In VitroCell.Dev.Biol.

--Plant32:272-289,October-December1996
9 1996SocietyforIn VitroBiology
1054-5476/96 $05.00+0.00

Review

P L A N T H O R M O N E S AND P L A N T G R O W T H R E G U L A T O R S IN P L A N T T I S S U E C U L T U R E
THOMASGASPAR,CLAIREKEVERS,CLAUDEPENEL, HUBERTGREPPIN,DAVIDM. REID, ANDTREVORA. THORPE1
Universit( de Liege, Hormonologie v~g~tale, lnstitut de Botanique B 22, Sart Tilman, B-4000 Liege, Belgium (T. G., C. K.); Universit~de
Genc~ve,Physiologic v(g(tale, 3, place de l'Universit~, CH-1211 Getu)ve4, Switzerland (C. P., H. G.); Plant Physiology Research Group,
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada (D. M. R., T. A. T.)

(Received 21 March 1996; accepted 6 June 1996; editor R. H. Smith)

SUMMARY
This is a short review of the classical and new, natural and synthetic plant hormones and growth regulators (phytohormones) and highlights some of their uses in plant tissue culture. Plant hormones rarely act alone, and for most processes-at least those that are observed at the organ level--many of these regulators have interacted in order to produce the final
effect. The following substances are discussed: (a) Classical plant hormones (auxins, cytokinins, gibberellins, abscisic acid,
ethylene) and growth regulatory substances with similar biological effects. New, naturally occurring substances in these
categories are still being discovered. At the same time, novel structurally related compounds are constantly being synthesized. There are also many new but chemically unrelated compounds with similar hormone-like activity being produced.
A better knowledge of the uptake, transport, metabolism, and mode of action of phytohormones and the appearance of
chemicals that inhibit synthesis, transport, and action of the native plant hormones has increased our knowledge of the role
of these hormones in growth and development. (b) More recently discovered natural growth substances that have phytohormonal-like regulatory roles (polyamines, oligosaccharins, salicylates, jasmonates, sterols, brassinosteroids, dehydrodiconiferyl alcohol glucosides, turgorins, systemin, unrelated natural stimulators and inhibitors), as well as myoinositol. Many
of these growth active substances have not yet been examined in relation to growth and organized development in vitro.
Key words: abscisic acid; auxins; brassinosteroids; cytokinins; ethylene; gibberellins; jasmonates; natural/synthetic growth
active substances; oligosaccharins; phytohormones; polyamines; salicylates; sterols; systemin; turgorins.

INTRODUCTION

In the area of cell behavior, topics such as the cytology, nutrition,

primary and secondary metabolism of cells in culture, morphogenesis
(including xylogenesis, organogenesis, and somatic embryogenesis)
and pathology have been studied for nearly 50 years (Thorpe, 1990).
In vitro methods are being used increasingly as an adjunct to traditional breeding methods for the modification and improvement of
plants since the 1960s. Techniques contributing to this area include
in vitro pollination and fertilization, coupled with embryo rescue;
embryo culture; generation of haploids via anther and microspore
culture, and less frequently by gynogenesis; the use of cell cultures
in the presence or absence of selecting agents or mutagens; the exploitation of somaclonal variants; and the use of protoplasts, directly
or following fusion. More recently, the use of vector-independent and
vector-dependent gene transfer methods are playing an important
role in plant modification.
In this review, information on natural and synthetic plant growth
regulators and hormone-like substances (phytohormones) will be presented. The emphasis will be on the compounds themselves rather
than on the tissue culture processes in which they are used. The

Plant tissue culture, used here to include all aspects of the in vitro
culture of cells, tissues, and organs, is generally dependent for its
success on the inclusion of plant hormones and plant growth regulators (referred to subsequently in this article as phytohormones) and/
or other growth active substances, as one of the five classes of required substances, in the medium (Gamborg et al., 1976). Such tissue cultures have been used in five broad areas of research and
application (Thorpe, 1990). These are studies on cell behavior, plant
modification and development, production of pathogen-free plants
and germplasm storage, clonal propagation, and secondary product
formation. These three latter more applied areas are being pursued
widely. However, in all of the above areas good progress is being
made with a variety of plant species (Vasil and Thorpe, 1994), with
micropropagation being the most advanced commercial activity (Debergh and Zimmerman, 1991).
t To whomcorrespondenceshould be addressed.

