Plant Cell and Tissue Culture – A Toolin Biotechnology

Karl-Hermann Neumann • Ashwani Kumar •Jafargholi Imani

Plant Cell and TissueCulture – A Toolin BiotechnologyBasics and Application

Second Edition

Karl-Hermann Neumann (deceased)Hungen, Germany

Ashwani KumarDepartment of BotanyUniversity of RajasthanJaipur, India

Jafargholi ImaniInstitute of PhytopathologyJustus-Liebig University GiessenGiessen, Germany

ISBN 978-3-030-49096-6 ISBN 978-3-030-49098-0 (eBook)https://doi.org/10.1007/978-3-030-49098-0

# Springer Nature Switzerland AG 2009, 2020This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or partof the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission orinformation storage and retrieval, electronic adaptation, computer software, or by similar or dissimilarmethodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in thispublication does not imply, even in the absence of a specific statement, that such names are exemptfrom the relevant protective laws and regulations and therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this bookare believed to be true and accurate at the date of publication. Neither the publisher nor the authors or theeditors give a warranty, expressed or implied, with respect to the material contained herein or for anyerrors or omissions that may have been made. The publisher remains neutral with regard to jurisdictionalclaims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG.The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface to First Edition

This book is intended to provide a general introduction to this exciting field of plantcell and tissue culture as tool in biotechnology, without overly dwelling on detaileddescriptions of all aspects. It is aimed at the newcomer, but will hopefully alsostimulate some new ideas for the “old hands” in tissue culture. Nowadays, with thevast amount of information readily available on the internet, our aim was rather todistill and highlight overall trends, deeming that a complete report of each and everytissue culture investigation and publication was neither possible, nor desirable. Forsome techniques, however, detailed protocols are given. We have tried to be asthorough as possible, and regret if we have inadvertently overlooked any pertinentliterature or specific development that belong in this work.

The three authors have been associated for many years, and have worked togetheron various aspects in this field. Without this close interaction, this book would nothave been possible. At this opportunity, we wish to reiterate our mutual appreciationof this fruitful cooperation. An Alexander von Humboldt Stiftung fellowship toAshwani Kumar (University of Rajasthan, Jaipur, India) to work in our group at theInstitut für Pflanzenernaehrung der Justus Liebig Universität, Giessen, supportedthis close cooperation and the completion of this book, is gratefully acknowledged.

Such a book takes time to grow. Indeed, its roots lie in a 3–4 week lecture andlaboratory course by one of us (K.-H.N.) about 30 years ago as visiting professor atAin Shams University, Cairo, Egypt, which later led to the development of agraduate training unit at the University of Giessen, Germany, and other universities.So, also older key literature, nowadays risking being forgotten, has been considered,which could be of help for newcomers in this domain.

Thanks are due to our publisher for all the help received, and for patiently waitingfor an end product that, we feel, has only gained in quality.

Giessen, GermanyMarch 2009

K. -H. NeumannA. KumarJ. Imani

v

Preface to Second Edition

The first edition of the book was intended to provide a general introduction to thisexciting field of plant cell and tissue culture as tool in biotechnology, without overlydwelling on detailed descriptions of all aspects. After a decade of the first edition,and despite the enormous amount of information readily available on the Internet,our aim was to distill and highlight overall trends of tissue culture that currentlyrepresent a basic tool in plant biotechnology in the second edition. Thus, a completereport of each and every tissue culture investigation and publication was neitherpossible nor desirable. The second edition was written to emphasize the latestadvances in the developing field of genetic engineering, especially the thoroughlyrevised Chap. 13. For some techniques, however, detailed protocols are provided inthis book. The second edition of this book was written by two authors (Dr. A. Kumarand Dr. J. Imani), because Prof. Neumann, who had played a crucial role in firstedition, is unfortunately no longer alive. These two authors have been associatedwith each other for many years and have worked together on various aspects in thisfield. Without this close interaction, the second edition of this book would not havebeen possible. An Alexander von Humboldt Stiftung fellowship to Ashwani Kumar(University of Rajasthan, Jaipur, India) to work in our group at the Institut fürPflanzenernaehrung and Dr. Imani at the Institute for Phytopathology, Justus LiebigUniversität, Giessen, which supported this close cooperation and the completion ofthis book, is gratefully acknowledged. We have tried to be as thorough as possible,and regret if we have inadvertently overlooked any pertinent literature or specificdevelopment that belongs in this work.

