Table Of ContentIN VITRO EMBRYOGENESIS IN PLANTS
CurrentPlantScience and Biotechnology in Agriculture
VOLUME20
ScientificEditor:
RJ.Summerfield,The UniversityofReading,DepartmentofAgriculture,
P.O.Box236,ReadingRG62AT,Berkshire,UK
ScientificAdvisoryBoard:
B.K.Barton,AgracetusInc., Middleton,Wisconsin, USA
F.C.Cannon,UniversityofMassachusettsatAmherst,Amherst,Massachusetts, USA
H.V.Davies,ScottishCropsResearchInstitute,Dundee, Scotland, UK
1.Denecke,UniversityofYork, York, UK
1.Hamblin,TheUniversityofWesternAustralia,Nedlands, W.A.,Australia
1.LymanSnow,RutgersUniversity,NewBrunswick,NewJersey, USA
C.P.Meredith,UniversityofCaliforniaatDavis,California, USA
1.Sprent,UniversityofDundee,Dundee,Scotland, UK
D.P.S.Verma,TheOhioStateUniversity, Columbus, Ohio, USA
AimsandScope
The book series is intended for readers ranging from advanced students to senior research
scientistsandcorporatedirectors interestedinacquiringin-depth,state-of-the-artknowledge
about research fmdings and techniques related to all aspects ofagricultural biotechnology.
Althoughtheprevious volumes in theseries dealtwithplantscienceand biotechnology,the
aim is now to also include volumes dealing with animals science, food science and
microbiology. While the subjectmatter will relate more particularly to agricultural applica
tions, timely topics in b~si~~sc\enc¢ iUldbiotechnology will alsobeexplored. Somevolumes
will report progress in rapidly advancing disciplines through proceedings of symposia and
workshops while others will detail fundamental information ofanenduring nature that will
bereferencedrepeatedly.
In Vitro Embryogenesis
in Plants
Edited by
TREVOR A. THORPE
Plant Physiology Research Group, Department of Biological Sciences,
The University of Calgary, Calgary, Alberta, Canada
....
"
Springer Science+Business Media, B.V.
Library of Congress Cataloging-in-Publication Data
In vitro embryogenesis in plants / edited by Trevor A. Thorpe.
p. cm. -- (Current plant science and biotechnology in
agriculture ; v. 20)
Includes bibliographical references and index.
ISBN 978-94-010-4217-8 ISBN 978-94-011-0485-2 (eBook)
DOI 10.1007/978-94-011-0485-2
1. Botany--Embryology. 2. Plant tissue culture. 3. Plant cell
culture. 4. Plant micropropagation. I. Thorpe, Trevor A.
II. Series: Current plant science and biotechnology in agriculture
20.
QK665.155 1995
581.3·3'0724--dc20 94-32558
ISBN 978-94-010-4217-8
Printed an acidjree paper
AII Rights Reserved
© 1995 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1995
Softcover reprint of the hardcover 1s t edition 1995
No part ofthe material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system, without written permission from the copyright owners.
Table of Contents
Preface VB
1. In Vitro Embryogenesis: Some Historical Issues and Unresolved
Problems
W. Halperin
2. Asexual Embryogenesis in Vascular Plants in Nature
K.K. Sharma and T.A. Thorpe 17
3. Zygotic Embryogenesis in Gymnosperms and Angiosperms
V. Raghavan and K.K. Sharma 73
4. Culture ofZygotic Embryos
M. Monnier 117
5. Morphogenic Aspects ofSomatic Embryogenesis
S.A. Merkle, W.A. Parrott and B.S. Flinn 155
6. Structural and Developmental Patterns in Somatic Embryogenesis
E.C. Yeung 205
7. Physiological and Biochemical Aspects ofSomatic Embryogenesis
K. Nomura and A. Komamine 249
8. Molecular Biology ofSomatic Embryogenesis
D. Dudits, J. Gyorgyey, L. Bogre and L. Bake 267
9. Haploiq Embryogenesis
A.M.R. Ferrie, C.E. Palmer and W.A. Keller 309
10. Somatic Embryogenesis in Herbaceous Dicots
D.C.W. Brown, K.1. Finstad and E.M. Watson 345
11. Somatic Embryogenesis in Herbaceous Monocots
S. KrishnaRaj and I.K. Vasil 417
12. Somatic Embryogenesis inWoody Plants
D.1. Dunstan, T.E. Tautorus and T.A. Thorpe 471
List ofContributors 541
Subject Index 543
v
Preface
The capacity of somatic plant cells in culture to form embryos by a process
resembling zygotic embryogenesis is one of the most remarkable features of
plants. This best exemplifies the concept oftotipotency: that all normal living
cells possess the potential to regenerate entire organisms. This concept, first
proposed by the German plant physiologist Haberlandt in 1902, remained
unproven until the 1950s when Reinert in Germany and Steward in the USA
independentlysucceededinregeneratingcarrotplantletsfromculturedcells(see
Chapter 1 for a historical perspective). Researchers using cell cultures have
broughtsomaticembryogenesisto theforefront ofplanttissuecultureactivity,
in part because of the importance of micropropagation to agriculture,
horticultureandforestry. Ofthemethodsusedforclonalpropagation, somatic
embryogenesisispotentiallythemostimportant, as itiscapableofprovidinga
larger number of plants in a shorter period of time than organogenic
approaches. Although thephenomenonhas beenreported in numerousspecies
(see Chapters 10-12), there is as yet no commercial production ofplantlets by
this method. Somatic embryogenesis is not restricted to in vitro conditions, as
it is a phenomenon (apomixis) that has been known for a long time, and is
widespread in nature. As a matter offact, apomixis has been recorded in 94
families in the angiosperms. Therefore, a discussion on this topic has been
included (Chapter 2).
