions that can reduce the growth of cultures (minimum growth) or can even stop completely their growth (cryopreservation). Cryopreservation protocols for shoot tips of pineapple and pollen of bromeliads are described in Chapters 18 and 19.

10 Future Perspectives

Plant cell, tissue, and organ cultures have been applied to a range of different purposes including micropropagation, which is the most extended and successful application at commercial level and surely will continue in the future, and genetic engineering of important crops to confer tolerance mainly to pests and herbicides enabling the increase in production and yield with less applications of toxic insecticides and herbicides in millions of hectares worldwide. A significant impact is predicted in the production of different transgenic crops resistant or tolerant to drought, salinity, or cold under these stress conditions in the near future. Additionally, genetic transformation will be certainly a strategic tool for facing the global warming and its consequences by generating transgenic plants resistant to abiotic factors. Genetic engineering is still expected to contribute to the development of transgenic crops with increased nutritional or nutraceutical value or resistant to diseases caused by fungi, bacteria, or viruses. Plant metabolic engineering contribution to the development of more metabolically efficient crops [62, 63] or with modified biochemical pathway leading to the production of commercial secondary 10 Victor M. Loyola-Vargas and Neftalí Ochoa-Alejo

metabolites has been slow and modest, but it should have great promise to regulate the biosynthesis of target diverse secondary metabolites of industrial and pharmaceutical interest [64, 65]. Much more difficult is to evaluate quantitatively the impact that tissue culture has had or will have on plant breeding and crop improvement using embryo rescue, double-haploid generation, or somatic hybridization, but of course they will be contributing to get improved hybrid crops to increase productivity. Somaclonal variation in tissue cultures has been employed to rescue or recover interesting materials that have led to the generation of new varieties [66] and undoubtedly will continue to be applied in the future for the isolation of somaclones bearing polygenic novel traits in which the mechanisms underlying complex agronomical characteristics are unknown. Genome editing techniques have opened a new and wide ave- nue for the second green revolution and certainly will allow the creation of new and novel plant varieties with useful agronomic traits through the fine manipulation of specific genetic changes in important crop species [67]. The development of high-throughput genome and transcriptome sequencing techniques, the application of protein separation and sequencing, and the improvement of extraction, separation, and identification of metabolites, as well as the availability of data in public databases, have helped to decipher genome organization, gene function and regulation, and predic- tion of protein function and to know the set of metabolites produced in different plant species. Omics have therefore become fundamental tools for the study of basic biological processes in plants. Integration of omics is desirable for a better understanding of whole biological phenomena. It is evident that omics will be of great benefit to investigate in vitro morphogenetic processes and should facilitate the establishment of more efficient in vitro plant regeneration protocols if master control genes of differentiation and development are identified and characterized. On the other hand, the combination of different omics should enable the metabolic engineering of interesting biochemical pathways in order to manipulate specific characteristics for the optimization and production of secondary metabolites of industrial and pharmaceutical importance.

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Cell Culture the Fundaments Chapter 2

Micropropagation in the Twenty-First Century

Jean Carlos Cardoso, Lee Tseng Sheng Gerald, and Jaime A. Teixeira da Silva

Abstract

Despite more than a century of research on effective biotechnological methods, micropropagation continues to be an important tool for the large-scale production of clonal plantlets of several important plant species that retain genetic fidelity and are pest-free. In some cases, micropropagation is the only technique that supports the maintenance and promotes the economic value of specific agricultural species. The micropropagation of plants solved many phytosanitary problems and allowed both the expansion and access to high-quality plants for growers from different countries and economic backgrounds, thereby effectively contributing to an agricultural expansion in this and the last century. The challenges for micropropagation in the twenty-first century include cost reduction, enhanced efficiency, developing new technologies, and combining micropropagation with other systems/propagation techniques such as microcuttings, hydroponics, and aeroponics. In this chapter, we discuss the actual uses of micropropagation in this century, its importance and limitations, and some possible techniques that can effectively increase its wider application by replacing certain conventional techniques and technologies.

Key words Actual applications, Breeding, Combined techniques, Cost reduction, Micropropagation, Plant tissue culture, Large-scale production, Secondary metabolic production, Somatic embryogenesis

1 Introduction

One of the first reports on plant tissue culture was by Gottlieb Haberlandt [1], who used individual cell culture and totipotency; this was followed by other attempted cultivation of isolated root tips under aseptic conditions. Philip White (1934) reported unlimited growth in excised root tips in liquid culture media containing inorganic salts, sucrose, and yeast extract [2]. Further, in 1939, he reported unlimited growth of proliferated masses in the same culture media from tumor-like excised tissues of leaves and stems from a Nicotiana hybrid [3]. In the same year, Philip White also reported spontaneous shoot growth from tumor tissues of Nicotiana in a liquid medium [4]. Subsequently, on the basis of these studies of White, Folke Skoog and collaborators [5, 6] established the

Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo (eds.), Plant Cell Culture Protocols, Methods in Molecular Biology, vol. 1815, https://doi.org/10.1007/978-1-4939-8594-4_2, © Springer Science+Business Media, LLC, part of Springer Nature 2018 17 18 Jean Carlos Cardoso et al.

importance of relationship among auxins and cytokinins during in vitro stimulation/inhibition of roots and shoots from tobacco callus culture, and these observations formed the foundation of modern plant biotechnology and are still in use [7]. It is also important to include studies by Knudson [8], who experimented with nonsymbiotic germination of orchids and reported the need for a type of sugar, either as glucose or fructose, for the germination and width enhancement of zygotic embryos. Toshio Murashige and Folke Skoog, in a paper published in Physiologia Plantarum [9], described a culture medium that is currently most widely used in plant tissue culture and pointed to a culture medium that could provide all essential nutrients for the rapid growth and large yields for in vitro pith tissues of tobacco. They showed that the inclusion of a tobacco leaf extract in the mineral culture media modified from White [10] resulted in a significant increase in the fresh and dry weight of tobacco tissues. This medium, called the “revised culture medium” published by Murashige and Skoog (MS), was developed based on changes in the fresh and dry weight of tobacco callus (variety Wisconsin 38) after the addition of single or multiple nutrients to the culture medium. After more than half a century, MS medium and its modifications continue to be one of the main culture media used for micropropagation of a wide variety of plant species, with satisfac- tory results. The formulation of MS culture medium, considered a structural pillar of actual micropropagation systems being currently used, is an example of the importance of basic studies that facilitate new advances and support modern, agriculturally important, biotechnology techniques. Such techniques help to overcome genetic barriers of interspecific hybridization [11] and facilitate the production of transgenic plants by plant tissue culture techniques [12] or the development of double-haploid technology for obtaining hybrids of important crops [13], apart from accelerating the large- scale production of pest-free plantlets with genetic uniformity similar to other clonal propagation techniques. Although many new techniques are being developed, modern biotechnology also continues to be extremely dependent on the development of more efficient protocols for plant tissue culture and micropropagation [12] as these techniques offer many advantages. These include controlled environmental conditions for cultivation that allows high repeatability of results irrespective of local climatic conditions; high rates of regeneration; the production of shoots from small tissues or organs (normally less than 1.0 cm in length); rapid, efficient, and large-scale disease-free production of plantlets; an aseptic environment with a better control of cultivated organism as plants or plant microorganisms that have no or low rates of contaminants; automation of different processes; better control of tissue or organ development using plant growth regulators; single-cell or few-cell origin organogenesis that eases the pro- Plant Micropropagation 19

duction of solid mutants or transgenic plants rather than chimeras; easy maintenance of replicates of important cultures under in vitro conditions that help avoid the loss of important genotypes; cryopreservation of tissues or organs for long-term conservation; and obtaining plants that cannot survive in uncontrolled environmental conditions such as haploid plantlets. Most of these techniques owe their origin to theories proposed by Haberlandt, while the evolution of micropropagation techniques that have made modern biotechnology possible can be attributed to various investigators. Nonetheless, certain aspects of micropropagation were considered to be challenging during the twenty-first century. These include increasing the efficiency of the most micropropagation techniques, limited innovation in new techniques developed, the maintenance of recalcitrant species for micropropagation, and the high cost of micropropagation that could lead to substitution by cheaper techniques. In this chapter, we address some aspects of real micropropagation systems with the aim of reducing the costs of micropropagated plantlets, the use of dedicated techniques, and some new techniques that could either replace existing ones or potentially alter the final products from large-scale micropropagation in the twenty-first century. Finally, we also offer some perspectives and difficulties that are likely to be encountered during micropropagation in this century.

2 Cost Reduction of Micropropagated Plantlets

The high costs of micropropagated plants are related to the use of aseptic culture conditions in a highly controlled environment that aims to produce high-quality plantlets in large quantities and ensures their genetic fidelity and pest-free status. The essential control of environmental conditions constitutes the major nonre- curring costs of a laboratory [14, 15] and includes infrastructure construction and maintenance; equipment required for preparing, sterilizing, and stocking culture media and associated products; equipment used for maintaining an aseptic environment in growth and transfer rooms; and materials and equipment required to control environmental factors, mainly in the growth room, such as lamps for artificial light and air conditioning to maintain temperature in a narrow range for the ideal development of plants cultivated in vitro. Other costs include electrical energy required to maintain environmental control, labor to maintain the production of micropropagated plants, high-quality plant material as starter cultures, a greenhouse for acclimatization, royalties paid to the breeder if commercial laboratories use new or protected cultivars, and, finally, costs associated with marketing, sales, and the logistics of delivery of plantlets from a laboratory to the final client [14–16]. 20 Jean Carlos Cardoso et al.