We also acknowledge numerous publications which have been the source ofsome pictures in this book.

Acknowledgments are due to our publisher for all the received help and patiencewhile waiting for the manuscript.

Giessen, GermanySpring 2020

A. KumarJ. Imani

vii

Acknowledgments

Figures 3.2–3.5, 3.8, 3.10, 3.12, 3.13, 3.16, 4.1, 4.4, 5.2, 5.4, 5.5, 5.7, 6.3, 7.3, 7.5–7.9, 7.11, 7.15, 7.16, 7.33, 8.1, 8.3, 8.15, 9.2, 12.1, 13.3 and Tables 2.1, 3.3–3.8, 5.1,6.1–6.3, 7.1, 7.3, 7.5, 7.8, 12.1 are published with kind permission of Verlag EugenUlmer. Further, we are indebted to the following authors and their publishers forgetting permissions to use their figures in our book in Chap. 13. Hayta, S., Smedley,M.A., Demir, S.U. et al. (2019) An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) PlantMethods 15, 121 (Figs. 13.11 and 13.12); He, L., Hannon, G. (2004) MicroRNAs:small RNAs with a big role in gene regulation. Nat Rev Genet 5, 522–531. https://doi.org/10.1038/nrg1379 (Fig. 13.27); Moises Zotti, Ericmar Avila dos Santos,Deise Cagliari, Olivier Christiaens, Clauvis Nji Tizi Taning, Guy Smagghe (2018)RNA interference technology in crop protection against arthropod pests, pathogensand nematodes. Pest Management Science 74, 1239–1250 (Fig. 13.29); Liu, S.,Jaouannet, M., Dempsey, D., Imani, J., Coustau, C. et al. (2020) RNA-basedtechnologies for insect control in plant production. Biotechnology Advances 39(107463), 1–13 (Fig. 13.30); Karimi, Mansour et al. (2002) GATEWAY™ vectorsfor Agrobacterium-mediated plant transformation. Trends in Plant Science 7(5),193–195 (Fig. 13.31); Yuriko Osakabe, Keishi Osakabe. Genome editing withengineered nucleases in plants. Plant and Cell Physiology 56, 389–400(Fig. 13.34); Yuriko Osakabe, Keishi Osakabe. Genome editing with engineerednucleases in plants. Plant and Cell Physiology 56, 389–400 (Fig. 13.35); Erkes, A.,Reschke, M., Boch, J., Grau, J. (2017) Evolution of transcription activator-likeeffectors in Xanthomonas oryzae. Genome Biol Evol 9(6), 1599–1615(Fig. 13.36); Joung, J.K., Sander, J.D. (2013) TALENs: a widely applicable tech-nology for targeted genome editing. Nat Rev Mol Cell Biol 14(1), 49–55(Fig. 13.37); Khatodia, S., Bhatotia, K., Passricha, N., Khurana, S.M., Tuteja,N. (2016) The CRISPR/Cas genome-editing tool: application in improvement ofcrops. Front Plant Sci 7, 506 (Fig. 13.38); Belhaj, K., Chaparro-Garcia, A., Kamoun,S., Nekrasov, V. (2013) Plant genome editing made easy: targeted mutagenesis inmodel and crop plants using the CRISPR/Cas system. Plant Methods 9(1),39 (Fig. 13.39); Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Nekrasov,V. (2013) Plant genome editing made easy: targeted mutagenesis in model andcrop plants using the CRISPR/Cas system. Plant Methods 9(1), 39 (Fig. 13.41);

