In vitro Plant Recalcitrance: An Introduction

Author(s): Erica E. Benson

Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 36, No. 3 (May - Jun., 2000), pp.
141-148
Published by: Society for In Vitro Biology
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In Vitro Cell. Dev. Biol.-Plant 36:141-148, May-June 2000
C 2000 Society for In Vitro Biology
1054-5476/00 $10.00+0.00

SPECIAL SYMPOSIUM: IN VITRO PLANT RECALCITRANCE

IN VITRO PLANT RECALCITRANCE:AN INTRODUCTION

ERICAE. BENSON*

Plant Conservation Biotechnology Group, School of Science and Engineering, University of Abertay Dundee, Bell Street, Dundee DD1 1HG, UK

(Received 22 July 1999; accepted 11 November1999; editor P. Ozias-Akins)

SUMMARY

In vitro recalcitrance is the inability of plant cells, tissues and organs to respond to tissue culture manipulations. With
respect to plant regeneration, recalcitrance can be a major limiting factor for the biotechnological exploitation of
economically important plant species and it can also impair the wider application of in vitro conservation techniques. This
first paper introduces a compilation of Symposium papers, collectively entitled "Do we understand in vitro plant
recalcitrance?", presented at the 1999 Congress of the Society for In Vitro Biology. The Symposium reviewed recalcitrance
in the context of genetic predeterminism, molecular markers and gene expression patterns, whole and explant physiology,
stress physiology, habituation, neoplastic progression and plant cancer. The symposium contributors present fundamental
and applied investigative approaches which have the potential to enhance our current understanding of in vitro
recalcitrance and to assist in overcoming the problems associated with nonresponsive plant cultures. This introductory
paper presents the general concept of recalcitrance in relation to whole-plant and explant physiology and considers basic
aspects of tissue culture manipulations in the context of recalcitrance problems.

Key words: recalcitrance; in vitro plants; tissue cultures; morphogenesis; totipotency.

INTRODUCTION
Recalcitrance is the inability of plant cells, tissues and organs to
respond to tissue culture. It can occur at all stages of a culture
regime and we know little regarding its causal factors. The success
of a plant biotechnology project can largely depend upon the ability
to regenerate whole plants from in vitro cultures. Thus, the 1999
Society for In Vitro Biology Congress (New Orleans, LA) hosted the
Symposium "Do we understand in vitro plant recalcitrance?" and
the following papers were delivered by the symposium speakers.
This paper will not present detailed, specific accounts of
recalcitrant species, but instead its aim is to provide a general
introduction to recalcitrance and to set the scene for the other
Symposium papers. Thus, McCown will discuss the concept of
'genetic predeterminism' in woody plants and other long-lived
perennial species. As it is essential that we develop investigative
tools to study recalcitrance, Cairney et al. (1999; 2000) describe the
elegant application of gene expression studies (using differential
display and DNA arrays) to the in vitro study of embryo
development in loblolly pine. Importantly, these authors highlight
the need to link a basic knowledge of plant physiology to molecular
gene expression studies. Benson considers the 'stress physiology' of
recalcitrance and evaluates the role of free radicals in in vitro stress
and ageing. The Symposium papers conclude with the article by
*Authorto whomcorrespondenceis to be addressed:Email e.e.benson@
tay.ac.uk
Gaspar et al., who propose the fascinating concept that disturbances
in the regulation of metabolic pathways of in vitro-grown plants
promote neoplastic progression which ultimately results in the
irreversible loss of totipotency in habituated plant cancer cells.
Although in vitro recalcitrance is a major problem in plant
biotechnology programs it is rarely, if ever, considered in any detail.
The 1999 Congress of the Society for In Vitro Biology provided an
important forum to open discussions on the subject and it is hoped
that this series of papers will stimulate further interest. Each paper
may be considered in its own right; however, it is the hope of the
contributors that the reader will consider the series of papers as a
whole, as it offers a unique and integrated overview of some current
theories of in vitro plant recalcitrance.
FACTORSWHICH INFLUENCERECALCITRANCE
Tissue culture responses are influenced by three main factors:
* 'whole plant' physiology of the donor;
* in vitro manipulations;
* in vitro plant stress physiology.
Integrating our knowledge of whole plant physiology with an
understanding of tissue culture responses (including the optimiza-
tion of tissue culture factors) is an essential first step towards
overcoming recalcitrance. It is also important to consider that the
culture environment per se may evoke stress responses which
promote recalcitrance problems.

141

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142 BENSON
\NHOLE
PL.ANT
PHYSIOLOGY: CHOOSING THE EXPLANT
Ideally, the donor plant should be healthy and well-character-
ized; unfortunately, explant choice can be limited, for example
when the donor population is very small (e.g. endangered species)
or when it is essential to work with recalcitrant mature tissues.
