Table Of ContentKarl-Hermann Neumann
Ashwani Kumar
Jafargholi Imani
Plant Cell and
Tissue Culture
– A Tool in
Biotechnology
Basics and Application
Second Edition
–
Plant Cell and Tissue Culture A Tool
in Biotechnology
(cid:129) (cid:129)
Karl-Hermann Neumann Ashwani Kumar
Jafargholi Imani
Plant Cell and Tissue
–
Culture A Tool
in Biotechnology
Basics and Application
Second Edition
Karl-HermannNeumann(deceased) AshwaniKumar
Hungen,Germany DepartmentofBotany
UniversityofRajasthan
Jaipur,India
JafargholiImani
InstituteofPhytopathology
Justus-LiebigUniversityGiessen
Giessen,Germany
ISBN978-3-030-49096-6 ISBN978-3-030-49098-0 (eBook)
https://doi.org/10.1007/978-3-030-49098-0
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Preface to First Edition
Thisbookisintendedtoprovideageneralintroductiontothisexcitingfieldofplant
cellandtissuecultureastoolinbiotechnology,withoutoverlydwellingondetailed
descriptions of all aspects. It is aimed at the newcomer, but will hopefully also
stimulatesomenewideasforthe“oldhands”intissueculture.Nowadays,withthe
vast amount of information readily available on the internet, our aim was rather to
distillandhighlightoveralltrends,deemingthatacompletereportofeachandevery
tissue culture investigationandpublicationwasneitherpossible,nordesirable. For
some techniques, however, detailed protocols are given. We have tried to be as
thorough as possible, and regret if we have inadvertently overlooked any pertinent
literatureorspecificdevelopmentthatbelonginthiswork.
Thethreeauthorshavebeenassociatedformanyyears,andhaveworkedtogether
on various aspects inthis field. Without this close interaction, this bookwouldnot
havebeenpossible.Atthisopportunity,wewishtoreiterateourmutualappreciation
of this fruitful cooperation. An Alexander von Humboldt Stiftung fellowship to
AshwaniKumar(UniversityofRajasthan,Jaipur,India)toworkinourgroupatthe
Institut für Pflanzenernaehrung der Justus Liebig Universität, Giessen, supported
thisclosecooperationandthecompletionofthisbook,isgratefullyacknowledged.
Such a book takes time to grow. Indeed, its roots lie in a 3–4 week lecture and
laboratorycoursebyoneofus(K.-H.N.)about30yearsagoasvisitingprofessorat
Ain Shams University, Cairo, Egypt, which later led to the development of a
graduatetrainingunitattheUniversityofGiessen,Germany,andotheruniversities.
So,alsoolderkeyliterature,nowadaysriskingbeingforgotten,hasbeenconsidered,
whichcouldbeofhelpfornewcomersinthisdomain.
Thanksareduetoourpublisherforallthehelpreceived,andforpatientlywaiting
foranendproductthat,wefeel,hasonlygainedinquality.
Giessen,Germany K.-H.Neumann
March2009 A.Kumar
J.Imani
v
Preface to Second Edition
The first edition of the book was intended to provide a general introduction to this
excitingfieldofplantcellandtissuecultureastoolinbiotechnology,withoutoverly
dwelling 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 currently
representabasictoolinplantbiotechnologyinthesecondedition.Thus,acomplete
report of each and every tissue culture investigation and publication was neither
possible nor desirable. The second edition was written to emphasize the latest
advances in the developing field of genetic engineering, especially the thoroughly
revisedChap.13.Forsometechniques,however,detailedprotocolsareprovidedin
thisbook.Thesecondeditionofthisbookwaswrittenbytwoauthors(Dr.A.Kumar
and Dr. J. Imani), because Prof. Neumann, who had played a crucial role in first
edition, is unfortunately no longer alive. These two authors have been associated
witheachotherformanyyearsandhaveworkedtogetheronvariousaspectsinthis
field.Withoutthiscloseinteraction,thesecondeditionofthisbookwouldnothave
beenpossible.AnAlexandervonHumboldtStiftungfellowshiptoAshwaniKumar
(University of Rajasthan, Jaipur, India) to work in our group at the Institut für
PflanzenernaehrungandDr.ImaniattheInstituteforPhytopathology,JustusLiebig
Universität,Giessen,whichsupportedthisclosecooperationandthecompletionof
thisbook,isgratefullyacknowledged.Wehavetriedtobeasthoroughaspossible,
and regret if we have inadvertently overlooked any pertinent literature or specific
developmentthatbelongsinthiswork.
We also acknowledge numerous publications which have been the source of
somepicturesinthisbook.