Given these factors, large variations in the cost of micropropagated plantlets are to be expected, and these will be based on the location of the laboratory and the type and quantity of technology and automation used [17]. Micropropagated plantlets are normally more costly than those obtained using other sexual and asexual techniques of propagation [18]. Greater costs related to the actual technologies used for plant micropropagation in agriculture, horticulture, floriculture, and forestry continue to be the main problem that prevents the expansion of this technique in the developing and undeveloped countries [15, 16, 18–20]. Kozai [21] proposed that a 90% reduction in the cost of production would be required for worldwide commercialization of micropropagated plantlets and concluded that only robotization/automation could lead to such a drastic reduction in costs. In a commercial laboratory, with few automated processes, intensive labor use accounts for the main proportion (60–70%) of the cost of micropropagated plantlets, followed by electrical energy costs (10–25%), which are used for culture medium preparation and to maintain environmental control and aseptic conditions in the growth room. Variations in the costs depend on the region, the type of plant produced, the technology used, and its efficiency [15, 22]. Due to high labor costs, many large-scale commercial micropropagation companies transfer plantlet production to developing countries to lower costs and increase competitiveness in the world of micropropagated plantlets. High labor costs are the principle reason for switching to automation of certain processes in some plant tissue culture laboratories. The use of bioreactor systems would result in a significant cost reduction of micropropagated plants due to a reduction in the number of workers required to perform labor-intensive processes such as preparation of culture media and transplantation of plant shoots. Few studies have evaluated the yield as plantlets per worker, and such data can result in a significant cost reduction. For example, a cost analysis of Phalaenopsis micropropagation showed that the greatest cost was skilled labor required for transferring plantlets (61.7%), followed by electrical energy used for air conditioners (16.9%) to maintain a stable temperature [15]. According to Charanasri (1989), in Thailand, the mean yield of a single worker is 100,000 plantlets per year. Similarly, based on personal experience (JCC) in a commercial laboratory in Brazil, ten workers (5 days a week, working 8 h/day) have the capacity to produce around 1.0–1.2 million plants per year (100,000–120,000 plantlets/worker/year) during micropropagation of Anthurium, gerbera, and orchid plantlets on agar culture media. Other factors that affect efficiency and increase costs include a low rate of multiplication, the quality of shoot clusters produced for transplantation, problems with microbial contamination, and Plant Micropropagation 21 the loss of plants during different phases of micropropagation, such as plant death during acclimatization. According to Chen [15], an increase in multiplication rate from 1.5 to 2.5 could lead to a 50% cost reduction in micropropagated Phalaenopsis; this increase in efficiency is one of the most important factors that affect costs. Based on this information and the fact that the cost of one micropropagated plantlet is significantly higher per unit than plants propagated sexually or asexually by other methods [16, 18, 23], micropropagation as a technique was found to be of limited use and could only be applied to some important horticultural and forest species and conditions, as described below: (a) Species with important limitations related to sexual propagation, where seeds are unavailable (e.g., most cultivars of banana and pineapple); plants with dormancy limitations (strawber- ries), those with limitations in genetic material and quantity from the mother plant (e.g., Anthurium); and plants with long juvenile phases due to sexual propagation (most fruit trees), those associated with or not with high heterozygosity where maintenance of the main characteristics of interest in some commercial cultivars by propagation of seeds alone is difficult (Anthurium, eucalyptus, orchids, pineapple). (b) Species with few or limited techniques of alternate methods of vegetative or asexual propagation, for example, banana or pineapple. In these plants, mother plant developmental period is long, a limited number of new shoots are produced upon standard vegetative propagation, and the rooting of new shoots occurs directly from adult plants. In contrast, micropropagation produces more efficient and rapid mass propagation of new shoots. (c) When limited numbers of mother plants are available to start propagation: during eucalyptus clonal propagation, mini- cuttings are the main propagation technique used by the industry. However, as soon as new clones are developed from seeds (when only one or few mother plants are available), micropropagation is predominantly used by the industry to rapidly mass propagate and increase the number of mother plants. Similar procedures were applied to a high number of commercial species, as a part of the propagation process, but it is not the main technique for large or mass propagation of the culture. Some examples include potatoes, Anthurium and other ornamental plants, eucalyptus, tree fruits, and sugarcane. (d) The removal of important diseases in old mother plants propagated asexually generally causes huge damage to the culture, as in potato tubers. Mass propagation of potato tubers is nearly impossible due to the presence of a high number of viruses and 22 Jean Carlos Cardoso et al.

other bacterial and fungal diseases after several generations of propagation. It is important to note that many micropropagated plants have this benefit because most plants with bacterial and fungal infections do not survive in vitro or are discarded at some time point after inoculation. The removal of viruses and some bacteria may require additional treatment with antibiotics, antiviral agents, or heat treatment, along with shoot meristem culture. (e) To support available biotechnological tools that aim to breed and develop new cultivars and hybrids, e.g., for somatic hybridization of different species by protoplast fusion (Citrus spp.), obtaining transgenic plants using direct or indirect transformation (soybean, maize), and double-haploid production (maize). (f) In some dioecious plants, micropropagation could be used to propagate only plants of a specific sex, such as in papaya (Carica papaya), where only hermaphrodite and female plants are required for fruit production. (g) This technique can be used to conserve noncommercial species and reforestation of areas with native vegetation, especially in plants where propagation of species is very limited or unknown. This scenario is mostly futuristic and will be possible because of new advances in mycorrhization and somatic embryogenesis. The high cost of plantlets produced by micropropagation is the major limitation for their use and expansion in extensive plant propagation [23]. Thus, cost reduction represents the most important advance for this technique in this century. Furthermore, micropropagated plantlets display many advantages compared to other systems of vegetative propagation and produce high-quality (physiological and genetic) plants that are free of pests and diseases and better vegetative development and yield [23, 24]. For example, in potatoes, an important food source, combining micro-tubers produced in vitro with soil cultivation under plastic polytunnels produces conventional sized tubers. This represents a significant increase in the final productivity of edible tubers because this type of propagule is practically free of diseases and pests, which considerably increases yields [25]. Another alternative is the combined use of micropropagation to produce disease- free mother plants followed by propagation using low-cost microcuttings under greenhouse conditions to achieve the large- scale production of disease-free tubers [26]. Essentially, new techniques such as aeroponics (soil and substrate-less growth) increases potato yield from multiplication systems; however, this requires further verification [27]. Plant Micropropagation 23

Thus, cost reduction of micropropagated plants could be a definitive solution to increase the production of commercial species in vitro and is a separate issue from the usefulness of this technique when used as part of a larger propagation system, e.g., potato, sugarcane, strawberry, and some important ornamentals. The cost of micropropagated plantlets can be reduced by using two alternatives. The first method (option A) aims to reduce costs associated with the micropropagation technique, i.e., using automation to reduce manual labor costs, while the second option (option B) is to increase the efficiency of micropropagation systems, i.e., increasing the multiplication factor of each explant inoc- ulated in vitro. Opinions differ on option A or B as being a better method to reduce costs. However, in the twenty-first century, only a combination of A and B would significantly and efficiently reduce the price of micropropagated plantlets. The use of low-cost techniques, but with low efficiency, (e.g., low multiplication rate) or vice versa, is not compensatory. In Phalaenopsis micropropagation, only efficiency (option B) was considered to be effective. Therefore, increasing shoot multiplication was identified as the main approach to reduce plantlet costs [15] because higher multiplication rates drastically reduce the time required to obtain a specified number of plantlets in the laboratory, which reduces both labor costs and electrical energy consumption. In Coffea arabica, the high cost (US$ 0.60) of in vitro plantlets, compared to the costs for propagating plants by seedlings (US$ 0.20), is the major limiting factor preventing the use of somatic embryogenesis for large-scale production [18]. Many techniques for reducing costs are available, but most of them are not applicable to commercial laboratories for various rea- sons. For example, the use of a specific culture medium for only one cultivar of a micropropagated species is hard to achieve in commercial laboratory conditions, unless this cultivar represents one of the main cultivars in the laboratory or has high potential and is irreplaceable. Based on personal experience (JCC) in commercial laboratories with gerbera (cut gerbera daisies), a species for which many new cultivars are marketed every year and introduced in the laboratory for micropropagation, important problems include bacterial contamination or low rates of multiplication. In this case, together with field tests for the development and evaluation of ornamental characteristics, a cultivar is also grown under in vitro conditions for evaluating general growth. Additionally, propagation rate, the presence of endogenous bacteria in culture, rooting capacity, and signs of hyperhydricity are also monitored [28]. These tests are typically performed for certain types of culture media that are used 24 Jean Carlos Cardoso et al.

for groups of gerbera cultivars. If a new cultivar shows low rates of propagation or some specific developmental failure, such as extremely hard root induction or high hyperhydricity in shoots (after some tentative in vitro reintroduction) in all the culture media tested, according to the experience of JCC, this cultivar will most likely be eliminated from the laboratory and field production rather than investing in the development of a specific culture medium or procedures for this particular cultivar. This process is simple to understand from a business point of view as using a new and specific culture medium for only one or few cultivars not only increases the direct cost of the micropropagated plantlet but also involves additional labor that also increases overall cost. In addition, the use of many different culture media is expected to result in greater risk of human error in media preparation as each medium would require a unique formula for preparation. Thus, the development and preparation of specific media for one or few cultivars are only possible in cases where the cultivar is irreplaceable and has very low growth rates, or if is one of the most important commercial cultivars of that laboratory. One example of a plant that requires specific procedures for cultivating a specific cultivar isAnthurium andraeanum cv. White Beauty, which is considered recalcitrant to in vitro organogenesis regeneration, as it requires the use of leaf segments from juvenile explants instead of adult tissues [29]. This cultivar is the only one with completely white spathes instead of regular green-white spathes, which result in vigorous growth and excellent horticultural and ornamental characteristics under greenhouse conditions. Interestingly, in gerbera cultivars that normally show physiological problems in vitro, propagated plantlets continue to manifest these problems under field conditions, probably due to epigenetic variations. It has been noted that under commercial conditions, some gerbera cultivars with problems in the laboratory, such as high hyperhydricity, are harder to acclimatize and show early plant death. However, interestingly, the reintroduction of the same cultivar under in vitro conditions, using healthy mother plants from the field, can result in normal responses of tissues and good in vitro development in the same culture medium that previously caused problems in growth (unpub- lished observations, JCC). Essentially, new culture media are introduced in a commercial laboratory only when it represents an alternative that results in a significant increase in regeneration, shoot, or root production for a specific minimum number of cultivars. Most laboratories use one or a few types of culture media for each micropropagated species. MS medium continues to be the predominantly used salt mixture because of its high applicability and suitability for various species and for multiple varieties of the same species. However, other salt formulations that could be used Plant Micropropagation 25

for some species include the wood plant medium [30] for some woody species such as eucalyptus [31]. In order to reduce the cost of culture media preparation, some laboratories have adopted the use of pre-prepared culture media that are formulated by companies that sell biochemical products, instead of preparing stock solutions for each salt and subsequent preparation of the culture media. Many different formulations of culture media are available on the market, and this availability represents an important source of reduction in labor required for culture media preparation and in error risk during stock solution and culture media preparation. Differences in culture media in a laboratory entail the addition of different plant growth regulators and certain complex mixtures and additives that are required for each culture and stage of micropropagation. Commercially available culture media are attractive alternatives to tissue culture laboratories as these can be custom- formulated based on the nutritional requirements of the species and their genotypes. Other alternatives to reduce micropropagation costs are discussed below.