Thus, the suboptimal selection of an explant can be an important
recalcitrance determinant. The following sections will present and
discuss the relevance of a range of factors (i.e. taxonomic, life cycle,
in vitro responses, environment, stress) to recalcitrance phenomena.
Taxonomic relationships and prediction of in vitro responses. The
first tissue culture systems were developed primarily for compliant,
herbaceous, dicotyledonous species (Pierik, 1987). The ever-
increasing need to apply biotechnology to a diverse species range
necessitates greater consideration of the specific requirements of
different groups of plants to in vitro manipulations. Phylogeny can
have an immediate impact on tissue culture recalcitrance; for
example, the gymnosperms have a highly varied response to tissue
culture, with some species being extremely successful and others
far less so (Bonga and Von Aderkas, 1992). Marked differences in
in vitro responses are observed between monocotyledonous and
dicotyledonous species and the former can be especially recalci-
trant. There are a number of key phylogenetic differences in the
morphology of these two angiosperm groups. Monocotyledonous
shoot meristems are usually basal in origin, and their vascular
tissues are randomly scattered throughout stems which frequently
lack cambium tissues and the meristematic cell types that are
especially culture-responsive. Progression in the successful devel-
opment of tissue culture systems for several cereal monocots is well
documented (Vasil, 1987; Blackhall et al., 1999). It is of primary
importance to successful monocot tissue culture to select explant
tissues from meristematic regions (immature embryos, basal leaf
meristems, seed scutella, basal plates, basal bulb scales) which may
be in very different locations to those of dicotyledonous plants. For
example, Stanilova et al. (1994) showed that the morphogenetic
potential of explants from two endangered monocotyledonous plant
species (Leucojumasetivum and Lilium rhodopaeum) was influenced
by the explant selection. Basal bulb scales had a higher population
of meristematic cells; however, the leaf sheaths of young leaves
were preferred for organogenic micropropagation as they possessed
the higher regeneration capacity and could be removed without
destroying the plants.
Plant development and explant choice. The mode of reproduc-
tion (sexual, asexual) of the donor plant is important in terms of
biotechnological application, explant selection and culture method.
Three main options may be considered: (1) gametes; (2) seed; and
(3) vegetative tissues.
Anther, microspore and pollen culture. These cultures are
important for haploid production (Pierik, 1987; Jaihne-Gartner and
Lirz, 1999) as the normal development of anthers and microspores
can be altered in vitro to produce callus and embryos. Establishing
haploid cultures can present special recalcitrance problems and the
most important factor is the stage of reproductive development of
the donor plant. Optimal responses are associated with: the time at
which the flowers are taken; donor plant vigor; photoperiod; light
intensity and quality; temperature; and nutrition (Jiihne-Gartner
and Lirz, 1999). It is necessary to determine which developmental
stages of the microspores are the most responsive in vitro. The
manipulation of pollen reproductive programming using exogenous
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signals (e.g. cold treatments and nutrient limitation) can assist
cultureestablishment.In the case of barley spikes, this can include
the application of dark, cold pre-treatments(3-4 wk at 40C) at
relativelyhigh humidities(fora review,see Jiihne-Gartner and Larz,
1999).
Seedand zygoticembryoculture. This is the growthof matureor
immatureseed germplasmin vitro,with the aim of initiatingcallus
cultures(e.g. in the case of cereals)or to obtaina viable whole plant
which may be used for other manipulations, most usually
micropropagation. A majoradvantageof using seeds and embryos
as an explant source is that juvenile tissue is usually more
responsive to tissue culture. As is the case for other explants,
developmental stage and reproductive physiology are key to
determiningseed and zygoticembryocultureresponsesbut specific
parameterswhich influence the success of embryoculture include:
genotype;growthstatusof the donorplant;and developmentalstage
of the embryoon isolation (Pierik, 1987). Embryoculture is most
frequentlyused for:shorteningbreedingcycles; preventingembryo
abortion;and as a startingpoint for vegetativepropagation.A most
importantapplicationof seed and zygotic embryoculture is in in
vitro plant conservation.Seed and embryogermplasmis used for
cryopreservation(Engelmann,1991) and is a startingpoint for the
clonal propagationof endangeredspecies (Gonzalez-Benitoet al.,
1999). Fay (1992) reviewed specific factors associated with
initiating cultures from seeds. Thus, the removal of testas under
sterile conditions can enhance germinationand in vitro culture
establishment.Orchidspresentparticularrecalcitranceproblemsas
in vitro germination is dependent upon associated symbiotic
mycorrhiza. Frequently, seed components (scutella, nucellus
tissues, cotyledons)are used as a source of juvenile explants for
initiating somatic embryogenic cultures. Santaremet al. (1997)
studied the effects of different explant and in vitro factors (pH,
explantorientation,woundingand solidifyingagent)on the capacity
to producesomaticembryosfromimmaturesoybeancotyledonsand
found that abaxial explant orientation (abaxial side facing the
medium)enhancedresponsiveness.