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Sharma, M., Schmid, M., Rothballer, M., Hause, G., Zuccaro, A., Imani, J. et al.(2008) Detection and identification of bacteria intimately associated with fungi ofthe order Sebacinales. Cell Microbiol 10, 2235–2246. https://doi.org/10.1111/j.1462-5822.2008.01202.x (Fig. 13.42); Reitz, M., Bissue, J.K., Zocher, K., Attard,A., Hückelhoven, R., Becker, K., Imani, J., Eichmann, J., Schäfer, P. (2012) Thesubcellular localization of Tubby-like proteins and participation in stress signalingand root colonization by the mutualist Piriformospora indica. Plant Physiology 160(1), 349–364. https://doi.org/10.1104/pp.112.201319 (Fig. 13.43); Sharma, M.,Schmid, M., Rothballer, M., Hause, G., Zuccaro, A., Imani, J. et al. (2008) Detectionand identification of bacteria intimately associated with fungi of the orderSebacinales. Cell Microbiol 10, 2235–2246. https://doi.org/10.1111/j.1462-5822.2008.01202.x (Fig. 13.44).

Ashwani Kumar sincerely acknowledges encouragement and continued supportof Prof. Dr. Sven Schubert, Director of the Institute of Plant Nutrition, Justus LiebigUniversität, Giessen, after the demise of Prof. Dr. K.H. Neumann in 2009, and theaward of Alexander von Humboldt Fellowship which enabled his frequent researchvisits and also helped in shaping the book. Dr. Jafargholi Imani acknowledges thesupport of Prof. Dr. Karl-Heinz Kogel, Director of the Institute of Plant Pathology,Justus Liebig Universität, Giessen. We thank our research students, fellow workers,and colleagues whose works we have quoted and who have provided support to thiswork directly or indirectly. We also thank Dr. Andrea Schlitzberger, Project Coordi-nator, book production Germany and Asia, and Mr. Bibhuti Sharma from Springer.It was pleasure to work with Springer, and we thank them for bringing out the bookso nicely.

x Acknowledgments

In Memoriam Prof. Dr. Karl-Hermann Neumann(1936–2009)

Prof. Dr. Karl-Hermann Neumann passed away on October 13, 2009. He was bornon 22 May, 1936, in Morgensdorf, near Leitmeritz (Sudetenland), which is now apart of the Czech Republic. After the end of Second World War, his family migratedto Bernburg in Saxonia-Anhaltinia (later German Democratic Republic, GDR). Inthis place, his father acquired another farm, where he grew up. In 1956, followingsome problems with Communist administration of the GDR, he had to move to theFederal Republic of Germany, where he finished his schooling. In 1957, he enteredJustus Liebig Universität to study agriculture. He spent one semester as a foreignstudent with a stipend at “Den Kgl. Veterinaer og Landbohojskole” in Copenhagen,Denmark, majoring in agricultural chemistry; he completed his studies again atGiessen, as “Diplomlandwirt” in 1960. In the same year, he received scholarshipfrom Cornell University in Ithaca, NY, USA. He enrolled here in a graduate school,with botany as major and biochemistry and physical chemistry as minor. He alsostarted working for Ph.D. under the supervision of Prof. F.C. Steward, FRS. Thetopic of his study was “Function of some heavy metals (iron, magnesium andmolybdenum) on the growth and metabolism of carrot tissue cultures” (mainlyprotein metabolism and photosynthesis), which also became major for his Ph.D.thesis later on. Since the scholarship was given to him for only 1 year, he returned toGermany and completed his Ph.D. under the supervision of Prof. H. Linser in 1962from the University of Giessen. After spending few years with Prof. Linser whiledoing postdoc, he finished his Habilitation (equivalent to D.Sc.) studies in 1969. He

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got promoted as “Privatdozent” at the agricultural faculty of Justus Liebig Univer-sity, Giessen, Germany. In 1972, he became Professor for Biochemistry and CellBiology of Plants at the Faculty of Nutrition of the Justus Liebig University.

“While working with Prof. Steward and Prof. Linser he was also intensivelytrained in systemic thinking and multidisciplinary research approaches whichinfluenced his own scientific career to a considerable extent,” wrote Arnholdt et al.(2010).