272

GROWTHACTIVESUBSTANCES
INCULTURE

273
CH=::CH--COOH

CH:-COOH

Indolyl-3-acetic acid (IAA)

Indolyl-3-acrylic acid (IAcrA)

CH2--CH2--CH:~H

CHa----COOH

Indolyl-3-butyficacid (IBA)

4-Cl-indolyl-3-acetic acid(4-CI-IAA)

~ N

~N ~

CH2--C--NH--CHCOOH
[I
)
O CHzCOOH

CH=--C--I~OSO3H

S-glucose

H
Glucobrassicin

lndolyl-3-acetylaspartate

CH2--COOH

Cl~

Cl

E
~ - / " ~ OCH2__COOH
2,4-Dichlorophenoxyacetic acid (2,4-D)

l-Naphthaleneaceticacid(NAA)

O
OCH~

~OCH2--COOH

CI

Dicamba

NH2~
CI

COOH

CH~--COOH

CI

Pichloram

Benzo(b)selenienyl-3 acetic acid

(BSAA)
FIG.1. Somenaturalandsyntheticauxins.

274

GASPARETAL.
.. CH2OH
CH3

NH ~

trans-Zeatin

NH ~ ~

CH3

2-iP
N6-(2-isopentyl)adenine
,. CH~OH
CH3

NH ~

tCHj

/ CH2OH
CH3

NH ~

~~_~CH2OH
OH OH

dihydro-Zeafin

Zeatin riboside
/ CH:-~
NH

Kinetin
6-furfurylaminopurine

BA
6-benzylaminopurine
or benzyladenine

PBA
6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine
FIG.2. Somenaturalandsyntheticcytokinins.

275

GROWTHACTIVESUBSTANCESIN CULTURE

SUBSTITUENT
ABBREVIATION
R,

NH

OH
--CH

R
R~

R2

R3

BA

Rib

[9R]BA

Rib-5'-P

[9R-5'PIBA

Glc

[9G]BA

(oOH)BA

Rib

(oOH)[9RIBA

CH3S

GIc

(oOH)[2MeS 9G]BA

OH

(mOH)BA
---CH

Rib

(mOH)[gRIBA

FIG.3. Naturallyoccurringaromatic cytokinins(after Strnad et al., 1992).

latter approach can be found elsewhere, e.g., most recently in Vasil

and Thorpe (1994) and Krikorian (1995). It is our belief that the type
of information to be presented here will be of benefit to all those who
use tissue culture technology as a tool in basic and/or applied research. However, to be as concise as possible, we have limited the
number of references (often citing reviews and our own research);
but at the beginning of each section on a specific phytohormone, the
reader is directed to one or two more in-depth reviews.

PHYTOHORMONES:GENERALCOMMENTS

Auxins, abscisic acid, cytokinins, ethylene, and gibberellins are

commonly recognized as the five main classes of naturally occurring
plant hormones. Auxins, cytokinins, and auxin-cytokinin interactions are usually considered to be the most important for regulating
growth and organized development in plant tissue and organ cultures,
as these two classes of hormones are generally required (see Evans
et al., 1981; Vasil and Thorpe, 1994). However, abscisic acid, ethylene, gibberellins, and other hormone-like compounds have regulatory roles which must not be ignored in culture systems. For instance, although one may not need to add abscisic acid, ethylene,
and gibberellins to cultured cells to ensure organogenesis or cell
proliferation, this does not mean that these plant hormones are of no
importance. Rather, these hormones are being synthesized in the
tissues and are playing an active, but hidden role in growth and
development. The added auxins and cytokinins will interact with
these other endogenous plant hormones. As well, so-called recalci-

trant tissues may well respond to exogenous application of these other

classes of hormones.
Synthetic compounds that act like natural plant hormones are
called "plant growth regulators" (Davies, 1995). Many such plant
growth regulators have been discovered with a biological activity
which equals or exceeds that of the equivalent endogenous hormones.
In addition to these useful compounds, there are now quite a number
of chemicals which interfere (generally inhibit) with the synthesis,
transport, or action of endogenous hormones. These inhibitors are
extremely helpful in the study of the role of plant hormones in in
vitro cultures.
In addition to the classical plant hormones, new natural growth
substances with regulatory roles in tissue cultures have been discovered in the last few years (Gross and Parthier, 1994). Examples
of these are polyamines, jasmonates, brassinosteroids, oligosaccharins, sterols, phosphoinositosides, salicylic acid, and systemins.
The effects of natural and synthetic plant growth regulators are
rarely specific in their ultimate influence on growth and development,
and the responses of cells, tissues, and organs in vitro can vary with
cultural conditions, the type of explant, and the genotype. Usually a
combination of two or more growth regulators of different classes is
required, either applied simultaneously or sequentially (e.g., see Evans et al., 1981). One must be aware that besides exerting a direct
effect on cellular mechanisms, many exogenously applied synthetic
and natural regulators may modify the synthesis, destruction, activation, sequestration, transport, or sensitivity to endogenous growth
substances of the same or other types (Beale and Sponsel, 1993;

276

GASPAR ET AL.