Acknowledgmentsareduetoourpublisherforallthereceivedhelpandpatience
whilewaitingforthemanuscript.
Giessen,Germany A.Kumar
Spring2020 J.Imani
vii
Acknowledgments
Figures3.2–3.5,3.8,3.10,3.12,3.13,3.16,4.1,4.4,5.2,5.4,5.5,5.7,6.3,7.3,7.5–
7.9,7.11,7.15,7.16,7.33,8.1,8.3,8.15,9.2,12.1,13.3andTables2.1,3.3–3.8,5.1,
6.1–6.3,7.1,7.3,7.5,7.8,12.1arepublishedwithkindpermissionofVerlagEugen
Ulmer. Further, we are indebted to the following authors and their publishers for
gettingpermissionstousetheirfiguresinourbookinChap.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.) Plant
Methods15,121(Figs.13.11and13.12);He,L.,Hannon,G.(2004)MicroRNAs:
smallRNAswithabigroleingeneregulation.NatRevGenet5,522–531.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)
RNAinterferencetechnologyincropprotectionagainstarthropodpests,pathogens
and nematodes. Pest Management Science 74, 1239–1250 (Fig. 13.29); Liu, S.,
Jaouannet, M., Dempsey, D., Imani, J., Coustau, C. et al. (2020) RNA-based
technologies for insect control in plant production. Biotechnology Advances 39
(107463),1–13(Fig.13.30);Karimi,Mansouretal.(2002)GATEWAY™vectors
for Agrobacterium-mediated plant transformation. Trends in Plant Science 7(5),
193–195 (Fig. 13.31); Yuriko Osakabe, Keishi Osakabe. Genome editing with
engineered nucleases in plants. Plant and Cell Physiology 56, 389–400
(Fig. 13.34); Yuriko Osakabe, Keishi Osakabe. Genome editing with engineered
nucleasesinplants.PlantandCellPhysiology56,389–400(Fig.13.35);Erkes,A.,
Reschke, M., Boch, J., Grau, J. (2017) Evolution of transcription activator-like
effectors 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 of
crops.FrontPlantSci7,506(Fig.13.38);Belhaj,K.,Chaparro-Garcia,A.,Kamoun,
S., Nekrasov, V. (2013) Plant genome editing made easy: targeted mutagenesis in
model 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 and
crop plants using the CRISPR/Cas system. Plant Methods 9(1), 39 (Fig. 13.41);
ix
x Acknowledgments
Sharma, M., Schmid, M., Rothballer, M., Hause, G., Zuccaro, A., Imani, J. et al.
(2008) Detection and identification of bacteria intimately associated with fungi of
the 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) The
subcellular localization of Tubby-like proteins and participation in stress signaling
androotcolonizationbythemutualistPiriformosporaindica.PlantPhysiology160
(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.etal.(2008)Detection
and identification of bacteria intimately associated with fungi of the order
Sebacinales. 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 support
ofProf.Dr.SvenSchubert,DirectoroftheInstituteofPlantNutrition,JustusLiebig
Universität, Giessen,after thedemiseofProf. Dr. K.H.Neumann in2009, andthe
awardofAlexandervonHumboldtFellowshipwhichenabledhisfrequentresearch
visits and also helped in shaping the book. Dr. Jafargholi Imani acknowledges the
supportofProf.Dr.Karl-HeinzKogel,DirectoroftheInstituteofPlantPathology,
JustusLiebigUniversität,Giessen.Wethankourresearchstudents,fellowworkers,
andcolleagueswhoseworkswehavequotedandwhohaveprovidedsupporttothis
workdirectlyorindirectly.WealsothankDr.AndreaSchlitzberger,ProjectCoordi-
nator,bookproductionGermanyandAsia,andMr.BibhutiSharmafromSpringer.
ItwaspleasuretoworkwithSpringer,andwethankthemforbringingoutthebook
sonicely.
In Memoriam Prof. Dr. Karl-Hermann Neumann
–
(1936 2009)
Prof.Dr.Karl-HermannNeumannpassedawayonOctober13,2009.Hewasborn
on 22 May, 1936, in Morgensdorf, near Leitmeritz (Sudetenland), which is now a
partoftheCzechRepublic.AftertheendofSecondWorldWar,hisfamilymigrated
to Bernburg in Saxonia-Anhaltinia (later German Democratic Republic, GDR). In
this place, his father acquired another farm, where he grew up. In 1956, following
someproblemswithCommunistadministrationoftheGDR,hehadtomovetothe
FederalRepublicofGermany,wherehefinishedhisschooling.In1957,heentered
Justus Liebig Universität to study agriculture. He spent one semester as a foreign
studentwithastipendat“DenKgl.VeterinaerogLandbohojskole”inCopenhagen,
Denmark, majoring in agricultural chemistry; he completed his studies again at
Giessen, as “Diplomlandwirt” in 1960. In the same year, he received scholarship
fromCornellUniversityinIthaca,NY,USA.Heenrolledhereinagraduateschool,
with botany as major and biochemistry and physical chemistry as minor. He also
started working for Ph.D. under the supervision of Prof. F.C. Steward, FRS. The
topic of his study was “Function of some heavy metals (iron, magnesium and
molybdenum) on the growth and metabolism of carrot tissue cultures” (mainly
protein metabolism and photosynthesis), which also became major for his Ph.D.