His first major achievement was replacement of coconut milk in White’s nutrientmedium used in Steward’s laboratory at Cornell with an artificial nutrient mediumalso named as Neumann’s Lösung or Neumann’s liquid medium of defined chemicalcomposition. “All later investigations can be traced back to the three experimentsperformed in the 1960s and the medium was named as Neumann’s Lösung orNeumann’s medium. One of the experiments focussed on the photosynthesis ofcultured carrot explants, which was based on work done at Cornell; second onnucleic acid metabolism of carrot cultures; and third on somatic embryogenesis incarrot cells in a defined nutrient medium. The work on photosynthesis wasaccelerated after Ludwig Bender and Ashwani Kumar joined his group in the1970s and both held Professorships subsequently.” A. Kumar joined as Alexandervon Humboldt Fellow while serving at the University of Rajasthan, Jaipur. “Theresults, covering cytology as well as biochemistry aspects, were published in numberof papers. This line of work came to a close with studies on somatic embryogenesisof autotrophic cultures under normal atmosphere (Dr. Eva Plescka).”

“The work on nucleic acid started with studies on metabolic turnover of bothDNA and RNA, followed by comparative studies of DNA organization of severalplant species by Cot hybridization indicating about 15% identity of unique andrepeated DNA. This represents the basic genetic information, which can be used todistinguish higher plants from other biological systems. Here also first results turnedup on the occurrence of metabolic DNA localised in repeated fractions and broadlyassociated with differentiation. This work was done in cooperation with DrA. Schafer, Dr. E. Duerssen and Professor Savedra of Chilie.”

“Later, Dr. B. Arnholdt-Schmidt, now a Professor (Universidade de Évora:Évora, PT) joined the group and was mainly concerned with DNA methylationand amplification as related to differentiation. Based on these early studies in the1990s, gene technology was taken up resulting in the insertion of the information ofa coat protein of Hepatitis B virus into the carrot genome which was also expressedin mature carrot roots at harvest. Dr. Jafargholi Imani took the lead with thecooperation of medical virologist (Professor W. Gerlich, Giessen, Germany) fromthe University. Clinical studies with respect to immunization after oral applicationcould not be performed till now. Here also the results and experience of many studieson the cell cycle and its synchronization of haploid and diploid Datura cultures andothers (Dr. J. Blaschke, Dr. R. Kiebler) was utilized by using synchronised culturesfor insertion of foreign DNA into carrot cells, preferably during S-phase,” he wrotein his autobiography published in Kumar and Soporty (2008).

He further wrote: “Many studies were performed on somatic embryogenesis,mainly with petiole explant of carrots, including histology, protein, and nucleic acid

xii In Memoriam Prof. Dr. Karl-Hermann Neumann (1936–2009)

organization and metabolism. These studies resulted from cooperation ofDr. B. Grieb and Professor Li of the University of Huehot, P.R. China. Carbonmetabolism and hormonal system were also studied by Dr. E. Pleschka andF. Schaefer, and published in a number of papers. A broad research programme onthe ploidy level and its significance on development and secondary metabolism wasinitiated by Dr. Forche and Dr B. Zeppernick; both associated with our group forseveral years. The results of these exciting studies were published in several papers.”

“Quite interesting were the cooperative investigations on diurnal variations of theconcentrations of several phytohormones in intact plants, in pot experiments, as wellas in cultured cells. In cultured cells, in constant environment (including continuousillumination) clear maxima of IAA as well as several cytokinins was observed 24 h aday for several days.” “All this work would not have been possible without thededicated help of my associates especially Frau Christa Lein, who had the sameposition and function in my laboratory as Mrs M. Mapes had in F.C. Steward’slaboratory at Cornell,” said Prof. Dr. Neumann in his autobiography.

In 1995, a small book on cell and tissue culture (Pflanzliche Zell—undGewebekulturen) was published in German by Ulmer Verlag, Stuttgart, Germany.In this book results obtained till then were discussed in context to ideas of the time.This book was subsequently developed into first edition of the present book(Neumann et al. 2009).

He further wrote: “In 1972, I became Professor in the faculty of nutrition at theJustus Liebig University Giessen (Plant nutrition, biochemistry and cell biology ofplants) and I worked there till my retirement in 2001. During this period I spent sometime abroad mainly as a visiting Professor or in a similar position for severalcountries of Asia and Africa, from where students came to my laboratory to workfor a Ph.D. or Post doctorate. My longest cooperation was with Professor AshwaniKumar, University of Rajasthan, India (since 1977 till now) as he wrote this in 2009just before his death.”