2.1 LEDs, CCFLs, Light-emitting diodes (LEDs) and cold cathode fluorescent lamps and Laser Light (CCFLs) have gained greater application in plant tissue culture due for Tissue Culture to the ability to control their spectral composition and the ability to reduce radiant heat while offering high light intensity, allowing such light systems to be placed close to plants on a growth shelf [32–35]. Heat-emitting incandescent and fluorescent lamps not only increase cooling costs, important for commercial plant tissue culture laboratories and enterprises, but also limit the density of cultures that can be commercially cultivated per unit area. These aspects, together with their short life-spans, relative to LEDs and CCFLs, reduce their competitive nature in intensive commercial crop production that uses tissue culture operations, although they still have lower unit costs, making them the continued standard in tissue culture laboratories [32, 35]. However, lower manufactur- ing costs, for example, in China, now allow individual LED lamps and LED boards to be produced at competitive prices [34]. For laboratories with limited space (e.g., plant production on the space station [36]) or that need highly specialized experiments, such as the simultaneous use of specific light intensity of spectral quality,

together with photoautotrophic micropropagation (i.e., CO2 enrichment), small CCFL units [35] or LED boards are the ideal treatment unit for small-scale lab-based experiments, even if their unit costs may be higher than the use of conventional fluorescent lamps. The history of the use of LEDs in horticulture, and plant tissue culture, is not that long, since practical blue LEDs were only first invented in the early 1990s [37]. The ability to change the red to blue LED ratio, and to alter the light intensity of each bulb, and thus of a LED board, allows for an excellent experimental system 26 Jean Carlos Cardoso et al.

to study in vitro organogenesis of horticultural and ornamental crops, including strawberry [38], chrysanthemum [39], Zantedeschia [40], grapevine [41], orchids like Cymbidium [42], and papaya [43], or even of photosynthetic parameters of tomato in a greenhouse setting [44]. Despite these advantages and increasing use of LEDs, Ooi et al. [45] claim that “the electrical-to-optical power conversion of LEDs remains inefficient beyond a certain electrical current,” suggesting instead that laser light could be used to deliver individualized light spectral quality beyond what LEDs (or CCFLs) can offer, showing the growth of model plant Arabidopsis thaliana with lasers being equivalent to growth under LEDs and with comparable costs. As horticultural production becomes more competitive, researchers and industry need to begin to assess the cost-benefit ratio, depending on available size, bud- get, labor, and expected profits, as in rapeseed plant factories employing LEDs [46]. Researchers and commercial producers are cautioned, however, at the possible risk of LEDs increasing endoreduplication in tissue cultures, leading to somaclonal variants, as occurs with Phalaenopsis [47].

2.2 Photoautotrophic The control of environmental conditions, especially temperature Micropropagation and light, is essential for micropropagation in growth rooms prob- Systems, the Use ably because these two factors have the greatest influence on plant of Natural Light, development. The use of artificial light continues to be the main and Greenhouse method to control the intensity, periodicity, and quality of light in Micropropagation growth rooms. For temperature control, most laboratories use air conditioners for cooling growth rooms. In general, approximately 15–25% of the cost of a micropropagated plantlet accounts for environmental control to sustain development in micropropagated plants, e.g., the production of normal chlorophyll plants with leaves, stem, and roots. Interestingly, it is known that in vitro grown plants are not photosynthetically active, because of two main factors. The first is the low intensity of light from the lamps used in laboratories (LED or fluorescent lamps). The second is a

low concentration of CO2 inside the flask and in the growth room caused by a combination of rapid consumption of CO2 by plantlets and the poor air exchange between in vitro conditions and the external environment [48, 49]. In addition, maintaining a narrow temperature (22–27 °C) in the growth room increases micropropagated plantlet costs and reduces the efficiency of acclimatization under greenhouse conditions, as the latter shows wider fluctua- tions in temperature and other climatic conditions such as low air humidity which can to plant death on these conditions by excessive loss of water for the environment. In order to compensate the absence of photosynthesis under in vitro conditions, conventional micropropagation uses heterotrophic or photomixotrophic systems that externally add a source of sugar (carbon), normally sucrose, to maintain Plant Micropropagation 27 regeneration, multiplication, rooting, or other in vitro development responses. This addition of sucrose also leads to many problems that eventually increase the costs of micropropagated plantlets. In addition, plants cultivated in vitro have high photosynthetic ability due to the presence of chlorophyll and develop photoautotrophy [48, 49].

To address the problems of low CO2 concentration and the internal accumulation of ethylene in culture flasks, some companies have developed perforated polypropylene caps with filters. Other types of caps as plastic film, cotton balls, and filter caps have also been used to increase internal-external air exchange. However, the use of such caps alone does not result in suffi- ciently increase CO2 for photoautotrophy [49]. Additionally, any increase in air exchange could also result in microbial contamination, which reduces the efficiency of micropropagation. Nonetheless, modifications in the type of flasks being used can increase air exchange and CO2 supply. For example, Tanaka et al. [50] developed the “Culture Pack” to replace commonly used flasks for film cultures. Film vessels composed of a fluorocarbon polymer film, the “Vitron,” were successfully used in the photoautotrophic micropropagation of Eucalyptus urograndis [51] and Spathiphyllum [52]. Kozai [48] proposed that photoautotrophic micropropagation systems can be established only when certain conditions are ful- filled. First, plant growth should occur in sucrose-free culture medium, the culture should be cultivated under in vitro conditions with high PPFD (photosynthetic photon flux density), and cultures should be cultivated under in vitro conditions in a CO2- enriched environment. Xiao et al. [53] described that this system could be enhanced by decreasing relative humidity in flasks by sub- stituting agar-based gelling agents with a fibrous or porous support material with high air porosity. Under these conditions, photoautotrophic micropropagation could be successfully established for different species. The use of this technique proposed by Kozai has several advantages including reduced contamination (probably because of the sucrose-free culture medium), a decrease in plant loss and physiological disorders, and consequently, costs. Further cost reduction can be achieved by increasing automation/ robotization and the use of larger vessels, as these measures drastically decrease the number of transplants and labor required [17, 54]. However, the main disadvantages of using automation are high initial costs compared to conventional micropropagation systems and difficulties associated with the creation of a modified and controlled atmosphere necessary for a growth room, i.e., controlled CO2 enrichment, or the use of light bulbs that increase the PPFD to permit photoautotrophy. Well-developed systems for

CO2 enrichment are available and are used in horticulture. However, the use of higher light intensity and PPFD increases 28 Jean Carlos Cardoso et al.

costs attributable to electrical energy, and because more light implies more heat, longer hours of air conditioning are needed to maintain temperature. Nevertheless, as discussed in Subheading 2.1, the use of LED, laser, or CCFL light bulbs with high PPFD and with a wavelength designed to stimulate photosynthesis could be a solution. In addition, the higher costs for establishing photoautotrophic micropropagation can be compensatory, if micropropagation efficiency is increased and labor for plantlet production is

reduced [17]. Photoautotrophic micropropagation (high CO2 and PPFD and an increase in the number of air exchanges) enhanced the growth and survival of acclimatized Doritaenopsis plants as a result of biochemical and physiological changes [55]. Similar environmental conditions also stimulated autotrophy and an increase in the rate of biomass production in “golden” papaya [56]. Another alternative for photoautotrophic micropropagation is the use of a greenhouse environment with natural light and atmo-

spheric CO2 concentration. This could be considered as the most low-cost disposable source of light in terms of intensity and quality (i.e., replacing artificial light), far exceeding that required for photosynthesis and photomorphogenesis for in vitro cultivation. The feasibility of using a natural environment under greenhouse conditions, where in vitro cultures would be performed using various strategies to control the environment, has been scientifically tested on a reasonable number of micropropagated species. In many developing countries, the micropropagation of commercial orchids such as Vanda, Phalaenopsis, Dendrobium, Cattleya, and Oncidium has been achieved under greenhouse conditions several years ago. Normally, growers seed orchids in glass bottles containing a culture medium based on a mixture of fruit pulp and charcoal and maintain these bottles in horizontal positions covered with plastic film (to avoid excessive air humidity) with a net shade cutting more than 80% of incident sunlight but with approximately 10–20% of natural light inside in the greenhouse. This minimal entrance of light avoids excessive temperature elevation as light transforms to heat under greenhouse conditions. Under these high net shading greenhouse conditions, plants receive between 8000 and 15,000 lux of natural light, which is approximately 5–15 times more intense than the light in a growth room with LED or cold fluorescent lamps in commercial micropropagation laboratories. Such greenhouse micropropagation has been used in the final phase of orchid shoot rooting, before acclimatization. Cardoso et al. [57] used this greenhouse micropropagation technique for gerbera cultivation with a PPFD of 100 μmol m−2/s. They compared this to an identical phase in the growth room during rooting and observed significant increases in leaf number and diam- eter, rooting percentage, and number, as well as fresh and dry weight in plantlets cultivated under greenhouse conditions. The plants cultivated in sucrose-free medium achieved the same efficiency only Plant Micropropagation 29 during acclimatization when cultured under greenhouse conditions in the previous stage (pre-acclimatization). The use of pre-acclimatization in micropropagated plantlets (rooting phase in greenhouse conditions) drastically reduces the time required in the growth room and facilitates an increase in the efficiency of micropropagation in commercial laboratories. The efficiency of yield of in vitro Solanum tuberosum mini- tubers was not different in a controlled growth room and in a non- controlled room, similar to a greenhouse that used natural light, in long-term experiments (2009–2014) covering different seasons of growth [58]. These results show the potential of this technique not only for rooting orchids but also for the micropropagation of other ornamentals [57], tubers [58], and fruit species, as banana [59–61] and pineapple [62] apart from improving the use of laboratory space, especially for a growth room as it will only be used during specific phases of micropropagation such as establishment and multiplication. The rooting phase normally requires more space than other phases of micropropagation as only a few plants can be accom- modated in each flask, and each plant requires space to increase weight, leaf number, and roots in preparation for acclimatization. For example, the rooting phase of gerbera requires two to three times more space than its multiplication phase to achieve adequate rooting of shoots obtained in the previous phase. A working estimate is that each flask in the multiplication phase requires two flasks for rooting and elongation, while the last phase uses approximately 60–70% of the useful space in the growth room. By transferring these flasks in the rooting phase to greenhouse conditions, it is possible to double or triple the usable space in the growth room for multiplication, thereby significantly expand- ing the capacity of the laboratory to produce more shoots. The following is a simple calculation for a laboratory with a full capacity of 10,000 flasks where each flask can support five initial shoots in the multiplication phase and ten shoots in the rooting stage. Using a multiplication factor of 4:1 for simultaneous rooting and multiplication, the use of a greenhouse rooting system with pre- acclimatization will enhance the laboratory capacity to 180,000– 200,000 shoots/month (9–10,000 flasks × 5 initial shoots × a multiplication factor of 4:1, 90–100% of the laboratory for multiplication). Comparatively, a conventional system that uses the growth room for multiplication and rooting will only accommo- date 60–70,000 shoots (3330 flasks × 5 initial shoots × a multiplication factor of 4:1) as most of the space will be used for shoot elongation and rooting. Other advantages of pre-acclimatization under greenhouse conditions, apart from cost reduction of micropropagated plantlets, are the presence of natural light and cheaper methods of 30 Jean Carlos Cardoso et al.

temperature control, as fogs or pad fan systems which reduce temperature by increasing cold air humidity, instead of artificial light and temperature control by air conditioners. Additionally, plants rooted under greenhouse conditions begin photosynthesis before those rooted in growth rooms [55], probably owing to the cultivation in sucrose-free medium and better anatomical adaptation for acclimatization [57]. Furthermore, pre-acclimatization under greenhouse conditions increases survival rates of acclimatized plantlets [59, 62, 63]. The main difficulty associated with this technique involves good planning and project implementation to maintain a greenhouse in close proximity to the growth room to ease the transport of flasks from the laboratory to the greenhouse. This technique also requires a greenhouse for in vitro culture with specific environmental conditions. Verticalization, which is normally used in laboratories with artificial lights, is harder to achieve in a greenhouse and results in great variations in the intensity and quality of natural light received by the plants in vitro. This less controlled environment could also increase microorganism contamination in flasks, and it is harder to control photoperiod in the greenhouse without supplementary artificial light. A successful alternative for micropropagation is the use of tubular skylights that redirect natural light to a growth room without heat, instead of using a greenhouse environment [60].