NI-ICONH~
1,3-diphenylurea
CI
2 C1-4PU or PPU
N-(2-ehloro-4-pyridyl)-N'-phenylurea

121

NI-ICONH~
t21
/

2,6 C1-4PU
N-(2,6-dichloro-4-pyridyl)-N'-phenylurea

N~S/

NHCONH~~>

Thidiazuron (TDZ)
N-phenyl-N'- 1,2,3-thiadiazol-5-ylurea
FIG. 4. Phenylureas with cytokinin activity.

Davies, 1995, p. 28-30). Any large change in growth and development requires the concerted and cooperative activities of many plant
hormones. Below we briefly present information on each class of
hormone and hormone-like substance.

AUXINS
(Bandurski et al., 1995 on metabolic aspects; Brock and Kaufman,
1991 on general physiology).
Auxins exert a strong influence over processes such as cell growth
expansion, cell wall acidification, initiation of cell division, and organization of meristems giving rise to either unorganized tissue (callus) or defined organs (generally roots) and promote vascular differentiation. In organized tissue, auxins appear to be key players in
maintaining apical dominance, affecting abscission, promoting root

formation, and tropistic curvatures, delaying leaf senescence, and

fruit ripening (Addicott, 1982; Sabater, 1985; Chandler and Thorpe,
1986; Liu and Reid, 1992; Aloni, 1995; Tamas, 1995).
Naturally occurring auxins, precursors and conjugates. The most
commonly detected natural auxin is indole-3-acetic acid (IAA), but
depending on the species, age of the plant, season, and the conditions
under which it has been growing, other natural auxins have been
identified such as 4-chloroindole-3-acetic acid, indole-3-acrylic
acid, indole-3-butyric acid (IBA) (Fig. 1). Although it has been
known for some time that IAA is commonly formed from L-tryptophan
via indole-3-pyruvic acid or tryptamine, other pathways exist (Bandurski et al., 1995). Auxin precursors, which might also have auxinlike properties by themselves, can sometimes replace IAA and may
be more effective than auxin itself in stimulating growth or inducing
organized development.
Within plant tissues, IAA and other naturally occurring auxins
combine with small molecules (alcohols, amino acids, sugars) to produce ester, amide, or glycoside conjugates (Bandurski et al., 1995).
This appears to be a mechanism for storing auxins in cells and stabilizing the level of free auxin by metabolizing the excess. Auxin in
conjugated molecules is protected from oxidative breakdown and may
be later enzymatically released when required. Indoleacetylaspartic
acid and conjugates with sugars, such as glucobrassicin (in Crucifereae), are commonly found (Fig. 1). There have been several reports
on the successful use of IAA conjugates as growth regulators in plant
cultures (Bandurski et al., 1995).
Auxin-like growth regulators. Synthetically-prepared IAA and IBA
are commonly used in plant culture media. They tend to be denatured
in media and rapidly metabolized within plant tissues. These attributes can be useful when developmental phases in progress require
less auxin (in rooting for instance, Gaspar et al., 1994), or in automatically changing the auxin:cytokinin ratio (organogenesis from
some callus). Several indole derivatives, both naturally occurring and
synthetic, are active in culture. For example, indole-3-acetaldehyde,
indole-3-acetamide, indole-3-acetonitrile, indole-3-acrylic acid, indole-3-1actic acid, indole-3-proprionic acid, indole-3-pyruvic acid,
and tryptophan, as well as phenylacetic acid have all been shown to
support callus growth and shoot formation in tobacco callus cultures
(Maeda and Thorpe, 1979).
Commonly used synthetic auxins in tissue culture are 2,4-dichlorophenoxyacetic acid (2,4-D; often used for callus induction
and suspension cultures), and 1-naphthaleneacetic acid (NAA;
when organogenesis is required; Fig. 1). Among others, dicambra
(3,6-dichloro-o-anisic acid) and picloram (4-amino-3,5,6-trichloropyridine-2-carboxylic acid) (used commercially as selective herbicides) are often effective in inducing the formation of embryogenic tissue or in maintaining suspension cultures (Gray and
Conger, 1985; Hagen et al., 1991). BSAA [benzo(b)selenienyl-3
acetic acid] is another synthetic auxin with powerful auxin-like
activities (Lamproye et al., 1990; Gaspar, 1995).
Regulation of levels and activity of auxins. Auxin activity is dependent on the free availability of boron. In boron-deficient plants,
both the translocation of IAA and nuclear RNA synthesis in response
to auxin treatment can be inhibited. Boron may affect rooting by
controlling IAA-oxidase activity (Jarvis et al., 1983). The responsiveness of tissues to auxins can also be modified by other hormones
such as cytokinins (Aloni, 1995) and ethylene (Liu and Reid, 1992).
For the regulation of at least some processes involving cell growth
and cell division, it is thought that auxins need to become bound to