Vegetativepropagation. The capacity of plants to reproduce
asexually has been used to great advantagein plant biotechnology
through the application of clonal micropropagation(via somatic
embryogenesis,organogenesisand nodal culture). An understand-
ing of whole 'donor'plant physiologycan be criticalfor establishing
successful protocolswhich are dependentupon genotype,environ-
mental conditionsand donorplant physiology.Some families have
high regeneration capacities, but for others (especially woody
species) in vitro vegetativepropagationmay be more difficult. For
such systems, explant position along the stem of the donormother
plant is an importantdeterminantfor responsiveness.Thus, Hsia
and Korban (1996) showed that the nodal explant position of
different plastochrones(nodal explant position along the mother
stem) greatly influenced the in vitro regenerativecapacity of Rosa
spp. Higher numbersof shoots per explant were obtained from a
cultivarof Rosa hybridawhen the explantwas obtainedproximalto
the apical meristem rather than from distal nodes. Proliferating
shoots derived fromplastochronesproximalto the apical meristem
had a lower numberof leaves per explant and were shorterthan
those frommoredistal nodes. These researchersalso demonstrated
that the size, diameterand length of Rosa stem segmentsinfluenced
in vitro shoot establishment. Nodal stem segments with larger
diametersor longer internodeshad a better and faster bud-break
 SPECIALSYMPOSIUM:
IN VITROPLANTRECALCITRANCE:
development regardless of the plastochrone position. Hsia and
Korban(1996) concluded that these stem segments had a better
nutrient translocationfrom the donor mother plant and therefore
developed larger buds, which break sooner when placed in vitro.
This study clearly highlights the importanceof understandingthe
'pre-culture'physiologyof a donorplant and the effects of explant
status on in vitroresponses.
Wholeplantphysiology:the influenceof environment and life cycle
on in vitro responses. Plants have developed highly complex life
cycles and reproductivestrategies to competitively secure their
place in specific habitatsand ensuretheir survivalwhen challenged
by environmentalstress broughtabout by seasonal change. Whole
plant life cycle physiology is directly linked to reproduction,
vegetativedevelopmentand morphogenesis.It thus followsthat it is
one of the most importantfactors in determiningthe capacity of
explants to respond in vitro, and many examples of in vitro
recalcitrancecan be directly attributedto life cycle factors.
Plants can be placed into different life cycle 'groupings'
determined, in the first instance, by their centre of origin (i.e.
tropicalor temperatespecies) and, secondly, as to whetherthey are
annualsor perennials.Plantsfromthe humidtropicsand equatorial
regions may not have to contend with the environmentalextremes
associated with seasonal temperaturefluctuationsand the factors
which determine the different phases of their life cycles may,
therefore,be difficult to predict. In contrast,tropical plants from
arid and semi-arid zones must be physiologically adapted to
withstandthe environmentalfluctuationsof wet and dry seasons.
Temperate species have, through necessity, developed complex
seasonalresponseswhich allow themto survivethe winterextremes
of shortday lengths,low light intensities and reducedtemperatures.
Thus, temperatespecies undergopredictablecycles of dormancy,
senescence, quiescence and rejuvenationwhich can be manifestat
the whole-plant level or by seeds and vegetative organs (buds,
bulbs, tubers, etc.). It is not, therefore,surprisingthat temperate
trees such as Fagus sylvatica (Meier and Reuther, 1994; Nadel
et al., 1991) and Picea stichensis(Selby and Harvey, 1985) show
seasonal patternsin their responsesto tissue culture and that these
are dependent upon the explants being procuredat an optimum
time of the year. Even in the absence of seasonal cycles, species
from the humid tropics and equatorialregions can show rhythmic
growth patterns which greatly influence culture responsiveness.
Lardetet al. (1998) foundthat dormantand meristematicallyactive
buds of Hevea brasiliensis(rubber)and Theobromacacao (cocoa)
behaved differentlyin vitro. In both species, nodal explants taken
from dormant buds had a greater in vitro opening and shoot
elongation ability than buds taken from actively meristematic
branches(forT. cacao) and foliargrowthphases (forH. brasiliensis).
In vitro recalcitrancein long-lived perennials(especially woody
plants) is a major problem. A thoroughknowledge of the donor
plant's life cycle, including an understandingof the phases of
reproduction, rejuvenation and dormancy, is critical for the
developmentof strategiesto overcomerecalcitrance(see McCown,
2000).