2.3 Chemical The chemical sterilization of the culture media is an alternative to Sterilization of Culture the commonly used physical autoclaving system where high tem- Media peratures (121 °C) and pressure (1 kgf cm−2) are applied for 20–30 min. This process consumes electrical energy, labor, and time (normally 1–2 h for one round of autoclaving) to sterilize the culture medium and other products required for establishing an aseptic culture. The alternative is chemical sterilization, which uses a chemical substance with bacteriostatic/fungistatic or antibiotic/fungicide action to eliminate and avoid future contamination of the culture medium in flasks, thereby replacing the process of autoclaving. This technique differs from the use of antibiotics in the culture medium that is used to prevent microbial contamination from endogenous bacteria in certain types of explants, post-autoclaving contamination, or to prevent the growth of Agrobacterium tumefaciens after indirect genetic transformation of plant tissues. In all these cases, antibiotics are complementary to autoclaving and are added by filter sterilization after the culture medium has been autoclaved. Most products investigated and used for chemical sterilization are chlorine-based derivatives. Teixeira et al. [64] tested the use of NaOCl (using common bleach as the source) and observed Plant Micropropagation 31 that a concentration ranging between 0.0003 and 0.0005% of active chlorine results in 100% uncontaminated cultures with the highest average number of shoots and fresh weight. This study was conducted on pineapple (Ananas comosus) in vitro shoots using MS liquid culture medium, and interestingly, the average number of shoots obtained was the double (13.4 shoots/culture) that obtained with autoclaved culture media (6.6 shoots/ culture). Brondani et al. [65] also used NaOCl to establish nodal segments from different clones of Eucalyptus benthamii and found that concentrations between 0.001 and 0.003% resulted in a good percentage of establishment, viable shoots, and fungal decontamination that were similar to those observed with autoclaved culture medium. Similar results were obtained with gerbera cv. Essandre [66]. NaOCl is normally used to achieve aseptic explants from non- sterile conditions during the micropropagation establishment stage and has the advantages of being both cheap and easy to acquire. The disadvantage of this technique is that a more complex method to prepare flasks and culture media is required, which includes washing flasks with a solution containing NaOCl and the preparation of culture medium containing NaOCl. Most commercial laboratories wash culture flasks using an automatic washing machine to reduce labor and increase the yield of washes; however, it should be noted here that NaOCl is corrosive to metals. Other chemicals tested and used during the micropropagation of Anthurium andraeanum [67] and gerbera [68] include chlorine dioxide (ClO2), which is widely used and is recommended for food sanitation. Various commercial formulations containing stabilized ClO2 in a liquid phase facilitate its use in solution and its application in culture media preparation. ClO2 is added to the culture medium at 0.0025% before pH adjustment and results in microorganism-free in vitro growth during all stages of micropropagation of gerbera and better plant development compared to autoclaved culture media. This technique is simple to use and does not require any additional procedures for sterilization as flasks need to be washed with water and detergent before use. Finally, a gelling agent is added to the culture medium, dissolved at high temperature, poured into flasks, and flasks are capped. The culture medium is now ready for transplantation. Cultures were maintained sterile for up to 90 days after preparation of culture medium and in vitro cultivation of Anthurium [67], satisfying the time required to transplant more than 90% of in vitro cultured commercial species.

One disadvantage of this technique is that stabilized ClO2 increases the pH of the culture medium and requires the careful addition of HCl during pH adjustment to maintain a range between 5.8 and 6.2. 32 Jean Carlos Cardoso et al.

Other products that are free of chlorine can also be used for chemical sterilization of culture media, such as diethyl pyrocarbon- ate (DEPC) at 1 g/L [69] and hydrogen peroxide [70]. A distinct advantage of the chemical sterilization technique is the utilization of different types of flasks/vessels for micropropagation, such as polyethylene or other non-autoclavable plastic vessels that are cheaper than glass or autoclavable plastic flasks. In addition, this technique can be applied to bioreactor systems that use polyethylene bottles, instead of autoclavable plastic or glass bottles that are more expensive and difficult to autoclave. Cold sterilization with nontoxic chemical products also presents advantages compared to autoclaved products such as avoiding the degradation of nutrients, plant growth regulators, and other products added to the culture medium and prevents the formation of toxic substances caused by a high-temperature exposure of different components in the culture medium [71], resulting in better performance of in vitro plantlets.

2.4 Bioreactor The major obstacle to the micropropagation of conventional plants Systems to Reduce in a plant biofactory is the excessive use of labor, which increases the Cost costs of seedling/plantlet production in vitro. The term plant bio- of Micropropagated factory refers to a micropropagation laboratory that produces Plantlets plants in vitro in large quantities and whose production process is well-defined [72]. The most important advantage of using Rita® and BIT® bioreactors is the reduction in the demand for labor, which will, in turn, reduce plant costs by 20–40% or more. The process of plant micropropagation involves three main steps, namely, initiation, multiplication, and elongation rooting. Typically, in a biofactory using bioreactors, the initiation phase is similar to that of conventional methods, but bioreactors are used in the multiplication and elongation-rooting phases when the demand for flask and labor greatly increases [73]. Several types of bioreactors have been developed and used. The most common types of bioreactors are the aeration agitation bioreactor, roller drum bioreactor, spin filter bioreactor, air driven bioreactor, airlift bioreactor, gaseous phase bioreactor, oxygen permeable membrane aerator bioreactor, overlay aeration bioreactor, and the immersion by bubbles bioreactor. In all these models, the culture material remains continuously immersed in the culture medium. This continuous immersion causes problems that arise from hyperhydration of the shoots/plantlets. Depending on the species and the type of medium used, hyperhydration can cause serious physiological disturbances that affect the growth and development of the growing material, a phenomenon known as hyperhydricity [74]. In order to minimize this problem, Alvard et al. [75] developed a bioreactor model, called the temporary immersion bioreactor – BIT®. In this bioreactor, the culture medium remains in Plant Micropropagation 33

contact with the explant for a predetermined period after which the medium is drained, and the explant ceases to be in the direct contact with the culture medium [73, 74]. The model developed by Alvard et al. [75] was modified by Teisson et al. [76] and gave rise to the bioreactor system RITA®. The RITA® system has been used for a number of plant species with different types of explants and has shown very good results [73, 74]. The advantages of using temporary immersion bioreactors in a plant biofactory are enormous. In addition to reducing costs, the quality of the plants produced is much higher. This is not surpris- ing because, physiologically, the bioreactor process facilitates plant growth without stress. The plants thus produced are usually larger, well rooted, easily acclimatized, and do not suffer many losses. The advantages are summarized below [73, 74]: 1. Decreased demand for labor 2. Decreased use of appliances (laminar flow, tools for operation) and jars 3. Reduction in the space required for the micropropagation of plants 4. Reduction in electricity costs for plant cultivation 5. Total or partial elimination of the use of gelling agents that are one of the most expensive components in a plant micropropagation unit 6. A significant increase in the multiplication rate of the cultivated materials 7. Significant increase in the quality of the plants produced 8. Ease of acclimatization of cultivated materials 9. Decrease in contamination risk in carefully installed operations The disadvantages of using a bioreactor in a biofactory are rather few. Plant losses can be quite large if there is contamination, and the initial cost of an installation may appear too high when compared to that of traditional biofactories. Initial quality inspec- tion of plant material should be strict, and, possibly, latent bacterial indexing may become necessary before the use of this material in a bioreactor. The problem of hyperhydricity, which normally occurs in liquid culture, is easily addressed by adjusting the immersion time of the materials. Problems arising from sterilization in a bioreactor system can currently be solved by a set of chemical steriliza- tions and preparation of liquid culture media in industrial autoclaves [73, 77]. In conclusion, conventional plant micropropagation is a labor- intensive operation that results in a very high cost of plants produced by this technology. 34 Jean Carlos Cardoso et al.

The use of temporary immersion bioreactors is a semiauto- matic operation that not only solves this problem but also produces plantlets with a quality that is far superior to those produced by conventional micropropagation methods.

3 Increasing the Efficiency of Micropropagation for Other Applications

Micropropagation has great importance in improving techniques to obtain high-efficiency regeneration of cells, tissues, and organs; it is also useful in developing breeding techniques using biotechnological tools and for the production of secondary metabolites with medicinal properties. The main advantages of the use of micropropagation as a biotechnological breeding tool, instead of conventional breeding techniques, are attributable to the need of very small explants from the different parts of the plants to start in vitro culture. Other advantages are the cultivation and regeneration of plants from isolated cells, tissues, or organs, regeneration of complete plants from unicelular or few agglomerates of cells to avoid chimeras, excellent control over chemical and physical conditions, an aseptic environment, regenerated plants that are free of pests and diseases, reduction of genetic barriers commonly observed in some in vivo crosses, easy selection of regenerated plants using selective culture media, controlled systems for test isolated factors in plant development, and efficient protocols for large and rapid propagation of new genotypes obtained by breeding. Somatic embryogenesis (SE) is another technique that can be used to reduce costs and increase the efficiency of micropropagation techniques. Essentially, this technique is more actually limited to research on understanding genes and factors that control SE in plants [78] and is used in protocols applied to produce transgenic plants [79, 80]. Although SE and the production of synthetic seeds represent a large potential for application in several species and were considered a preferred method for propagation of woody plants [81], some problems that still challenge research in this century are the warranty of commercial production and the quantity and quality of synthetic seeds produced. The most important limitations of SE are the limited number of responsive species and genotypes [81] and the high epigenetic and somaclonal variations during the development in somatic embryogenesis (especially, during embryo-to-plantlet conversion). This limits its application in the commercial production of synthetic seeds in some important areas of agriculture, horticulture, and forestry. For example, Beyene et al. [82] detected the loss of CMD2, a monogenic resistance gene found in cassava (Manihot esculenta) cultivars resistant to cassava mosaic disease, when immature leaves and axillary buds were used for tissue regeneration by SE. Although somaclonal Plant Micropropagation 35

variation in SE-derived plants is detrimental for propagation, the technique could be used for breeding to obtain superior somaclones [83]. The combination of different techniques, SE, and the use of microcuttings (4–6 cm) rooting, from juvenile acclimatized SE-derived plantlets, have resulted in significant increases in the efficiency of Coffea arabica and have reduced the production costs of in vitro SE derived-plants, from US$ 0.60 to US$ 0.27, promoting mass propagation of coffee plantlets derived from SE technology [18]. The encapsulation of somatic-derived embryos with a calcium alginate gel is an alternative to storage and conservation that also increases germination of the somatic embryos. Further, somatic embryogenic cultures could be used for cryopreservation [84].