277

GROWTH ACTIVE SUBSTANCES IN CULTURE

0 ~CH2 9

CH3

0 ~/CH2

CH3

GA1

0 ~CH2

GA4

A,0H

GAs

. ~'m ~0H

T ~, "-coo.
CH3

GA7

CH3

~0H

GA9

15/3 - OH GA3
H

.,OH

CH3

CH3

GA32
.~

oC.,

CH3

20

12/9 -OH GAs

~H3

18CH.CH.,

ent-gibberellane
skeleton
F]c-. 5.

c"'

CH3

12a -OH GAs

~ 0 H

T t, "-coo.

OI3

GAs

c1"12

CH3

CH3

GA3

OH

0~

Gibberellin structures (after Pharis et al., 1992).

....o.

CH3

15/3-OHGA5

278

GASPARET AL.

0~O00H
2

6 ~~CH
O

2OH

O'COOH 7oCHo
: ooo
oOOH
ooH

ocooH

oo.

Fro. 6. Structuralformulae ol ABA and analogs. 1, ( + )-ABA;2, ( + )-ABA;3, ( - )-ABA;4, (+)-7'OHABA; 5, ( )-PA; 6, ( - )-ABA
alc; 7, (+)-ABA aid; 8, (
9, (-)-2',3'-DHABA; 10, (+)-2',3'-DHABA (after Robertson et al., 1994).

auxin-binding proteins. Specific high-affinity binding proteins have

been found (Venis and Napier, 1991). However, only if these proteins
can be shown to be involved in the physiological action of auxin, can
they be truly called receptors.
The levels of endogenous free auxins depend on the rate of their
anabolism, catabolism, transport, and conjugation (Bandurski et al.,
1995). Increased endogenous concentrations of IAA, via synthesis,
generally depend on the quantity of precursors. Auxin biosynthesis
can also be influenced by treatments which alter internal ethylene
levels (galaetose among others). However, modification of the oxidation or conjugation rate appears to be an important way by which
internal IAA levels are naturally regulated. Phenolic inhibitors of
IAA oxidase such as phlorogluciuol. (or its derivative phloridi~.in)~
catechol, r
acid, and rutin have been shown to enhance
auxin activities, being sometimes described as "aux~n-synergists"
(Bearder, 1980). Activators of lAA-oxldase can behave as auxinantagonists.
Synthetic auxins might affect the level of endogenous auxin by
modifying directly (enzyme synthesis) and indirectly via (through

effectors) IAA-oxidase. Some synthetic substances behave as antiauxins in part by acting as inhibitors of auxin transport, but their
action is complicated and by no means fully understood. The most
frequently used compounds in cultures are TIBA (2,3,5-tri-iodobenzoic acid), PTAA [(3, phenyl-l,2,4 thiadiazol-5-yl)thioacetic
acid], NPA (N-l-naphthylphthalamic acid), 2,4,6-T (2,4,6-trichlorophenoxyacetic acid) and PCIB (p-chlorophenoxyisobutyric acid)
(see George, 1993).
CYTOKININ$
(Binns, 1994; McGaw and Butch, 1995).
Two major properties of cytokinins that are use(u[ in culture are,
stirnuta~io~ of cell division (often together with auxins) and release
of lateral bud dormaney. They can also induce adventilious bud formation (in cuttings and cultures) (Fabijan el al., 1981; Krikorian,
1995). Cell division is regulated by the joint action of auxins and
cytokinins, each of which influences different phases of the cell cycle. Auxins affect DNA replication, whereas cytokinins seem to exert

279

GROWTHACTIVESUBSTANCESIN CULTURE

OOS is recycled i ~
-+
CH3"S'CH2"CH2"(~
"NH3
H

AT~

+
Xppi
Pi

L-METmONINE

COO"
!
+

C H 3 - ~ - C H 2 - C H 2 - C - N H3
I
I
ADENOSINE
H

S-ADENOSYLMETHIONINE(AdoMet)
ACCsynthase AVG inhibits.
wounding& IAA promote.