Maturationand rejuvenation. For long-lived perennial species
there are two choices for establishingin vitroculturesand explants
can be taken from either juvenile or mature tissues. Ideally,
cultures should be initiated from the former,but it is frequently
desirable or necessary to use adult phase plants as the explant
source. For many tree species it is still not possible to
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AN INTRODUCTION 143
micropropagatematuretrees that have a provenvalue in the field
(Bongaand Von Aderkas, 1992). Recalcitranceof matureexplants
is exhibitedat all stages of culture.Forthese systems,the selection
of explantsat a specific responsivestageof a maturetree'slife cycle
is the most importantfactor in overcomingrecalcitrance and a
number of strategies have been developed to help overcome the
problem.
Initially it is useful to comparethe in vitro responses of mature
andjuvenile tissues within a species. For example,Parraand Amo-
Marco (1998) comparedin vitro shoot proliferationin adult and
seedling materialof Myrtuscommunisand foundthat adult material
displayed symptomsof hyperhydricity,whereas this conditionwas
completely absent in seedling-derived explants, although these
shoots did develop apical necrosis. However, the hyperhydric
shootsfromadultmaterialproducedhighershootmultiplicationand
elongation rates comparedto the seedling-derivedmaterial. This
difference was sustained over five subcultures, each of 2 mo.
intervals.One explanationfor this may be that the maturetissue of
M. communis becomes rejuvenated in vitro, and indeed this
phenomenonis generallyexploited in tree tissue culture.However,
Parra and Amo-Marco(1998) noted that for mature tissues the
multiplicationrates were initially very high in the first subculture,
but became lower and stabilized during subsequent subcultures.
Thus in vitrorejuvenationin maturetissues may be limited for this
species and other factors must be important in maintaining
proliferation potential. This investigation shows that explants
taken from different life cycle stages can greatly influence
micropropagationpotential and that it is not always the case that
juvenile tissues are the mostresponsive.A moretypical responseof
juvenile and maturetissues was observedin Prunusdulcis (Miguel
et al., 1996), for which leaf explants of juvenile origin gave the
highest regenerationrates comparedto those of maturetissues.
Rejuvenation,throughgrafting,can also be used to overcome
recalcitrancein maturetrees. This can be performedon maturein
vivo systems or throughmicro graftingin vitro.A micropropagation
system developedfor Corylusspecies (Yu and Reed, 1993) clearly
demonstratesthe importanceof selecting explants at an optimum
seasonal stage; when this is combined with in vivo grafting an
amenable source of explants can be obtained from grafted
greenhouse-grownplants. By comparison,in vitroreinvigorationof
maturetrees of Olea europaeathroughmicrograftingonto juvenile
in vitro shoots resulted in the restoration of shoot and root
proliferation(Revilla et al., 1996).
Ewald (1998) gives a detailed account of advances in the
applicationof tissue culture methodsto adult larch and comments
that mostdifficultiesare due to phase changes in tree development.
Success is dependent on the rigorous preconditioningof plant
materialusing graftingand pruningand then linking these to the
strict optimizationof tissue culturemethodsbased on reinvigorating
and rejuvenating the explants. Ewald (1998) presents three
approaches to obtain cultures from adult larch: (1) serial
subculturing without hormones; (2) adventitious bud formation;
and (3) micrografting.
Procurementof explants from the new vegetative flushes of
maturetrees is an importantroute for establishingtissue cultures
from highly recalcitrant woody plant systems such as mature
gymnosperms.Litz et al. (1995) were able to produceembryogenic
callus from pinnae removed from the leaves of new vegetative
flushes of a mature cycad species Ceratozamiahildae. A recent
144 BENSON
review (Litz et al., 1999) highlights the considerable success that
has been made in producing embryogenic cultures from mature
phase tropical angiosperm and gymnosperm trees. Progress to date
is, however, the result of empirical studies and 'trial and error'
investigations and in the future more fundamental approaches
should be considered (see, e.g., Cairney, 2000).
IN VITRO ~MANIPULATIONS
The Use of Media and Culture Environment Manipulations to
Overcome Recalcitrance
An important approach to overcoming recalcitrance problems
relates to tissue culture manipulations per se. Hall (1999) has
summarized practical 'pointers' to successful tissue culture
involving the optimization of the following key media components:
-> INORGANICS
-= Macronutrients
= Ca, K, Mg, N, P, S
-+ Micronutrients
= B, Co, Cu, Fe, I, Mo, Mn, Zn
= ORGANICS
-> Vitamins
* thiamine (B1), pyridoxine (B6), nicotinic acid (B3)
-+ Myoinositol
- Amino acids
z=
glycine, proline
=> Carbon source
* sucrose, glucose
- PHYTOHORMONES
=* auxins, cytokinins, giberellic acids, abscisic acid
-= GELLING AGENT
* agar, agarose, gelrite, phytagel, alginate
=> OTHER FACTORS/ADDITIVES
=- pH, activated charcoal, AgN03, ethylene, polyamines,
antioxidants, jasmonates, salicylates, oligosaccharides
Physical factors such as culture vessel size and aeration, light
quality, day length and temperature also have a major influence on
culture responses.