3.1 Plant Breeding The development and evolution of micropropagation techniques Using Biotechnological also promotes their actual use as biotechnological tools for breed- Tools ing. The main techniques used are somatic hybridization by protoplast isolation and fusion, in vitro pollination and fertilization, embryo rescue, haploid and double-haploid production, polyploidization, mutation induction, and transgenic plant production. Most of these techniques were not possible under in vivo environmental conditions because of reproductive barriers and the limited capacity of isolated cells, tissues, and organs to survive outside the mother plant. Among these techniques based on plant tissue culture protocols, the most useful are transgenic plants and haploid and double-haploid production because of their potential or realist technologic applications. Transgenic plants offer the foremost applications of biotechnology in agriculture. Biotech crops (genetically modified or GM crops) represented 180 million ha in 2015, and 2 billion ha was cultivated in 20 years of cultivation, from 1996 (start of GM crop commercialization) to 2015 [85]. The production of transgenic plants has the main objective of generating more efficient plants, plants with environmental resistance to salinity, water deficit, and high temperature, those with resistance to pests and diseases, those with resistance to herbicides, those that produce essential amino acids and vitamins to solve malnutrition in undeveloped countries, those producing vaccines against animal and human diseases, those producing more resistant fibers for industry, those with new colors of flowers for the floriculture market, and those with rapid growth and high resistant wood for the timber industry and the better knowledge of genes and their function. GM crops could help sus- tainable agriculture under climate change in this century. However, onerous regulatory processes continue to be the main constraint in the adoption of new technologies from transgenic plants. 36 Jean Carlos Cardoso et al.

Golden rice, which is enriched by micronutrients, Clustered regularly Interspaced short palindromic repeats (CRISPR)-related technologies, and the expansion of GMO growing areas in Africa and China in this century, are to be one of the greatest advances in the future of transgenic plants [85, 86]. Haploid and double-haploid technologies are now used by many companies to obtain homozygous lineages with hybrid production, such as in maize (Zea mays). The production of haploids can be induced either in vivo or in vitro. In maize, the main method of haploid induction uses pollen grains from male lines, called inductors or inducers, which are crossed with female lineages that produce seedlings at 0.7– 16.8% of haploid-inducing frequency. For screening haploids in seeds, a marker gene R1-nj is used in the inductor male parent that has a purple scutellum and a “purple crown” of the aleurone in diploid zygotic embryos, instead of the white/green in haploids [87]. This technique is called parthenogenesis, but most of the haploids obtained would be through gynogenesis or pseudogamy. In the first scenario, the concept is related to the development of an embryo without fertilization or any stimulus, while in the second scenario of pseudogamy or gynogenesis, the development of embryos requires some stimulus (e.g.,, pollination) but without inheriting genes from the male parent [88, 89]. Parthenogenesis, using unpollinated ovules and ovaries, can also be attained in plants [90]. Gynogenesis or pseudogamy can be induced in vivo or in vitro using different techniques as follows: (1) using an inductor as described above for maize; (2) using crossings with pollen grains from distant species; (3) using crossings with irradiated pollen grains [91]; (4) using triploid pollen grains [92]; and (5) combining some of these techniques, e.g., irradiating pollen grains of distant species for use in crossings. Pollination can be achieved in vitro or in vivo followed by embryo rescue and in vitro cultivation of seeds or embryos. Importantly, seed/embryo rescue can also be carried out in vitro. Froelicher et al. [93] obtained haploid plants from rescued embryos of small seeds of Citrus clementina, tangor “Ellendale,” and “Fortune” mandarin, after irradiation with 150 and 300 Gy of gamma irradiation and crossing with pollen grains from “Meyer” lemon. Microspore culture is another technique used to obtain haploids and double haploids and entails cultivation of anthers or isolated microspores in a culture medium in order to switch from pollen grain formation to gametic embryogenesis induction via microspores; this is also a more common method of regeneration. The choice of the best technique for haploid production depends on the species and cultivars. For example, in Citrus clementina, microspore culture is very difficult [94] compared Plant Micropropagation 37 to anther culture; it is possible to produce large quantities of tri-haploids embryos from small quantities of anthers cultivated in vitro. Contrarily, in apple (Malus domestica), microspore culture was found to be ideal for embryo induction rather than anther culture [95]. In onion (Allium cepa), the best way to regenerate haploids is by parthenogenesis where flowers are cultivated, but many attempts with anther culture did not result in haploids in this species [96]. Developing haploid callus, embryos, or plantlets is generally associated with certain genotypes or species, and this represents one of the most important factors responsible for successful haploid embryogenesis. The second factor is the stage of the development of the microspore, and in most cases, the mid- to late-uninucleate stage is the most responsive. The in vitro technique of anther culture is limited by its high dependence on genotype responses that lead to haploid production and a low regeneration percentage in haploid plants [97]. This absent or very low rate of regeneration of haploids and double haploids (DH) limits the commercial application of the technique to only responsive species. Despite research on the efforts to understand the causes of recalcitrance in haploid and double-haploid induction and regeneration [98], many of the challenges have not yet been addressed to date. Developing effective protocols certainly increases the relative importance of haploid and double- haploid production, especially for economically important crops such as vegetables, fruits, ornamentals, forest plants, and other cultures that use hybrid seeds in large numbers for agriculture in the twenty-first century. The use of this technique also allows the development of completely homozygous lines in one growth cycle that can be used for hybrid production rather than conventional allogamous breeding for hybrid production using successive late and numerous self-fertilizations [99]. Actually, a centromere- specific histone 3 variant and correlated transgenes (CENH3- tailswap and other cenh3 null mutants) can help new discoveries to improve haploid production, by eliminating mutant chromosomes from the genome [99, 100], and will be applied mainly for haploid recalcitrant species [101]. Transgeny using combined double-haploid lineages accelerates programs of breeding in crops in which the main genotypes used for cultivation are hybrids [102, 103]. Haploids and double haploids are also used for molecular plant breeding, genetic studies, and other molecular techniques such as marker-assisted selection, QTL mapping, genome sequence programs, and studies on male sterility, among others [13]. Polyploidization is another technique used for the development of new cultivars; however, tetraploid cultivars have much larger organs than diploids. For example, in floriculture, the production of larger flowers could cater to a specific market. 38 Jean Carlos Cardoso et al.

Similarly, tetraploids could be used in crossings with diploids to produce triploids (3×). Triploid cultivars are predominantly used for seedless fruit production, such as in watermelon (Citrullus lanatus) and grape (Vitis sp.). Despite the presence of multiple strategies, micropropagation continues to be an important method to produce polyploidy plants, mainly due to certain important advantages. These include the possibility of using minimal quantities of very expensive antimitotic agents, such as colchicine or oryzalin, in the culture medium to induce polyploidy in cells but with decreased undesirable genetic variations, establishing solid polyploidy using organogenetic or embryogenetic processes from one or very small quantities of cell agglomerates that result in complete polyploid plantlets rather than common chimeras obtained using other types of in vivo treatment that require larger amounts of tissues/organs (e.g., seeds), possible rapid multiplication of numerous individual genotypes from only one or few initial mother plants, asexual propagation of fruits and flowers, and easy application and clonal propagation of new triploid genotypes. The techniques used to produce polyploids include protoplast isolation and fusion, which are mainly used for obtaining somatic hybrids (allopolyploids) from different species with the total number of chromosomes being the sum of each species. Autopolyploids can also be produced using leaf, internode, or hypocotyl segments exposed to antimitotic agents for varying periods and concentrations followed by the regeneration of plantlets from these tissues. Other applications are rescue and in vitro cultivation of immature embryos from crossings between 2× and 4× plants to obtain triploid genotypes and endosperm culture to obtain triploid plantlets. A combination of techniques used for in vitro immature ovule culture (50–60 days after pollination) using rescue and in vitro cultivation of ovules in culture media obtained from various crosses between 2× and 4× grape cultivars is reported to be a good strategy for obtaining triploids in grape [104, 105]. Triploids could also be obtained from diploid plants by using cultivation and regeneration of plants from endosperm tissues. This method has been successfully applied to several species and represents an important tool for the generation of new triploid genotypes [106].

3.2 The Use The in vitro milieu provided by a plant micropropagation vessel of Micropropagation offers the perfect platform for the mass production of secondary for Secondary metabolites, primarily from medicinal and aromatic plants, many of Metabolite Production which are used in food, flavorant, perfumery, and medicinal and pharmaceutical industries. This is because in vitro conditions are sterile, avoiding competition by microbiological agents while also providing the most suitable optimized conditions for cellular or tissue/organ growth. Researchers have always sought ways to Plant Micropropagation 39 assess optimal conditions, including lighting, temperature, and the inclusion of plant growth regulators, media, and vessels, to assess the optimal production of secondary metabolites from MAPs [107]. One of the most common means to mass produce secondary metabolites is through the use of callus or cell suspension cultures (CSCs) in bioreactors, eliciting the production of the desired compound(s) in continuous liquid culture, while the second popular method is the mass production of organs, such as leaves, on in vitro plants, that are then harvested en masse. Biofarming or biotransformation involves feeding precursors to cell or tissue cultures, allowing them to undergo a series of biochemical reactions to produce secondary metabolites of interest. Selected noticeable examples of key MAPs are discussed next. Callus induced from the roots, stems, leaves, and cotyledons of Rhodiola sachalinensis could produce salidroside (555 mg/L), about five- to tenfold higher than wild plants [108]. Cannabinoid content, especially of ∆9-tetrahydrocannabinol, was stable in plantlets derived from leaf-induced callus cultures of Cannabis sativa induced in the presence of 0.5 μM 1-naphthaleneacetic acid (NAA) and 1.0 μM thidiazuron (TDZ) [109]. The addition of 200 μM methyl jasmonate (MeJA) to Taxus canadensis and Taxus cuspidata CSCs induced the production of paclitaxel (taxol) and other taxoids, but paclitaxel never exceeded 20% of the total taxoid production [110]. MeJA, when combined with TDZ, increased the production of centelloside from Centella asiatica about 2.4- fold more than when just MeJA was used [111]. Another popular chemical elicitor, salicylic acid, allowed for the production of hypericin and pseudohypericin to be doubled in a Hypericum per- foratum CSC [112]. The optimization of withanolide production (withanolide A, withanolide B, withaferin A, and withanone) from a Withania somnifera CSC was possible by adjusting the levels of sucrose in a basal CSC that included 40% (w/v) Gracilaria edulis extract, 200 mg/L L-glutamine, 1 mg/L picloram, and 0.5 mg/L kinetin [113]. In some circumstances, physical treatments can improve secondary metabolite production, for example, exposure of Rosmarinus officinalis stem-induced callus to heat (45 min of 50 °C) increased rosmarinic acid production 1.7-fold relative to non-heat treatment [114]. Stress induced by ultraviolet light can also serve as a useful physical treatment to elicit secondary metabolite production. Exposure of Camptotheca acuminata callus cultures to 3 days of UV-B increased camptothecin levels 11-fold more than no UV induction [115]. Genetic transformation is also a valuable method to produce secondary metabolites in plant tissue cultures. For example, the use of Agrobacterium rhizogenes to produce hairy roots in American ginseng, Panax quinquefolium, allowed for the production of ginsenosides [116]. Similarly, the ability to genetically engineer 40 Jean Carlos Cardoso et al.

jasmonate-responsive transcription factors would allow for the controlled production of specific metabolites through chemically induced control of specific metabolic genes [117].