“One great challenge before me was to establish a research farm in the south ofFrankfurt in 1979 to pursue investigations mainly concerned with irrigation and thequality of irrigation water. What a change! After more than 20 years doing basicresearch, I had to turn to practical problems to continue studies on biochemical andcell biological problems as before. Here Dr. Buno Pauler, a research associate, was agreat help, especially in the statistical evaluation of the data obtained from theexperimental work of about 15 years. The work on irrigation and salinity wasextended to studies on sugar beet cultivation in saline conditions in Egypt. Thiswork was done together with Prof. A. Raafat and Dr. seyed Eisa, Ain ShampsUniversity, including work on biological remediation of saline fields in cooperationwith Professor Kumar and Dr. Shekhawat, University of Rajasthan, Jaipur, India. InEgypt, some work was concerned with the control of Orobanche infection of fababeans based on hormonal studies (Prof. N. Al. Gamrawy and Dr Salem, CairoUniversity); mango malformation (Prof. A. Raafat and Dr El Deep), concentratingon the hormonal system. At the University, I occupied the chair of dean severaltimes, and was also the director of the department as well as the chief of

In Memoriam Prof. Dr. Karl-Hermann Neumann (1936–2009) xiii

examinations.” He has published around 150 research papers and produced over20 Ph.D. students during his entire career.

Prof. Neumann’s special interest in systemic and applied research was probablybest mirrored by the small edition dedicated to him by his former scientist colleaguesat his retirement with the title “From Soil to Cell—a Broad Approach to Plant Life”(Bender and Kumar 2001).

“Prof. Neumann was truly an international scientist with global vision who caredso well, beyond national boundaries, for his many students, research associates andfellows from all over the world. We are sure that all of them, having had the privilegeto work and learn in his team will keep an honourable memory of his generouspersonality, scientific acumen and lifetime achievement” (Arnholdt-Schmidt et al.2010).

References

Arnholdt-Schmitt B, Kumar A, Imani R et al (2010) In memoriam Prof. Dr. Karl-Hermann Neumann (1936–2009). Plant Cell Tiss Organ Cult 100:121–122.https://doi.org/10.1007/s11240-009-9644-5

Bender L, Kumar A (2001) From soil to cell: A broad approach to plant life. GiessenElectron. Library GEB, pp 1–5. http://geb.uni-giessen.de/geb/volltexte/2006/3039/pdf/FestschriftNeumann-2001.pdf

Neumann K-H (2008) Professor Dr Karl-Hermann Neumann. In: Kumar A, SoporySK (eds) Recent advances in plant biotechnology and its applications. IK Inter-national, New Delhi

xiv In Memoriam Prof. Dr. Karl-Hermann Neumann (1936–2009)

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2 Historical Developments of Cell and Tissue CultureTechniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3 Callus Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.1 Establishment of a Primary Culture from Explants of the

Secondary Phloem of the Carrot Root . . . . . . . . . . . . . . . . . . . 263.1.1 Sterile (Aseptic) Technique: Microbe Eradication . . . 323.1.2 Initial Contamination . . . . . . . . . . . . . . . . . . . . . . . 333.1.3 Latent Contamination . . . . . . . . . . . . . . . . . . . . . . . 333.1.4 Introduced Contamination . . . . . . . . . . . . . . . . . . . . 333.1.5 Surface Sterilizing of Plant Material . . . . . . . . . . . . . 33

3.2 Fermenter Cultures (See Also Chap. 10) . . . . . . . . . . . . . . . . . 343.3 Immobilized Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . 363.4 Nutrient Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.5 Evaluation of Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 433.6 Maintenance of Strains: Cryopreservation . . . . . . . . . . . . . . . . 443.7 Some Physiological, Biochemical, and Histological Aspects . . . 46References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4 Cell Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614.1 Methods to Establish a Cell Suspension . . . . . . . . . . . . . . . . . 614.2 Cell Population Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . 63References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