Growth regulators and hormones. One of the most important
approaches for overcoming in vitro recalcitrance problems is to
optimize the plant growth regulator (PGR) regime (for a detailed
review on in vitro plant growth regulators and hormones see Gaspar
et al., 1996). Factors which must be considered when using PGRs to
overcome recalcitrance include:
* optimizing standard PGR regimes specifically for recalcitrant
genotypes;
* applying potent PGRs (e.g. thidiazuron);
* applying novel PGRs (e.g. jasmonates);
* mitigating the inhibitory interactive effects of exogenous and
endogenous hormones (e.g. ethylene).
Optimization of standard PGR regimes provides an empirical
approach to recalcitrance; however, cultures are under the control
of both endogenous and exogenous plant growth regulation and the
'balance' between the two can have important effects. Lardet et al.
(1998) discussed the importance of endogenous PGRs relative to
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bud responsegradientsin nodes takenfromdifferentstem positions.
Responsiveness increased from the apical to the basal section in
H. brasiliensis and T. cacao and this affected the in vitro
proliferationof nodal segments. It is known that shoot dormancy
in T. cacao is controlledby endogenouslevels of abscisic acid ABA
and shoot rejuvenationis controlled by endogenous cytokinins
(Orchardet al., 1980). Thuswholeplant, explantand in vitrofactors
must be consideredwhen developingPGR regimesfor recalcitrant
tissue cultures.
Achieving the correct exogenous balance of the key hormones
auxin and cytokinin can be critical and their appropriate
application can effectively overcome certain recalcitrance pro-
blems. By carefullyoptimizingauxin and cytokininlevels, Guohua
(1998) promotedand enhanced shoot organogenesisand somatic
embryogenesisin cassava genotypesfor which plant conversionand
embryogenesiswere previously extremely low and unacceptably
slow. Similarly,by phasingthe applicationof 6-benzyladenineand
naphthaleneaceticacid, Heatley and Smith (1996) were able to
regeneratewhole plants fromshoot apices of A. hypogaea.
Recalcitrancecan also be mitigatedby the applicationof potent
synthetic PGRs such as thidiazuron (Murthy et al., 1998).
Thidiazuron (N-phenyl-N'-1,2,3,-thidiazol-5-ylurea; designated
TDZ) is a highly effective regulatorof in vitro morphogenesisand
can be used to induce a wide range of differentiated and
dedifferentiatedresponses. To date, the mode of action of TDZ is
unknownbut Murthyet al. (1998) speculate that it may modulate
endogenoushormonalactivity,eitherdirectlyor as a resultof stress.
TDZcan display both auxin- and cytokinin-typeactivities and this
is most likely due to it having both phenyl and thidiazolgroups.It
has been most effectively used for in vitro shoot regenerationin
woody species and can be far more effective in promoting
morphogeneticresponses comparedto other cytokinins, although
it mustbe cautionedthat it can impairgrowthat superoptimallevels
(Murthyet al., 1998). Improvementof shoot organogenesis in
Mentha spp. which have previously been difficult to regenerate
reliablywas achieved throughthe applicationof mannitoland TDZ
(Faureet al., 1998).
Thereis increasinginterestin the applicationto culturemediaof
compoundswhich can not be strictly defined as plant hormones
(e.g. jasmonates,polyamines,salicylates, antioxidants),but which
do exert growth-modulating effects (see Gasparet al., 1996), and
such approaches may provide novel means of overcoming
recalcitranceproblems.Ascorbic acid has been found to enhance
organogenesisin both young and old tobacco callus (Joy et al.,
1988) and was found to completelyreverse the inhibitionof shoot
formationby gibberellicacid in youngcallus, althoughresponsesin
older callus were less effective. Polyamineshave been effectively
applied to enhance in vitro rootingin woodyspecies such as hazel
(Rey et al., 1994), and their application offers the possibility of
enhancing rooting in difficult systems. Jasmonic acid has
stimulatory effects on growth when incorporated into culture
media (Ravnikaret al., 1992) and was found to enhance in vitro
plantlet shoot and root proliferation and protoplast cell wall
formationin Solanumtuberosumcultures. The steroid lactone 24-
epi-brassinolidehas been applied in culture media to enhance in
vitro stem elongation in sweet pepper (Franck-Duchenneet al.,
1998) and is consideredby this groupto be a potentiallyimportant
PGR which can be used to enhance in vitroregenerationprocesses
in recalcitrantagriculturalplants.