4 Conclusions and New Perspectives for Micropropagation

The priorities for increasing the importance of micropropagation techniques in this century require addressing some complex challenges. A reduction in costs is a clear requirement for the wider use of micropropagation in various important commercial species that are used in agriculture, horticulture, and forestry. Other priorities include increasing the efficiency of micropropagation techniques to reduce the main costs incurred during micropropagation of plantlets. Somatic embryogenesis and bioreactor systems could be techniques that replace conventional micropropagation using agar sucrose-based media. Advances in photoautotrophic systems are largely dependent on new sources of light, such as LEDs, laser, or use of the natural light and represent a solution for sucrose-free culture that has several benefits with respect to reducing problems of contamination and increasing the efficiency of micropropagation. Greenhouse micropropagation is a realistic alternative to micropropagation in developing countries. Nonetheless, advances are required in this area, not only for cultivating new species under these conditions but also for changing the environmental condi-

tions using CO2 injection systems and low-cost efficient alternatives to reduce the temperature as mist fogger systems [118] and heat tube exchangers for heat on cold nights [119] and complementary light sources for maintaining photoperiod, e.g., LED light technologies increase the rate and quality of survival of acclimatized plantlets [120]. In addition, combining efficient micropropagation with other propagation techniques and production systems, such as microcutting propagation from disease-free in vitro plantlets under greenhouse conditions, can be an effective solution for efficient propagation with reduced costs and easy access to high-quality plantlets with wide application in plant production [18, 121]. Many species remain very recalcitrant to in vitro cultivation and require targeted research to better understand such phenomena and to solve technological problems that can ultimately be transformed into new technologies. Furthermore, micropropagation also continues to be an important technique for breeding purposes because it offers a diverse range of advantages compared to limitations of common and natural reproductive barriers in conventional breeding techniques. Increases in the importance of the use of cells, tissues, callus, and plants as a bioreactor for the production of secondary metabolites will be a solution for the production of biochemicals for pharmaceutical Plant Micropropagation 41

and medicinal purposes. A combination of transgenic technologies and the overexpression of target genes are already in use to promote secondary metabolite production [122]. Plant tissue culture will increase its use for this purpose, and MAP CSC is a reality for the production of secondary metabolites [123]. New software and traceability also emerged in many commercial laboratories, which increases management of plantlets produced in large-scale micropropagated plant production. Also, 3D printing can improve products and systems for plant tissue culture, e.g., the development of functional culture vessels [124].

Acknowledgments

JCC thanks CNPQ for the research fellowship No. 304174/2015-7.

Contribution Statements

LTSG contributed writing item 2.4 Bioreactors Systems to Reduce the Cost of Micropropagated Plantlets. JATS contributed writing items 2.1 LEDs, CCFLs, and Laser Light for Tissue Culture and 3.2 The Use of Micropropagation for Secondary Metabolite Production and with final revision of all the text. JCC contributed writing all other items in the chapter and final revision of the text.

References

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Cellular and Morpho-histological Foundations of In Vitro Plant Regeneration

Diego Ismael Rocha, Lorena Melo Vieira, Andréa Dias Koehler, and Wagner Campos Otoni

Abstract

In vitro plant regeneration systems have turned into invaluable tools to plant biotechnology. Despite being poorly understood, the molecular mechanisms underlying the control of both morphogenetic pathways, de novo organogenesis and somatic embryogenesis, have been supported by recent findings involving proteome-, metabolome-, and transcriptome-based profiles. Notwithstanding, the integration of molecular data with structural aspects has been an important strategy of study attempting to elucidate the basis of the cell competence acquisition to further follow commitment and determination to specific a particular in vitro regeneration pathway. In that sense, morpho-histological tools have allowed to recognize cellular markers and patterns of gene expression at cellular level and this way have collaborated in the identification of the cell types with high regenerative capacity. This chapter ties together up those fundamental and important microscopy techniques that help to elucidate that regeneration occurs, most of the time, from epidermis or subepidermal cells and from the procambial cells (pericycle and vascular parenchyma). Important findings are discussed toward ultrastructural differences observed in the nuclear organization among pluripotent and totipotent cells, implying that regeneration occurs from two cellular mechanisms based on cellular reprogramming or reactivation.

Key words Histology, Morphology

1 Introduction

The capacity of a plant cell to acquire competence and assume a new developmental fate after modulation of the growth conditions is the basis for the establishment of plant regeneration systems. Regeneration is a physiological process and refers here to the ability of a given cell be able to regenerate new tissues, organs, or the whole individual. This process is widely conserved and appears to be a fundamental adaptive strategy for plants [1]. It can be induced in nature or in vitro which has long been utilized for clonal propagation.

Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo (eds.), Plant Cell Culture Protocols, Methods in Molecular Biology, vol. 1815, https://doi.org/10.1007/978-1-4939-8594-4_3, © Springer Science+Business Media, LLC, part of Springer Nature 2018 47 48 Diego Ismael Rocha et al.

Under in vitro conditions, there are two main morphogenetic pathways extensively used for plant regeneration of higher plants: de novo organogenesis and somatic embryogenesis. These pathways have been exploited for the vegetative propagation and contributed to contemporary plant biotechnology allowing the sophistication of genetic transformation and cloning practices. Researchers have attempted to extend these practices to agriculturally important crops as a prerequisite for in vitro genetic manipula- tions. Several plant regeneration systems have been established with applications in tissue culture-based techniques and genetic transformation for many plant species, and an intensive work has been conducted to overcome some of the drawbacks of these systems [2]. Over the recent decades, a range of descriptive studies using light and electron microscopy has provided detailed characterization of morpho-histological events underlying the progression from somatic cells to the formation of apical meristems (de novo organogenesis) or somatic embryos (somatic embryogenesis). These microscopy studies have contributed to the better understanding the basic cellular mechanisms related to the induction of the different plant morphogenetic pathways. Here, we summarize our current understanding of the histological and ultrastructural changes involved to both de novo organogenesis and somatic embryogenesis induction. We also describe some microscopy techniques that may be helpful for further works.

2 Concepts and General Aspects of In Vitro Plant Regeneration

2.1 Developmental The establishment of efficient in vitro regeneration systems requires Pathways a high degree of developmental plasticity. For that, explants are cultured in vitro in nutrient media supplemented with plant growth regulators in order to enhance the regenerative capacity and induce a given morphogenetic pathway. De novo organogenesis refers to the formation of ectopic apical meristems and subsequently development of shoots and roots, in a monopolar pattern, temporally and spatially apart. The meristems are formed by a group of plant stem cells that can both renew themselves and differentiated generating tissues and organs through cell division and differentiation. Somatic embryogenesis, by its turn, is the process in which a bipo- lar structure similar to zygotic embryos (containing both shoot and root apical meristems differentiated simultaneously at opposite poles) is formed and subsequently generates whole plant bodies. Both de novo organogenesis and somatic embryogenesis pathways occur either directly from parental tissues or indirectly via the formation of a callus, being mainly influenced by the type of explant and the plant growth regulator signaling. Histology in In Vitro Cultures 49

The use of plant growth regulators is a landmark breakthrough in the history of plant tissue culture. Since the classical finding of Skoog and Miller [3], the balance between auxins and cytokinins exogenously applied in the culture media is still the guiding principle to determine the fate of regenerating tissue. In general, high ratios of cytokinin to auxin induces de novo shoot organogenesis, whereas opposite low ratios result in root development. An appropriate concentration and type of exogenous auxin are also crucial to induce somatic embryogenesis in many plant species. During early events of somatic embryogenesis induction, high levels of auxin are required in the culture medium to promote cell proliferation and embryonic callus formation [4]. After that, the explants are transferred to auxin-free medium for reestablishing the auxin gradients in the embryonic callus. This initiates a developmental program similar to zygotic embryogenesis and is also guided by polarized auxin distribution [5]. Among the synthetic auxins, 2,4-dichlorophenoxyacetic acid (2,4-D) is the most effective inducer of somatic embryos, possibly because it triggers both auxin and stress responses simultaneously [6].

2.2 Morphological Based on the requirements and effects of exogenous plant growth Phases regulators, both de novo organogenesis and somatic embryogenesis pathways can be generally divided into three morphological phases: (1) morphogenetic acquisition of competence, somatic cells acquire competence to assume a new developmental fate; (2) cell determination, competent cell or tissue becomes committed in response to exogenous plant growth regulator supplementation; and (3) morphological differentiation, morphogenesis proceeding independently of exogenous plant growth regulator supplementation [7–9]. Although these established morphological phases are not easily recognized in the morphogenetic pathways of some species, the first phase, competence acquisition, is certainly conserved and denoted as a key step in plant regeneration process [10–12]. The acquisition of competence is acquired by somatic cells that are able to respond a specific hormonal signal breaking their deter- mined cell fate. These competent cells originate the meristematic- like centers and assume a new developmental route [13]. According to the degree of dedifferentiation, these competent cells can be characterized as multipotent, totipotent, or pluripotent. The cellular totipotency corresponds to the ability of a single cell to produce different kinds of cell within a particular cell lineage [14]. The pluripotency is the cellular ability to give rise to complete organ formation but not the entirety of plant body (e.g., shoot or root formation), whereas totipotent cells can give rise to all the cell types constituting the plant body [15]. Both pluri- and totipotency terms have been used to describe the competent cells in organogenic and embryogenic developmental pathways, respectively [16]. 50 Diego Ismael Rocha et al.