Somatic embryogenesis is offundamental biological interest and has often
been compared to zygoticembryogeny, particularly with reference to 'normal'
development. As well, the culture ofexcised mature zygotic embryos has been
possible since the early 1900s and has led to the development ofimportant in
vitro techniques, in addition to application to problems in agriculture and
horticulture since the 1930s.Thuschaptersonzygoticembryogenesisin higher
vascular plants (Chapter 3) and on the culture ofzygoticembryos (Chapter4)
are included. The discussion ofthese subjects, as well as ofapomixis (Chapter
2) provides valuable information on aspects germane to the examination of
somatic embryogenesis. In addition, they can aid in appropriate in vitro
experimentation.
Sincethe 1960smuchinformationhasbeengatheredabouttherequirements
VB
Vlll
forthemanipulationofsomaticembryogenesisinvitro.Thishasincluded,inter
alia, knowledge ofempirical selections of the inoculum, the medium and the
culture environment; so that now, well-established principles are in place
(Chapter 5). Several reproducible morphogenic systems currently exist, of
whichcarrot, for historical reasons, is the mostimportant. These systemshave
allowedfor detailedexaminationsofthestructuraland developmentalpatterns
ofsomaticembryogenesis(Chapter6),aswellasforstudiesonthephysiological
andbiochemical(Chapter7),and themolecularbiological(Chapter8)basesof
the process. The development of the synchronous single cells embryogenic
system in carrot and the advance in molecular biological techniques are
allowing solutions to formerly unanswerable questions. Although much
remains to be learned and understood about the phenomenon, much progress
has been made on these various aspects of in vitro somatic embryogenesis,
particulary during the last decade.
The role ofhaploidy in plant breeding and in the study ofgenetics is well
accepted. Successful androgenesis and gynogenesis (Chapter 9) are playing an
importantroleinplantimprovement.Finally,thelastthreechaptersinthebook
attempt to summarize,mainlyintabularform, all the informationavailableon
somatic embryogenesis in herbaceous dicots (Chapter 10), herbaceous
monocots(Chapter 11)andwoodyplants,bothangiospermsandgymnosperms
(Chapter 12). The magnitude ofthis task can be grasped from the number of
references cited in these chapters: 491, 340 and 485, respectively. These tables
showthatspecieshither-toconsidered recalcitrant, suchas cerealsand grasses,
legumesandconifers,haveallrespondedfavourably sincethemid 1980s.These
chapters are destined to serve as excellent reference sources for data up to the
early 1990s, on somatic embryogenesis in all plants.
The various chapters ofthis book present together in one place and for the
first time detailed information on all aspects of embryogenesis in plants,
whetherzygoticorasexual,invivoorinvitro. Thedetailedreviewsaredirected
to professionalsandgraduatestudentsinallareasoftheplantscienceswhoare
interested in basic or applied aspects ofplantlet formation and regeneration.
Thebookwillbeuseful,therefore,forcellbiologists,physiologists,biochemists,
molecular biologists, geneticists, and breeders working in agriculture,
horticulture,orforestry, orindevelopmentalbotany,andmorphogenesis. Plant
tissue culture is playing an important part in plant biotechnology directly for
mass clonal propagation, and indirectly as a central tool or as an adjunct to
other methods, such as genetic engineering, in plant modification and
improvement. Inallofthisworkreliablemethodsofplantletregenerationfrom
selectedoralteredcellsarerequired. Invitrosomaticembryogenesisisdestined
for a pivotal role in this work, and this book contributes to the pursuit and
achievement ofthe goals.
Onapersonalnote,Iampleasedtoacknowledgetheencouragementreceived
from two ofmyformer Postdoctoral Fellows(IndraHarryand Kiran Sharma)
and one ofmy former graduate students (Richard Joy IV) to undertake this
project, when I first beganto considerit. They reinforcedmy viewofthe value
IX
ofand the need for this book, a view also endorsed by my colleagues, David
Reid and Edward Yeung. Drs. Harry, Sharma and Joy further critiqued my
initial attempts at an outline for the book. While the final table ofcontents,
authors, etc. are my own selections, I am grateful to all ofthe above for their
intellectualinput.Ialsowishtothankthepresentmembersofmylaboratoryfor
their help in indexing this book. Finally, I am indebted to the various authors
whohavecontributedtheirtimeandexpertisetotheproductionofthein-depth,
comprehensive and critical reviews which make up In Vitro Embryogenesis in
Plants.