thesislateron.Sincethescholarshipwasgiventohimforonly1year,hereturnedto
GermanyandcompletedhisPh.D.underthesupervisionofProf.H.Linserin1962
from the University of Giessen. After spending few years with Prof. Linser while
doingpostdoc,hefinishedhisHabilitation(equivalenttoD.Sc.)studiesin1969.He
xi
xii InMemoriamProf.Dr.Karl-HermannNeumann(1936–2009)
got promoted as “Privatdozent” at the agricultural faculty of Justus Liebig Univer-
sity, Giessen, Germany. In 1972, he became Professor for Biochemistry and Cell
BiologyofPlantsattheFacultyofNutritionoftheJustusLiebigUniversity.
“While working with Prof. Steward and Prof. Linser he was also intensively
trained in systemic thinking and multidisciplinary research approaches which
influencedhisownscientificcareertoaconsiderableextent,”wroteArnholdtetal.
(2010).
HisfirstmajorachievementwasreplacementofcoconutmilkinWhite’snutrient
medium used in Steward’s laboratory at Cornell with an artificial nutrient medium
alsonamedasNeumann’sLösungorNeumann’sliquidmediumofdefinedchemical
composition. “All later investigations can be traced back to the three experiments
performed in the 1960s and the medium was named as Neumann’s Lösung or
Neumann’s medium. One of the experiments focussed on the photosynthesis of
cultured carrot explants, which was based on work done at Cornell; second on
nucleic acid metabolism of carrot cultures; and third on somatic embryogenesis in
carrot cells in a defined nutrient medium. The work on photosynthesis was
accelerated after Ludwig Bender and Ashwani Kumar joined his group in the
1970s and both held Professorships subsequently.” A. Kumar joined as Alexander
von Humboldt Fellow while serving at the University of Rajasthan, Jaipur. “The
results,coveringcytologyaswellasbiochemistryaspects,werepublishedinnumber
ofpapers.Thislineofworkcametoaclosewithstudiesonsomaticembryogenesis
ofautotrophicculturesundernormalatmosphere(Dr.EvaPlescka).”
“The work on nucleic acid started with studies on metabolic turnover of both
DNA and RNA, followed by comparative studies of DNA organization of several
plant species by Cot hybridization indicating about 15% identity of unique and
repeatedDNA.Thisrepresentsthebasicgeneticinformation,whichcanbeusedto
distinguishhigherplantsfromotherbiologicalsystems.Herealsofirstresultsturned
upontheoccurrenceofmetabolicDNAlocalisedinrepeatedfractionsandbroadly
associated with differentiation. This work was done in cooperation with Dr
A.Schafer,Dr.E.DuerssenandProfessorSavedraofChilie.”
“Later, Dr. B. Arnholdt-Schmidt, now a Professor (Universidade de Évora:
Évora, PT) joined the group and was mainly concerned with DNA methylation
and amplification as related to differentiation. Based on these early studies in the
1990s,genetechnologywastakenupresultingintheinsertionoftheinformationof
acoatproteinofHepatitisBvirusintothecarrotgenomewhichwasalsoexpressed
in mature carrot roots at harvest. Dr. Jafargholi Imani took the lead with the
cooperation of medical virologist (Professor W. Gerlich, Giessen, Germany) from
the University. Clinical studies with respect to immunization after oral application
couldnotbeperformedtillnow.Herealsotheresultsandexperienceofmanystudies
onthecellcycleanditssynchronizationofhaploidanddiploidDaturaculturesand
others(Dr.J.Blaschke,Dr.R.Kiebler)wasutilizedbyusingsynchronisedcultures
forinsertionofforeignDNAintocarrotcells,preferablyduringS-phase,”hewrote
inhisautobiographypublishedinKumarandSoporty(2008).
He further wrote: “Many studies were performed on somatic embryogenesis,
mainlywithpetioleexplantofcarrots,includinghistology,protein,andnucleicacid