3 Structural and Ultrastructural Aspects of In Vitro Plant Regeneration

3.1 Morpho- Plant regeneration may be induced from different parts of plant histological Origins body (Fig. 1). Shoot tips or nodal segments carrying a single bud of In Vitro Plant have been used for clonal propagation to produce multiple shoots Regeneration (Fig. 1). These explants are referred as meristematic explants, and the regeneration occurs from the activation of the preexisting bud that begins to develop. The regeneration of shoot, root, or even the whole plant can also be induced from non-meristematic explants (Fig. 1). In this case, the formation of new organs is induced de novo from mature somatic tissues and occurs, primarily, at the cut surfaces of the explants [17]. Protoplast or haploid cells such as pollen and ovules have also been used to regenerate plants via somatic embryogenesis (Fig. 1) [8, 18–21]. It has long been known that mature somatic tissues may have low regenerative capacity. Thus, young juvenile tissues have been used as explants for in vitro regeneration of many plant species, as they are composed of cells in the beginning of their developmental path and are potentially totipotent [22]. The regeneration in grasses and mono- cotyledonous species, for example, has long been thought to be very difficult, and it has been obtained only from zygotic embryos, inflorescences, or tissue derived from juvenile shoots [23, 24]. According to Ikeuchi et al. [25], plants possess at least two distinct cellular strategies to begin the process of regeneration. One is through the reactivation of relatively undifferentiated cells and the other through the reprogramming of differentiated somatic cells. In both cases, regeneration relies on the phenomenon of cellular plasticity, which can be broadly defined as the ability to re-specify cell fate. Interestingly, most histological tracking studies of plant regeneration process have demonstrated that the morphogenesis responses typically originate from reactivation of pericycle and/or vascular parenchyma (Fig. 2b, c) [26–29] or from reprogramming of epidermal and/or subepidermal cells (Fig. 2a) [16, 30–33]. Pericycle has been proposed to be an extended meristem in the plant body ended [34, 35]. It is constituted by pluripotent cells and is able to acquire different cell fates depending on the conditions to which they are subjected (Fig. 2b, c). Several studies have reported the involvement of pericycle cells in the regeneration of embryos and/or buds mainly from root explants [29, 33, 36–39]. This is consistent with recent studies on the molecular mechanisms involved in in vitro morphogenesis. Many lines of evidence support the understanding that pericycle and vascular parenchyma cells are intrinsically prone to undertake different morphogenetic pathways [34, 40–42]. While it is clear the formation of the pluripotent pericycle-like cells, the mechanisms related to the reprogramming of epidermal cells and their capacity to produce pluri-/totipotent cell lineages Histology in In Vitro Cultures 51

Fig. 1 Pathways of in vitro plant regeneration. (a) Common types of explants used to induce regeneration in vitro. (b) Somatic embryogenesis obtained from leaf explants of Coffea sp. (c, d) Regeneration of a somatic embryo (d) from Passiflora gibertii protoplasts (c). (e) Regeneration of multiple shoots from nodal segment of Herreria salsaparrilha. (f) De novo shoot organogenesis obtained from hypocotyls of Bixa orellana. (g) Somatic embryogenesis induced from Eustoma grandiflorum root explants. Based on Ikeuchi et al. [25]

still remain elusive. The involvement of epidermal and subepidermal cells in plant regeneration has been reported in a number of morpho-histological studies, mostly derived from leaf and cotyle- don explants (Fig. 2a). It is believed that contact between the explant and the nutrient medium makes the epidermal and subepidermal cell layers more prone to perceive hormonal signaling that triggers the regeneration process. In fact, an accumulation of auxin was identified in protodermal cells of Arabidopsis zygotic embryo explants incubated on auxin-rich medium [43]. However, the molecular regulation related to the reprogramming of epidermal cells is still poorly understood. 52 Diego Ismael Rocha et al.

Fig. 2 Histological origins of in vitro plant regeneration. (a) Somatic embryogenesis from cotyledons of Bixa orellana. Transverse sections showing that epidermal cells (arrowhead) give rise to somatic embryos (asterisk). (b, c) De novo shoot organogenesis induction from hypocotyl (b) and root (c) segments of Passiflora edulis. Longitudinal and transverse sections of hypocotyl and root segments, respectively, are showing the regeneration from reactivation of procambial cells (pericycle and vascular parenchyma; arrowheads) giving rise to regenerated shoots (asterisk). Bars = 100 μm. Based on Ikeuchi et al. [25]

3.2 Direct In vitro plant regeneration may proceed directly from parental tis- and Indirect In Vitro sues without an intervening callus phase or indirectly after a callus Plant Regeneration: phase, referred to as direct or indirect regeneration mode, respec- Callus Histological tively. Previous studies have hypothesized that both processes are Features extremes of one continuous developmental pathway wherein callus represents a reprogramming step of unorganized mass of dividing dedifferentiated cells, which are capable of switching cell fate in response to hormonal signals [8, 44]. However, recent investigations suggest that there are various types of calli exhibiting different identities [45]. For instance, calli formed on Arabidopsis roots from pericycle or pericycle-like cells have a characteristic gene Histology in In Vitro Cultures 53

expression pattern reminiscent of partly organized root tip meristems [41]. Its development follows the initial steps of lateral root formation [40] and not a process of cellular reprogramming to an undifferentiated state, as previously thought [41]. Therefore, this callus tissue cannot be considered as a dedifferentiated but rather a misdifferentiated root meristem. The mechanisms behind the induction of each developmental pattern (direct and indirect) remain poorly understood. During the indirect system, both regenerative and non- regenerative clusters might be present in the callus (Fig. 3a, d). It is usually easy to distinguish these clusters based on the morphological and cellular features [46]. In general, regenerative clusters present yellow or dark-yellow color, nodular features, and smooth surface (Fig. 3a, d). Unlike, non-regenerative zones are generally described as rough, friable, and translucent with disorganized cellular system (Fig. 3a, d). At cellular level, regenerative clusters are generally constituted by small and isodiametric meristematic-like cells with dense cytoplasm, numerous mitochondria, evident stained nuclei and nucleoli, small vacuoles, and a higher metabolic activity (Fig. 3b, e). Conversely, non-regenerative zones are constituted by different cell shapes and highly vacuolated cells with few organelles that can be interpreted as signals of low metabolic activity (Fig. 3b) [8, 46, 47].

3.3 Ultrastructural The ability of a given cell to regenerate a new tissue, either by cel- Aspects of In Vitro lular reactivation or cellular reprogramming, is accompanied by Plant Regeneration nuclear changes and chromatin reorganization. Many studies have described the ultrastructural changes related to the transition of differentiated somatic cells into a pluri- or totipotent state, and some ultrastructural features were found to be a marker of cells undergoing changes in cell fate [11, 15, 30, 48, 49]. Analyses performed by Rocha et al. [49] on Passiflora edulis cotyledons during somatic embryogenesis process showed that at initial stages, the protodermal cells had large and spherical nuclei with conspicuous nucleoli and the presence of heterochromatin. Throughout the process, these cells acquired embryonic state and presented a large nucleus with only one evident nucleolus and less heterochromatin. The same pattern was also described in Brachypodium distachyon [11] which might be a conserved feature between eudicots and monocots. According to Verdeil et al. [15], the shape and structure of nucleus can be used to distinguish between pluripotent meristematic cells and totipotent embryonic cells. In pluripotent meristematic cells (the authors described shoot meristem cells), the nucleus was spherical containing several nucleoli and heterochromatin uniformly distributed within the nucleus (Fig. 3c). In the case of a totipotent embryonic cell, the nucleus was conspicuous and contained only one large nucleolus and less heterochromatin (Fig. 3e, f). The morphogenetic competence of a 54 Diego Ismael Rocha et al.

Fig. 3 Ultrastructure features of pluripotent meristematic cells and totipotent embryonic cells. (a–c) De novo organogenesis induced from root explants. (a) Organogenic callus. Note the presence of regenerated shoots (sh). (b) Histological section showing the meristemoid (me) formed at the surface of organogenic callus (ca). (c) Pluripotent meristematic cell of the meristemoid showing a dense cytoplasm with small vacuoles (v), many plasmodesmata (black arrows), and a large nucleus with some heterochromatin distributed in the periphery (white arrowheads). (d–f) Somatic embryogenesis of Brachypodium distachyon. (d) Embryogenic callus. Note the presence of somatic embryos (se). (e, f) Totipotent embryogenic cells showing a high nuclear cytoplasmic ratio, small vacuoles (v), amyloplasts containing starch, and conspicuous nuclei (n) containing only one large nucleolus (nu) and less heterochromatin. Abbreviations: d dyctiosome; r reticulum. Bars = b 100 μm, (c) and (f) 1 μm, (e) 5 μm

target cell increases as the nucleus euchromatin portion increases and therefore the property to give rise an entire individual. Larger quantities of euchromatin in relation to heterochromatin charac- terize the totipotency, whereas the increase in genetic silenced material (heterochromatin) characterizes the pluripotency [50]. Other ultrastructural features have also been used to character- ize both pluri- and totipotent cells. The pluripotent meristematic cells have high nuclear cytoplasmic ratio, dense cytoplasm with many fragments of small vacuoles, without the presence of an amyloplasts (Fig. 3c). It presents many plasmodesmata, due to the strong dependence and interaction with neighboring cells, creating a niche that maintains its cellular identity (Fig. 3c). Totipotent embryonic cells also have high nuclear cytoplasmic ratio and dense cytoplasm with small vacuoles fragmented (Fig. 3e, f). However, in some reports, it was also observed the presence of a high amount of amyloplasts and the absence of plasmodesmata that are rarely observed in the cell wall (Fig. 3e, f), modified by deposition of cal- lose, giving in this way the isolation of its immediate neighboring cells [15, 48]. Histology in In Vitro Cultures 55

4 Localization of Cellular and Molecular Targets

4.1 Histochemical The culture of plant explants in presence of a specific balance of Approach to Study plant growth regulators can trigger the regeneration process. Not In Vitro Plant all tissues can respond to the hormonal signaling, but some cells Regeneration will acquire competence changing their developmental fates. This process increases the heterogeneity of cell population within the explant, at a structural, molecular, and biochemical levels. Thus, histochemical methods have been used to obtain a detailed description of the cytological and biochemical state of individual cells or tissues involved or not in the regeneration process, elucidating structural and biochemical variations between them [11, 33, 51–55]. Histochemical analyses are based mainly in two approaches: histological techniques as well as physical and chemical methods which provide valuable information to identify and localize specific compounds of a particular chemical group within the plant cells and tissues [56]. The ultimate goal of these techniques is to cor- relate the anatomical or developmental stage of the tissues or cells to their biochemical state [57]. In the plant regeneration process, histochemical monitoring has also been used to determine the essential factors involved in the morphogenetic differentiation allowing the recognition of regions and/or tissues with high metabolic activity [11, 26, 33]. Furthermore, it allows following the dynamics of mobilization, synthesis and metabolism of storage compounds during the regeneration process. Storage reserves may have an important role during in vitro morphogenesis, mainly, if zygotic embryos, cotyledons, or endosperm are used as explants. In this section, we included few basic and common histochemical protocols for localization of cell components that have been shown to be important for plant regeneration.

4.1.1 Starch Starch is considered a primary source of energy for cell growth and proliferation [58]. A high amount of starch has been observed during in vitro regeneration process of many different species [31, 32, 59–61]. However, histochemical analyses have shown differences in the starch distribution among the population of cell types within the explant. In many species, cells or tissues mitotically active and involved in the regeneration process presented a lower abundance of starch in comparison with the adjacent vacuolated callus cells (Fig. 4a–d), which may be related to the differences in energy requirements and consumption between these cell populations [30, 61]. Different protocols can be used for localization of starches. Below we described two simplified staining procedures: 56 Diego Ismael Rocha et al.

Procedure I: Lugol’s 1. Use fresh materials or fixed tissues embedded in paraffin or Reagent methacrylate. 2. Place a drop of Lugol’s reagent onto the tissue and allow react- ing for 5 min. 3. Gently wash 1–2 min in running water. 4. Mount the slides as usual. The positive reaction of the test results in starch grains stained in brown, purple, or black (Fig. 4a, b).