ACC oxidase
(EFE)
Co++,low 02 inhibit.
CH2--CH 2

ETHYLENE

CH 2
|

C\cooNee

cb 2
Ascorbate
HC~
02
Dehydroascorbatc

~ malonyl
"~ansferase
n-MALONYLACC
(MACC)

FIG. 7. Biosyntheticpathway of ethylene. ACC = 1-aminocyclopropane-l-carboxylicacid; AVG = aminoethoxyvinylglycine;EFE

= ethylene formingenzyme.

some control over the events leading to mitosis (Vesely et al., 1994)
and cytokinesis. Thus, auxin and cytokinin levels in cultures need
to be carefully balanced and controlled. In intact plants, cytokinins
promote lateral bud growth and leaf expansion, retard leaf senescence, promote chlorophyll synthesis, and enhance chloroplast development (Kuhnle et al., 1977; Nood~n and Leopold, 1988). Little
is known about their mode of action.
Naturally occurring cytokinins. Cytokinins have been identified,
either as free compounds, glucosides, or ribosides. The most commonly used cytokinins in plant tissue culture are zeatin, 2-iP, dihydro-zeatin, and zeatin riboside (Fig. 2). These natural cytokinins
contain an isoprenoid side chain attached to the N6-position of the
adenine. Cytokinins with an aromatic ring substituting at N6 (6-benzyladenine and its glycosides) have been identified recently (Fig. 3).
Cytokinin-like growth regulators. The most commonly used cytokinins are the substituted purines: kinetin and BA (Fig. 2). Adenine
(vitamin B4) , adenosine, and adenylic acid may have cytokinin activity, although less than that of the cytokinins. Adenine can be used
to bring about or reinforce response normally attributed to cytokinin
action. The fungicide benomyl, which has a structure similar to that
of adenine-based cytokinins, can also be used as a cytokinin but may
have damaging effects under certain conditions (Woo and Wick,

]995).

Many substituted ureas (Fig. 4) have cytokinin activity (Krikorian,

1995). In some plants, thidiazuron (registered as a cotton defoliant)
is more effective than adenine-based compounds fordnducing axillary or adventitious shoots. Such shoots, however, tend not to elongate
sufficiently and are susceptible to rapid hyperhydricity on repeated
subcultures in the presence of this compound.
Regulation of activity and levels of cytokinin. The pool of active
cytokinins present at any one time is a product of a number of dynamic processes: biosynthesis, formation or mobilization of storage
forms (sequestered or O-glucoside forms), and deactivation (N-glucosylation, alanine conjugation and side chain removal by cytokinin
oxidase) (McGaw and Burch, 1995).
Many aspects of cell growth, cell differentiation, and organogenesis in tissue and organ cultures have been found to be controlled
by an interaction between cytokinins and auxins. The requisite concentration of each phytohormone varies greatly according to the kind
of plant being cultured, the cultural conditions, and the form of the
phytohormone used. Although both auxin and cytokinin are usually
required for growth and morphogenesis, auxin can inhibit eytokinin
accumulation, whereas cytokinins can inhibit at least some of the
actions of auxin.
Some of the effects of cytokinins on plant metabolism, physiology,
and development can be antagonized by abscisic acid. In many tis-

280

GASPAR ET AL.

Arginino-succinate- ~

Citrulline

i Ornithine cycle!

Arginine

Ornithine
S-adenosyl-methionine
(AdoMet)

DRVIO

Agmatine

9--~ MGBG
~P'
S-adenosyl-methionine
decarboxylated

N-Carbamoylputrescine
PUTRESCINE
I ~AG
CI_IA_L_
~
.... d-Pyrroline--~ GABA

Diaminopropane, - ~

Alanine

SPERMINE~
Aminopropylpyrroline
FtG. 8. Schematic outline of the pathwaysof polyamine synthesis and catabolism (see Hausman et al., 1994). AG, aminoguanidine;
CHA, cyclohexylamine;DFMA, a-difluoromethylarginine;DFMO, u-difluoromethylornithine;GABA,gamma aminobutyric acid; MGBG,
methylglyoxal-bis-guanylhydrazone.