 SPECIALSYMPOSIUM:
IN VITROPLANTRECALCITRANCE:
The productionof ethylene by plant tissue cultureshas received
considerableattentionas a possible factorin culturerecalcitrance.
Tissue culture can promoteinhibitoryinteractive effects between
exogenous and endogenous hormones and recalcitrance can be
associatedwith the overproductionand accumulationof ethylene in
the culture vessel. Ethylene is involved in many morphogenetic
responses (for a review, see Kumaret al., 1998) as it can both
promote and inhibit in vitro morphogenesis.The interaction of
ethylene with other PGRs is highly complex and is still little
understood.The synthesis and activities of auxins, cytokinins and
ethylene are thoughtto be closely related(Klee and Romano,1994)
and ethylene also shares a common precursor pathway with
polyamine biosynthesis via S-adenosyl methioine. The exogenous
applicationof PGRs such as TDZ (Hutchinsonet al., 1997), auxins
and cytokinins (Gude and van der Plas, 1985) can stimulate the
production of ethylene in tissue cultures. There are many
contrastingexamples which show that the regulationof ethylene
levels in tissue culturescan have both positive and negativeeffects
on in vitro morphogenesis and proliferative growth. Moreover,
previously recalcitrant cultures can become responsive by the
manipulationof ethylene productionand accumulationin culture
vessels, although the concentration of ethylene can greatly
influence its positive effects (Kumaret al., 1998). The growth-
inhibitingeffects of ethylene can be shown by applyinginhibitors
which interfere with the production of the hormone; as a
consequence a positive morphogenetic response is induced,
althoughit is importantto note that these effects can vary greatly
between species. Chile peppers (Capsicumannuum) are, unlike
manySolanaceousspecies, recalcitrantto tissue cultureat the shoot
elongationstage. Hyde and Phillips (1996) were able to enhance
shoot developmentand plant regenerationvia organogenesisin this
species followingthe additionof the ethylene inhibitorsilver nitrate
to the culture medium.
One final considerationin the developmentof PGR strategiesfor
in vitroculturesrelates to the potentialeffect that they can have on
the production of 'true-to-type'cultures. Exposure to PGRs for
extended periods may lead to epigenetic and genetic changes,
which could in turn compromisenormalgrowthand development
and indeed result in recalcitranceproblemsand genetic instability
(Harding,1996). This may be especially problematicwhen in vitro
techniques are used to conserve germplasmfromgenotypeswhich
have elite traits or for endangeredspecies. It is thus essential to
develop regimes which minimize the use of high levels of PGRs
(Edsonet al., 1996) and to avoid the prolongedor excessive use of
potent PGRs.
Otherculturefactors. Tissue culturemediawerefirst developed
for amenableherbaceousspecies (see Pierik, 1987); subsequently
manydifferentbasal media types have been formulatedfor specific
cultureapplicationsand species (fora review, see Hall, 1999). The
following sections summarizesome of the key parameterswhich,
when optimized,can significantlyenhance culture performance.
1. Basal media composition. Overcomingrecalcitrance can be
greatlyassisted by modifyingthe basal compositionof mediafor
difficult genotypes. This is exemplified by the case of woody
plant species (Bongaand Von Aderkas, 1992; McCown,2000).
Specific media components can have a profound effect on
certainculturesystems, e.g. Fe-ethylenediaminetetraacetic acid
is essential for in vitro androgenesisin Nicotiana spp. (Vagera
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AN INTRODUCTION 145
and Havrninek,1982). Nitrogen supplied in organic form as
amino acids (rather than as inorganic salts) produced an
importantbreakthroughfor the initial, successful development
of japonica rice tissue culture systems based on embryogenic
cell suspension cultures (Abdullah et al., 1986). Similarly,
changes in media composition have assisted the culture of
indica rice varietieswhichhave, until relativelyrecently,proved
far less responsive than japonica rices (Faruqueet al., 1998;
Blackhall et al., 1999).
2. Gelling agent. This influences in vitro responses and in some
systems a simple modificationof this additive improves the
culture of difficult genotypes. Agarose-solidifiedmedia were
foundto be effective for rice protoplastculture(Thompsonet al.,
1986); Gelrite enhances somatic embryo initiation in soybean
(Santaremet al., 1997) and reduces vitrification in Petunia
cultures (Zimmermanand Cobb, 1989); and Phytagelpromotes
adventitiousbud regenerationin pear (Chevereauet al., 1997).