5 Protoplast Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715.1 Production of Protoplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745.2 Protoplast Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

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6 Haploid Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836.1 Application Possibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836.2 Physiological and Histological Background . . . . . . . . . . . . . . . 866.3 Methods for Practical Application . . . . . . . . . . . . . . . . . . . . . 896.4 Anther and Ovary Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

6.4.1 Anther or Microspore Culture . . . . . . . . . . . . . . . . . 906.5 Haploid Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

7 Plant Propagation: Meristem Cultures, Somatic EmbryogenesisMicropropagation, and Transformation of Somatic Embryos inBioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077.1 General Remarks and Meristem Cultures . . . . . . . . . . . . . . . . 1077.2 Protocols of Some Propagation Systems . . . . . . . . . . . . . . . . . 115

7.2.1 In Vitro Propagation of Cymbidium . . . . . . . . . . . . . 1157.2.2 Meristem Cultures of Raspberries . . . . . . . . . . . . . . 1177.2.3 In Vitro Propagation of Anthurium

(following Geier 1986) . . . . . . . . . . . . . . . . . . . . . . 1207.3 Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

7.3.1 Basics of Somatic Embryogenesis . . . . . . . . . . . . . . 1297.3.2 Ontogenesis of Competent Cells . . . . . . . . . . . . . . . 1407.3.3 Genetic Aspects: DNA Organization . . . . . . . . . . . . 1427.3.4 The Phytohormone System . . . . . . . . . . . . . . . . . . . 1477.3.5 The Protein System . . . . . . . . . . . . . . . . . . . . . . . . . 1537.3.6 Cell Cycle Studies . . . . . . . . . . . . . . . . . . . . . . . . . 162

7.4 Practical Application of Somatic Embryogenesis . . . . . . . . . . . 1657.5 Artificial Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1717.6 Embryo Rescue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1727.7 Bioreactors for Micropropagation and Genetic

Transformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

8 Some Endogenous and Exogenous Factors in Cell CultureSystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1858.1 Endogenous Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

8.1.1 Genetic Influences . . . . . . . . . . . . . . . . . . . . . . . . . 1868.1.2 Physiological Status of “Mother Tissue” . . . . . . . . . . 1868.1.3 Growth Conditions of the “Mother Plant” . . . . . . . . . 188

8.2 Exogenous Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1928.2.1 Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . . 1938.2.2 Nutritional Factors . . . . . . . . . . . . . . . . . . . . . . . . . 194

8.3 Physical Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

9 Primary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2099.1 Carbon Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

9.1.1 Improving Photosynthesis . . . . . . . . . . . . . . . . . . . . 225

xvi Contents

9.2 Nitrogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

10 Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23310.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23310.2 Mechanism of Production of Secondary Metabolites . . . . . . . . 23710.3 Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24010.4 Plant Cell Cultures and Pharmaceuticals and Other

Biologically Active Compounds . . . . . . . . . . . . . . . . . . . . . . . 24510.4.1 Antitumor Compounds . . . . . . . . . . . . . . . . . . . . . . 24910.4.2 Anthocyanin Production . . . . . . . . . . . . . . . . . . . . . 256

10.5 Strategies for Improvement of Metabolite Production . . . . . . . . 26010.5.1 Addition of Precursors and Biotransformations . . . . . 26110.5.2 Immobilization of Cells (See Also Sect. 3.3) . . . . . . . 26410.5.3 Differentiation and Secondary Metabolite

Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26510.5.4 Elicitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

10.6 Organ Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27010.6.1 Shoot Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27110.6.2 Root Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27110.6.3 Adventitious Root Culture . . . . . . . . . . . . . . . . . . . . 273

10.7 Genetic Engineering of Secondary Metabolites(See Also Sect. 13.6.3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

10.8 Membrane Transport and Accumulation of SecondaryMetabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

10.9 Bioreactors (See Also Sect. 3.2) . . . . . . . . . . . . . . . . . . . . . . . 28310.9.1 Technical Aspects of Bioreactor Systems . . . . . . . . . 285

10.10 Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

11 Phytohormones and Growth Regulators . . . . . . . . . . . . . . . . . . . . . 309References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