Procedure II: Periodic This staining protocol is not starch-specific. It is used for identify- Acid-Schiff’s (PAS) ing all carbohydrates in a given prepared tissue section. Tissues Reaction fixed in aldehydes will react generally with Schiff’s bases to yield a false-positive background. 1. Use fresh materials or fixed tissues embedded in paraffin or methacrylate. For paraffin-embedded tissues, remove paraffin, and hydrate as usual. 2. Place slides in 0.5% periodic acid solution (prepare the solution just prior its use) for 10–15 min in order to oxidize it. 3. Wash three times for 3 min each in distilled water. 4. Stain in Schiff’s reagent for 30 min in the dark. 5. Rinse sections in distilled water for 5–10 min. 6. Place the slides in 2% sodium bisulfite for 3 min. 7. Wash in distilled water for 1 min. 8. Mount the slides as usual. The positive reaction to the Schiff reagent occurs by the purplish red stain of polysaccharides (e.g., cellulose, starch) (Fig. 4c, d). Due to the occurrence of false positive, the test is usually applied without the oxidation step in periodic acid as a control.

4.1.2 Proteins Proteins are involved in the regulation of cell expansion and the creation of biophysical characteristics necessary for morphogenetic processes [62]. Among the several chemical test used, Xylidine Ponceau is one of the most popular reagents for protein detection [63]. It has also been used to detect potential responsive morpho- genic sectors in the explant (Fig. 4e, f). Cells with intense staining by Xylidine Ponceau may suggest a high incidence of protein synthesis and high metabolic activity [7, 11].

Procedure: Xylidine 1. Use fresh materials or fixed tissues embedded in paraffin or Ponceau methacrylate. For paraffin-embedded tissues, remove paraffin, and hydrate as usual. 2. Place slides in Xylidine Ponceau solution for 15–30 min. 3. Wash in 3% acetic acid for 5 min. 4. Wash 2 min in distilled water. 5. Mount the slides as usual. The positive reaction occurs by the light purplish-red stain (Fig. 4e, f). Histology in In Vitro Cultures 57

Fig. 4 Histochemical analysis. (a–d) Brachypodium distachyon somatic embryogenesis. Embryonic callus (ec) stained with Lugol (a, b) and PAS (c, d). Note that starches are abundant only in the inner cell layers of the explant that are not involved in regeneration process. A higher magnification of the square area marked in a( ) and (c) is shown in (b) and (d), respectively, evidencing starch grains stained in black (c) and purplish red (d). (e–f) Histological sections of Bixa orellana organogenesis (e) and somatic embryogenesis (f) pathways stained with xylidine ponceau (XP) [75]. A strong positive XP staining is observed in cell population with high mitotic rate, such as pluripotent meristematic cells (black asterisk) in (e) and protodermis-dividing cells (pt) in (f). (g–i) Eustoma grandiflorum embryonic callus stained with acetocarmine/Evans Blue histochemical test. Somatic embryos (white asterisks) showed an intense red stained with acetocarmine (h). Non-embryogenic cells stained blue (i). Bars = (a, c–i)100 μm, (b, d) 50 μm 58 Diego Ismael Rocha et al.

4.1.3 Cellular A histochemical analysis to quickly diagnose the cellular morpho- Morphogenetic Potential genetic potential of the explants is the double staining of acetocarmine and Evans Blue test [64]. This test has been successfully used to detect the presence of pluripotent meristematic or totipotent embryonic cells in callus or cell suspension culture (Fig. 4g) [7, 64–68]. In general, cells with embryogenic/meristematic features such as small, isodiametric, and dense cytoplasm and high nucleus/ cytoplasm ratio stain an intense red with acetocarmine (Fig. 4g, h). Non-embryogenic cells stain blue (Fig. 4g, i).

Procedure: Acetocarmine/ 1. Rub and spread the cellular agglomerates (callus or cell suspen- Evans Blue Staining sion culture)Acetocarmine/Evans blue staining onto the slides. 2. Place a drop of Evans Blue staining 0.5% for 3 min onto the tissue. 3. Place a drop of Carmine acetic staining (0.1%) for 3 min onto the tissue. 4. Mount the slides in water or glycerin.

4.2 Integration The integration of molecular data with the structural aspects has of Cellular been a strategy to elucidate the origin of the cell competence in the and Molecular Data process of in vitro regeneration (Fig. 5). In this sense, it has been of In Vitro Plant growing the number of publications involving studies on tissue Regeneration by In culture protocols combined with gene cloning techniques, DNA Situ Hybridization and RNA sequencing and gene expression data. As part of the tools used for gene functional characterization of genes are the spatial-temporal localization of the genic products, the promoter activities studies, and target transcript location in induced tissues. This has been possible, thanks to the development of techniques as the transient expression of reporter genes under the control of specific promoters and by in situ hybridization. Particularly the improvement of the in situ hybridization techniques using nonradioactive RNA probe labeling with reporter molecule has facilitated studies of this nature, especially in non- model plants, which protocols of genetic transformation are still not available. The use of this tool has allowed to identify conserved patterns of gene expression at cell level as well as its variations between the different taxon and has collaborated for the identification of cell types with high regenerative capacity. Interestingly the use of this strategy has revealed the function of important genes during in vitro development. For instance, the SERK (Somatic Embryogenesis Receptor-Like Kinase) gene, isolated from an embryogenic culture of Daucus carota, was initially described as a marker gene of somatic embryogenesis [27], because of the differential expression between embryogenic and non- embryogenic. Indeed in the last 20 years, the role of this gene in the somatic embryogenesis has been proved in different embryogenic systems, because it marks competent cell groups for somatic Histology in In Vitro Cultures 59

Fig. 5 Structural characterization and analysis of SERK-like expression by in situ hybridization during somatic embryogenesis of Passiflora cincinnata. (a–c) Early developmental stage of somatic embryo formation. Strong signal of PcSERK in a meristematic multicellular clump at callus surface (c). (d–g) Somatic embryos developed (asterisks). (d) Morphological view. (e) Somatic embryos showing the typical meristematic features of totipotent embryonic cells such as small cells with dense cytoplasm and high nucleus/cytoplasm ratio. (f) Strong signal of PcSERK in somatic embryos. (g) Section hybridized with the sense probe. No signal above background was detected. Negative control. Bars = 200 μm

embryogenesis (Fig. 5) [11, 49, 69]. Recently, works involving the in situ hybridization technique have been added other attributed functions to these genes. In Cyclamen persicum, the localization of CpSERK1 and CpSERK2 transcripts in groups of meristematic cells present in both embryogenic and organogenic cell lines indi- cate that these genes are also markers of pluripotency [70]. A similar pattern was observed by Rocha et al. [49] in Passiflora edulis, detecting the presence of transcripts of the PeSERK1 gene in mitotically active cells from meristemoids in shoot-like structures and provascular elements. The in situ hybridization technique is based on the specific pairing of the two complementary molecules. The success of the technique aiming at gene expression studies requires a previous understanding of molecular biology and of the capacity of inter- pretation of the morphological and anatomical data of the material analyzed [71]. The knowledge of the structural and functional characteristics of the gene in study is fundamental for the construction of a specific probe. In this section, we attempt to show a basic protocol applied to the localization of transcripts from tissue culture-derived material. 60 Diego Ismael Rocha et al.

4.2.1 Procedure The protocol of in situ hybridization protocol shown in this chapter is based on protocols described by Dusi [72] and Brown [71]. Basically, this technique can be summarized in five fundamental steps: (1) construction and probe synthesis, (2) collection and preparation of samples for microscopy, (3) hybridization reaction, (4) post-hybridization, and (5) immunological detection.

Construction and Probe 1. For construction of specific probes, fragments of the cDNA Synthesis corresponding to mRNA of interest are inserted into a cloning vector containing the T7 and SP6 promoters, i.e., pGEM®-T Easy (Promega) or pSPT18/19. The orientation of the insert, in relation to transcription sites, should be considered and veri- fied by sequencing. 2. The linearized vectors with restriction enzyme or purified PCR fragments flanked by T7 and SP6 promoters are used as tem- plate for probe synthesis. 3. Sense and antisense single-stranded RNA probes are synthe- tized by in vitro transcription using the DIG RNA Labeling Kit (SP6/T7), following all recommendations of the manufacturer. 4. Check probe integrity and concentration on 1.5% agarose gel, using the labeled RNA control (vial 5 of the kit). 5. Confirm the probe labeling in nylon membrane by dot blot assay.

Sample Preparation 1. Collect samples in small pieces, and fix immediately in 4% para- formaldehyde, pH 6.8–7.1. Apply vacuum for 1 h. Change the fixative solution, and keep for 4–18 h at 4 °C, according to the characteristic of each material to be analyzed (see Notes 1 and 2). 2. Dehydrate the samples in 1-h series of ethanol gradient (10, 30, 50, 70, 90, and 100%) incubations. If necessary the samples can be stored at −20 °C in 70% ethanol until the next step or in 100% ethanol for months. Embedding the sample in Paraffin Histosec® (Merck Millipore): 1. Transfer the sample to ethanol: xylol solution at proportions 3:1, 1:1, and 1:3 and pure xylol for 1 h each in glass bottles. Gradually add pastilles of paraffin until it reaches a third of the volume, at 60 °C. Keep for 1 day. 2. Add new paraffin until the proportion 1:1 and keep for 1 day. Discard half of the solution xylol + paraffin, and complete the volume with melted paraffin. Then keep for 1 day. Repeat this procedure for two or three times until the xylol is completely eliminated. Histology in In Vitro Cultures 61

3. Carefully, insert the material in the desired orientation in small molds of paper, and slowly pour the melted paraffin. Let the paraffin solidify for 1 day. Store the blocks at −20 °C (see Note 3). 4. Serial sections (4–8 μm) are obtained in microtome, and they are fixed under adhesive slides or microscope slides previously treated with organosilane. Keep the slides at 42 °C overnight in the hot plate surface, and store at 4 °C until the next step.

Hybridization Reaction Remove the paraffin as follows (steps 1-4): 1. Pure xylol (two times): 5–15 min. 2. Xylol/ethanol (1:1): 5–15 min. 3. Pure ethanol: 10 min. 4. DEPC-treated water: 10 min (two times). 5. For each material analyzed, hybridize two slides: for one of them, use the antisense probe and for the other one the sense probe (negative control). 6. For each slide, denature 60 ng of probe and 60 ng of tRNA at 80 °C for 5 min. Put immediately on ice for 2 min, and then add 100 μL of hybridization buffer (Table 1). 7. Put the mix on top of the sections, and cover with Parafilm® sealing film, and place the slides in a humid chamber, and incu- bate at 42 °C (see Note 4).

Post-hybridization 1. Carefully remove the Parafilm®, and wash the slides with SSC buffer (Table 2) at the following concentrations: 4×, 2×, 1×, and 0.5× for 30 min at 42 °C, for until 16 h. 2. Remove excess liquid from the slides, and add 200 μL of block- ing solution (detection buffer 2—Table 3). Again, cover the

Table 1 Hybridization buffer

Final Components Amount concentration

1 M Tris–HCl pH 7.5 100 μL 10 mM 3 M NaCl 1 mL 300 mM Deionized formamide 5 mL 50%

50 mM EDTA pH 8.0 200 μL 1 mM Denhardt solution 50× 200 μL 1× 50% dextran sulfate 2 mL 10%

DEPC H2O 500 μL Aliquote in smaller portions. Store at −20 °C 62 Diego Ismael Rocha et al.

Table 2 SSC 20× solution