As a cautionarypointerto some recalcitranceproblems,note that
differences do exist between different commercialproducts of
the same gelling agent. The effects of different brands of one
gelling agent (agar)were investigatedfor the clonal propagation
of Ranunculus asiaticus, a species for which axillary bud
stimulationcan be problematic.Beruto et al. (1999a,b) found
that two brands produced successful growth,but proliferation
was poor on the third and the plants had symptoms of
hyperhydricityand produced malformed leaves. Clearly, in
developing culture protocolsfor plants which display abnormal
shoot morphologyand hyperhydricityit may be pertinentto test
the effects of gelling agents. Detailed studies indicate that the
differential properties of agar brands with respect to the
capacity to support normal in vitro growth are due to water
retention capacity which, in turn, influences mineral and
carbohydrateavailability (Beruto et al., 1999a,b; Spomer and
Smith, 1996).
3. Carbohydrate.As an energy source for heterotrophicgrowth,
sucrose is usually suppliedto plant tissue cultures.However,for
some species, glucose is the preferred substrate (Reed and
Chang, 1997). Optimizationof sucrose, togetherwith other key
macronutrientssuch as nitrogen,can have a profoundeffect on
the capacity of dedifferentiatedcells to proliferate in vitro
(Samoylovet al., 1998). Superoptimallevels of sucrosealso have
a negative effect and are involved in the manifestation of
recalcitrancesymptomsassociatedwith hyperhydricity(Zimmer-
man and Cobb,1989). The replacementof sucroseby glucose as
a carbon source enhances growthin certain species (Yu and
Reed, 1993). It is also importantto considerthat normalprimary
metabolismmay be impairedin certain types of culture. Plant
cells are obligate aerobes, but they can survive anaerobiosisfor
short periods and this leads to a switch from primary
carbohydratemetabolismto a fermentationpathwayresulting
in the productionof ethanol. Plant cell suspensioncultures can
be proneto anaerobiosis,as demonstratedby Perataet al. (1988)
who showed the toxic effects of ethanol productionby carrot
suspension cultures.
Moderation of sucrose levels and the application of sugar
alcohols, polyols and alternative sugars have a specific role in
somaticembryogensisand in assistingmaturationin the final stages
of embryo development. The application of these compounds
146 BENSON
(frequently in combination with ABA) can greatly improve
recalcitrance problems associated with embryo and organogenic
development in vitro and whole plant regeneration. Thus, ABA and
optimal sucrose levels increased the production of somatic embryos
and shoots in sugar beet callus (Saunders and Tsai, 1999) and
polyethylene glycol combined with ABA induced the later stages of
embryo development in Pinus taeda genotypes which were
previously unresponsive (Li et al., 1997).
1. Amino acids: Certain amino acids are associated with plant stress
responses and have been used as in vitro additives. Proline and
hydroxyproline accumulate in stressed plants. Their production
is associated with enhancing stress tolerance and they are used
to aid in vitro regeneration (Rao et al., 1995) and in the study of
stress responses (Devi Prasad and Potluri, 1996). In a
comparative study of androgenesis-recalcitrant and androgen-
esis-responsive barley genotypes, Ouedraogo et al. (1998) were
able to induce embryogenesis in the recalcitrant cultivar through
the application of 19 amino acids.
2. Microenvironment and physical parameters: Culture ventilation
can greatly influence normal growth and development and poor
ventilation promotes the deleterious accumulation of ethylene
and interferes with the humidity of the in vitro microclimate,
which can result in the problem of hyperhydricity. Gas-
permeable, transparent closures (e.g. Suncaps) are now available
and have proved most effective in avoiding the production of
vitrified (hyperhydric) plants (Majada et al., 1997). Alternatively,
more complex culture apparatus can be designed which allows the
measurement and control of in vitro microclimate parameters
(Demeester et al., 1995). As a cautionary point, certain types of
closure can have a negative, inhibitory effect on cultures. Selby
et al. (1996) showed that somatic embryo maturation in Sitka
spruce was inhibited by the production of butylated hydro-
xytoluene, a volatile antioxidant released from Parafilm.
3. Light: The quality, intensity and photoperiod of light can greatly
influence morphogenetic responses (Michler and Lineberger,
1987). D'Onofrio et al. (1998) found that somatic embryo
production was highest in quince cultures subjected to red light
treatment and decreased with the transition to red/blue and to
white light. These researchers concluded that embryogenic
competence could be correlated with the quality of light and that
phytochrome was involved. In contrast, light can also be
implicated in damaging photoxidation phenomena. Kevers et al.
(1995) have shown dark incubation to have a favorable growth-
promoting effect on white (nonchlorophyllous) hormone-habitu-
ated sugar beet callus and concluded that this is due to the
prevention of necrosis and oxidation phenomena.