12 Cell Division, Cell Growth, and Cell Differentiation . . . . . . . . . . . . 321References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

13 Genetic Problems and Gene Technology . . . . . . . . . . . . . . . . . . . . . 33713.1 Somaclonal Variations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

13.1.1 Ploidy Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33713.1.2 Some More Somaclonal Variations . . . . . . . . . . . . . 341

13.2 DNA Fingerprinting and Characterization of Germplasm . . . . . 34713.3 Estimations of Genetic Diversity . . . . . . . . . . . . . . . . . . . . . . 34713.4 Evaluation of Genetic Fidelity During Long-Term

Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34713.5 Molecular Marker-Assisted Selection (MAS) . . . . . . . . . . . . . 34813.6 Modes of Plant Gene Modification . . . . . . . . . . . . . . . . . . . . . 350

13.6.1 Classical Breeding (Wild Crossing) . . . . . . . . . . . . . 350

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13.6.2 Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35113.6.3 Gene Technology . . . . . . . . . . . . . . . . . . . . . . . . . . 352

13.7 Agrobacterium-Mediated Transformation in Mono andDicotyledonous Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36813.7.1 Generation of Transgenic Wheat Plants . . . . . . . . . . 36913.7.2 Generation of Transgenic Barley Plants . . . . . . . . . . 36913.7.3 Stable Root Transformation System for the

Functional Study of Proteins in Barley . . . . . . . . . . . 37213.7.4 Carrot Cell Culture and Transformation

Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37713.7.5 Uses of Transgenes to Increase Host Plant

Resistance to Plant Pathogens . . . . . . . . . . . . . . . . . 38013.7.6 Transgenic Carrot: Potential Source of Edible

Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38213.8 RNA Interference (RNAi) Technology . . . . . . . . . . . . . . . . . . 386

13.8.1 MicroRNA (miRNA) . . . . . . . . . . . . . . . . . . . . . . . 38613.8.2 Generation of Hypoallergenic Carrot by Means

RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38813.9 Precise Genome Editing Tools . . . . . . . . . . . . . . . . . . . . . . . . 391

13.9.1 Genome Manipulation Using the DNA RepairSystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

13.9.2 Zinc Finger Nuclease . . . . . . . . . . . . . . . . . . . . . . . 39813.9.3 Meganucleases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39813.9.4 Transcription Activator-Like Effector (TALE):

A Precision Tool for DNA Editing . . . . . . . . . . . . . . 39913.9.5 CRISPR-Cas9 System for Gene Knockout . . . . . . . . 40013.9.6 Structure and Functional Mechanism of Cas9

Nuclease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40513.10 CRISPR/Cas9 for Plant Genome Editing . . . . . . . . . . . . . . . . . 40513.11 Selectable Marker Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40913.12 Reporter Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

13.12.1 β-Glucuronidase (GUS) . . . . . . . . . . . . . . . . . . . . . . 41113.12.2 Green Fluorescent Protein (GFP) . . . . . . . . . . . . . . . 413

13.13 Antibiotics Resistance Genes . . . . . . . . . . . . . . . . . . . . . . . . . 41613.14 Elimination of Marker Genes . . . . . . . . . . . . . . . . . . . . . . . . . 418

13.14.1 Cre-lox Recombination-Based Systems . . . . . . . . . . 41813.14.2 Ac/Ds System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41913.14.3 Double Cassette System . . . . . . . . . . . . . . . . . . . . . 420

13.15 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421

14 Summary of Some Physiological Aspects in the Developmentof Plant Cell and Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . 437