1. In Vitro Embryogenesis: Some Historical Issues and
Unresolved Problems
WALTER HALPERIN
Contents
I. Introduction B. IsCellIsolation Requiredto
II. EarlyReportsofPlantIet InduceEmbryogenesis? 10
RegenerationinCarrotCultures C. Singlevs. MultipleCellOriginof
III. EarlyReportsofEmbryogenesisin Embryosand itsBearingonthe
CarrotCellCultures 3 ConceptofIsolation 11
IV. ControversiesandUnresolved D. DoBudsForminCarrot
Problems 5 Cultures? 13
A. AreThereSpecialChemical V. Embryosfrom PollenGrains 14
FactorswhichPromote References 15
Embryogenesis? 7
I. Introduction
Readers interested in a general discussion ofthe history ofplant tissue culture
cannotdobetterthan to readthefirst twochaptersofPhilipWhite's book, The
Cultivation ofAnimalandPlant cells [1). White was a pioneer in the 1930s and
1940s,alongwithR.J. GautheretandP. Nobecourt,indevelopingtissueculture
techniques.
Thefollowingdiscussionwillconcentrateoncarrotculturessincemostofthe
early work was on Daucus carata and it is still the most studied system.
Additional early work of significance which merits some discussion is the
discovery that haploid embryos can be derived from cultured anthers.
II. Early reportsofplantlet regeneration in carrot cultures
The story ofembryogenesis in vitro begins with observations by early workers
then plantlets were often regenerated in carrot callus cultures. The circum
stances under which plantlets appeared in these early studies were diverse and
offer only a few hints as to the controlling factors in organ regeneration. In
T.A. Thorpe(ed.). In VitroEmbryogenesisinPlants,pp. 1-16.
©1995 KluwerAcademicPublishers, Dordrecht.
2
1950, Levine [2] reported that carrot callus grown in a medium containing
indoleaceticacid(IAA) would regenerate rootsand shoots, inthatorder, when
IAAwasremovedfromthemedium. Sincenohistologicaldetailsweregiven,we
do not know whether preexisting primordia were merely released from auxin
inhibition by the removal of IAA or were initiated by the treatment. Levine
reportedthattheshootsbore"primitive"leavesofacotyledonoustypeandthat
thesewerefollowed bypinnatifidleavesresemblingthematureleavesofcarrot.
He did not know how entire plantlets were ultimately produced from these
isolated organ systems. In 1954, Wiggans [3] also observed carrot plantlet
formation, but under different circumstances. His medium contained adenine
sulfate. When the tissue was transferred to a medium lacking adenine, "buds"
appeared and these gave rise to plantlets. Wiggans did not describe leaf
morphology, but did state that tissue grown in the adenine-free medium
"exhibitedmuchlessauxinactivity than theoriginal tissuedid, therebymaking
budformation possible". Thus, Levineconcludedthat the removal ofauxinled
to root formation, followed by whole plantlets; whereas Wiggans concluded
that loweringofthe auxin contentofthe tissue led to bud formation, followed
byplantlets.Withoutexactlyrepeatingtheirwork,wecannotexplainwhyinthe
onecaserootsappearedfirstand in theothercaseshootsappearedfirst, orhow
entire plantletswere ultimately regenerated from these isolated organ systems.
Amoredetailed report on the formation ofcarrot plantlets in tissue culture
waspublishedintwopapersin 1958 byStewardandhiscoworkers[4, 5]. These
papers are frequently cited, but seldom read, judging by the errors which
continue to be propagated regarding their content. In the first paper [4], they
calledattentiontothemultiplicityofgrowthpatternsexhibitedbyculturedcells
and pointed out that certain patterns violated "classical concepts of cell
behaviour".Thisisahighlyoriginalandstimulatingpaper. Inthesecondpaper
[5], the authors discussed plantlet regeneration in their cultures and called
attention,inparticular, toproembryo-likeearlystages. Multicellularfilaments,
apparently derived from single cells, resembled the very early stage of
proembryo development in seed embryos ofDaucus. However, those who cite
this paperoverlook the fact that the authors report earlycessation ofembryo
likebehaviourasthefilamentsgrewintovascularizedclumps. Acambialregion
appeared and roots were initiated, exactly in the manner described by earlier
workerssuchasSterling[6],whostudiedtobaccostemexplants,andGautheret
[7], who made anexhaustivelydetailed study ofthe development and anatomy
ofsuch vascularized nodules. Steward and coworkers go on to state that when
their rooted clumps, growing in suspension culture, were transferred to agar
solidifiedmedium, budsformed atapointinthenoduleoppositetheroot, thus
forming a plantlet. That the shootoriginatedina budwasapparentlyinferred,
since the published data did not show histological sections establishing the
developmental sequence involved in shoot formation. A similar pattern of
plantlet formation, involving adventitious roots and presumptive buds
sequentially formed in a vascularized nodule, has been reported several other
times [8, 9].