4. Temperature: The control of temperature in tissue cultures is
firstly dictated by the place of origin of the donor plant (i.e.
tropical or temperate). However, in addition to the normal
maintenance regimes, temperature can influence specific culture
responses. Cold pre-treatments are used to enhance anther
culture responses (Kiviharju and Pehu, 1998; Peng et al., 1997)
and to enhance post-cryopreservation recovery in in vitro plants
(Reed and Chang, 1997). Heat treatments can also induce anther
culture response for some genotypes (Peng et al., 1997).
5. Microclimate changes: transferfrom in vitro to ex vitro conditions:
This is a most important response as poor acclimation of
transferred cultures can lead to 'late-stage' recalcitrance for a
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large number of plant systems. Unfortunately, this stage of
micropropagation can be a major block. On transfer from in vitro
conditions, plants are exposed to a vastly different microclimate
with a less humid atmosphere and higher light intensities and
have to undergo the switch from heterotrophic to fully autotrophic
growth. The root systems and stomata in these plants are not fully
functional and great care must be taken in the gradual process of
culture transfers (Thomas, 1998).
PLANTSTRESSPHYSIOLOGY
IN VITRO
The previous section reviewed the importance of applying certain
additives and regimes to in vitro systems which are known to have a
role in whole plant stress physiology. Thus, stress can have both
beneficial and deleterious effects on in vitro responses. Light,
temperature and seasonal fluctuations are important in whole plant
development and a knowledge of stress adaptation responses can
assist in the improvement of in vitro techniques. For example, Peng
et al. (1997) found significant correlations between asparagus
microspore callus yield, the percentage of uni-nucleate microspores
and cold-stress treatment. Transfer of explants to in vitro conditions
can also lead to negative responses and it is essential that the
contribution of natural defense mechanisms are taken into account
when developing strategies to overcome recalcitrance. Thus,
ethylene is implicated in stress, and an accumulation of the gas
can lead to decreases in cell proliferation (Kumar et al., 1998).
The oxidation of explants on culture initiation is a frequent cause
of early in vitro recalcitrance in tissues which have a high phenolic
content (Anthony et al., 1999). The problem is most prevalent in
woody plant tissues as they have high levels of phenols associated
with secondary thickening and lignification. It is also a cause for
concern in nonwoody species which produce secondary metabolites
that become oxidized on contact with culture media (Bhat and
Chandel, 1991). The oxidation of phenolic compounds in cut
explants is a wound response and culture techniques, such as
surface sterilization, may greatly exacerbate this. It is also probable
that specific components of the tissue culture media (e.g. metal
cations) may enhance phenolic oxidation. Symptoms include the
rapid browning or blackening of affected tissues and this can result
in tissue necrosis and, in the most extreme cases, explant death. A
number of culture manipulations have been developed to alleviate
the negative affects of stress. Thus, activated charcoal (Fridborg
et al., 1978) prevents tissue blackening and autoxidation and is
especially useful in anther cultures and for the establishment and
propagation of woody plant cultures.
Future studies of in vitro plant recalcitrance must address the
problem of stress and this will be considered in more detail by other
contributors to this Symposium series (Gaspar et al., 2000; Benson,
2000).
CONCLUSIONS
This paper has specifically addressed the importance of
integrating and combining our knowledge of whole plant and
in vitro physiology in order to achieve a better understanding and
control of recalcitrance. Plant reproduction, development and stress
all play a role in the natural life cycles of whole plants and it is
imperative that we explore the impact that these factors have on our
in vitro systems. The following series of papers makes a significant
 SPECIALSYMPOSIUM:
IN VITROPLANTRECALCITRANCE:
contribution to achieving this end. In this Symposium we have
posed the question "Do we understand in vitro plant recalcitrance?"
We do not, as yet, have well-defined answers or solutions. It is
likely, however, that we know far more about the problem than
current opinion would suggest. In the future, it will be essential to
take an integrated, interdisciplinary approach if we are to develop
effective and practical strategies to solve in vitro plant recalcitrance
problems.
ACKNOWLEDGMENTS
The authorthanksDrs. Scott Merkleand WayneParrottfor assistance in
organisingthe Symposium,the Symposiumparticipants(ProfessorThomas
Gaspar,Dr. John Cairneyand Dr. Brent McCown)and The Universityof
Abertay Dundee for financial support. On behalf of the Symposium
participants,the Convenors(Erica E. Benson and Scott Merkle)thank The
InternationalAssociation of Plant Tissue Culture, WESTVACOand the
InternationalPaperCompanyfor sponsorship.Dr. Keith Hardingis thanked
for reviewingthis paper.
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