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

xviii Contents

About the Authors

Ashwani Kumar, FBS, FPSI, FISMPP, FABP,FIFS born (1946) to Mr. Swami Dayal Tewari andMrs. Shanti Devi Tewari, at Bandikui, Rajasthan,India, received B.Sc. at Agra University and M.Sc.(Botany) at the University of Rajasthan. He wasawarded gold medal for standing first in order of merit.His Ph.D. (1971) was under the supervision of Prof.H.C. Arya and postdoc with Prof. Dr. K-H. Neumannand later on with Prof. Dr. Sven Schubert at JustusLiebig Universität, Giessen, Germany, with Alexandervon Humboldt Fellowship. His botanist father Prof.Swami Dayal Tewari (M.Sc. in Botany) was his firstteacher. Prof. Ashwani Kumar was also selected in theIndian Administrative Services (IAS: IPS) (1972), buthe opted for a career in botany being his family subject.His wife Mrs Vijay R. Kumar has also been Professor ofBotany at the University of Rajasthan on her ownqualifications. He was appointed as Assistant Professorin 1969, Associate Professor in 1985, and Full Professorfrom 1986 to 2007. Then he was an Adjunct Professoruntil 2016. He along with members of COC introducedIntegrated Biotechnology 5-year course in Rajasthan.

In recognition of his research contributions,Dr. Kumar was awarded Alexander von Humboldt Fel-lowship in 1977–1979, with resumption of fellowshipsuntil 2017, British Council Visitorship, UK (1986);Visiting Professorship at Toyama Medical and Pharma-ceutical University in Japan (1999–2000); Toyama Pre-fectural University Japan (2011); and INSA-DFG visitingProfessorship at Germany, 1997. He holds a diploma inGerman language and has a certificate in French lan-guage. His area of research includes photosynthesisin vitro and in vivo, biotic and abiotic resistance,

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ethnobotany, bioenergy, and presently understandingsalinity resistance in maize. He has also carried outresearch projects granted by UGC, USDA-ICAR,MNES, CSIR, DST, DBT, and FACT. He attended alarge number of national and international conferencesas invited speaker and served as chair or co-chair in theInternational Botanical Conference, Berlin, and EU Bio-mass conferences. He has published 220 research papersin national and international journals and 23 books, ofwhich 10 books are authored and 13 edited from reputedpublishers such as Springer and IK. He is a member of theeditorial board of Current Trends in Biotechnology andPharmacy. He has guided 39 research students to Ph.D. atthe University of Rajasthan, Jaipur, India. He published220 original research papers in National and Internatioaljournals. He is an elected Fellow of Botanical Society,Fellow of Phytopathological Society, Fellow of IndianSociety of Mycology and Plant Pathology, Fellow ofMendelian Association, Fellow of Association of Bio-technology and Pharmacology, and Fellow of IndianFern Society. He received V. Puri Medal as Botanistand Teacher’s Excellence award of CEE in 2015. Hehas been a consultant in the World Bank Project sanc-tioned to SPRI-HPPI, President Commonwealth HumanEcology Council (India Chapter), and presently PresidentIndian Botanical Society.

Jafargholi Imani (born 1955 in Aliabad-Gorgan, Iran)works as a scientific group leader at the Institute forPhytopathology, Justus Liebig University (JLU)Giessen, Germany. He has graduated in agriculturalscience and completed his Ph.D. under the supervisionof Prof. Dr. Karl-Hermann Neumann at JLU. He workedas postdoctoral fellow in the group of “Plant TissueCulture and Transformation” at the Institute of PlantNutrition, JLU, Giessen. Currently, he is administrativehead of field station and group leader in Plant TissueCulture and Transformation at the Institute of Phytopa-thology, Justus Liebig University, Giessen, Germany.He is continuing scientific works on the Plant TissueCulture and Genetic Transformation of several plantspecies. His area of research includes optimization ofgenome editing procedures in plants especially incereals and RNA-based technologies for pest control inplant production. He is also specialized in mutualistic

xx About the Authors

interaction between many crop plants and soil-borne andplant root-colonizing fungal endophytes that confersresistance to biotic and abiotic stresses and promotesgrowth in crop plants. He attended a large number ofnational and international conferences. He has published108 research papers in national and internationaljournals and is author of eight books. He is member ofWorking Group “German in Vitro Cultures e.V.”(ADIVK), International Association for Plant TissueCulture and Biotechnology (IAPTC&B), Society forPlant Biotechnology e.V., and Consultant at the EUMarie Curie Chair, Universidade de Évora, Portugal.In addition to teaching and supervising severalbachelor’s and master’s students in agrobiotechnology,he has guided 17 Ph.D. students at the Justus LiebigUniversity, Giessen, Germany.

About the Authors xxi