sues, including cultured tissues, cytokinins often promote ethylene

biosynthesis (Gamborg and LaRue, 1971; Wright, 1979; Abeles et
al., 1992, p. 79).
The ability to stimulate cytokinin oxidase appears to be a property
of a limited range of phenolic structures such as 2,6-dimethoxyphenol and acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone). The activity of acetosyringone is of particular interest because
this compound is reported to serve as a signal compound in the
interaction of the plant pathogen Agrobacterium tumefaciens with its
higher plant hosts. Acetosyringone and related phenolics released
by host plants stimulate the cytokinin production associated with the
tzs gene of the vir region in nopaline strains of Agrobacterium (John
and Amasino, 1988; Powell et al., 1988). Thus, the cytokinin production controlled by the vir region of Agrobacterium is regulated by
a compound that may play a role in cytokinin degradation by the
host plant.

Cytokinin antagonists. Cytokinin antagonists are compounds

structurally related to the phytohormone and may bind to common
sites. Selective suppression of multiple effects of cytokinins such as
callus growth and adventitious bud formation, with antagonists may
tell us more about the individual functions of cytokinins and possible
different binding sites. Both adenylate anti-cytokinins (pyrazolo[4,3d]pyrimidines, pyrrolo[2,3-d]pyrimidines) and nonadenylate anti-cytokinins (N-benzyl-N'-phenylureas, s-triazines, N-arylcarbamates)
have been developed (Iwamura, 1994). Cytokinin effects can also be
antagonized by metabolic inhibitors such as 8-azagnanine and 8azaadenine.
G1BBERELLINS
(Sponsel, 1995 on metabolism; Brock and Kaufman, 1991 on more
general physiology).

281

GROWTH ACTIVE SUBSTANCESIN CULTURE

CH=OH

Salicylic acid
(SA)

CH=OH

Salicyl alcohol-13-D-glucoside
Salicin
CO.OCH~

COOH
,- O-CO-CH,

Acetyl salicyhc acid

Aspirin (ASA)

Methyl salicylate
oil-of-wintergreen

CH2-COOH
I

CO- NH- CH2.COOH

HO~

COOH
CH2 -- C H 2 ~ O H

N-salicyloyl aspartic acid

Lunularic acid

CO - OCH3

CO - OCH3
O - Glucose

Methyl salicylate-O-13-glucoside
a Gaulterin
CO - OCH3

OH

CH 2

O - [GI.co~.xylose]

Monotropitoside
a Gaulterin

OH OH

Methyl salicylate arabinoglucoside

violutin
FIG. 9. Somenaturally occurring derivatives of salicylic acid.

282

GASPAR ET AL.

~~~/~/~/~/COOH
ILinolenic acid 1

Lipoxygenase

[ 13-Hydroxyperoxylinolenic acid I

Hydroperoxi
J
d
e
dehy~~''~
112-Oxo-phytodienoic acid [

~Reductase

HOOCk~v"

j3-oxidation

~z~k/~/\

CO0 H

[ Traumatic acid I

Jasmonic acid
FIG. 10. Formationofjasmonic and traumatic acids from linolenic acid.

Gibberellins (GAs) can promote flowering (particularly in species

that require long days and/or cold), cone initiation in some conifers,
seed germination, and stem elongation (by increasing cell division
and elongation). The ability to promote bolting and stem elongation
can be exploited in vitro for the elongation of the shoots of woody

species before rooting. Some GA effects are caused by increases or

decreases in the biosynthesis and activity of specific enzymes (e.g.,
increases in levels of aleurone hydrolytic enzymes).
When GAs are added to plant tissue culture media, they often
diminish or prevent the formation of roots, shoots, or somatic era-

GROWTHACTIVESUBSTANCESIN CULTURE
bryos, although the opposite has also been seen. Some endogenous
GAs may be necessary for normal callus growth (Lance et al., 1976b),
and inhibition of GA biosynthesis can influence development of cells
in liquid cultures (Ziv and Ariel, 1991). Some differences in the
inhibition or promotion of adventitious root and shoot formation by
GAs might be due to the fact that GAs inhibit meristemoid initiation
(Thorpe and Murashige, 1970) but are required for assisting the further growth and development of preformed organs. GAs may also alter
the availability of endogenous auxin. Growth of shoots in meristem
and shoot cultures may be enhanced by addition of GA (Fry and
Street, 1980).

Naturally occurring gibbereUins and regulation of levels and activity. About 90 naturally occurring GAs are known. Figure 5 shows
some typical structures and the scheme for the numbering of carbon
atoms in the ent-gibberellane skeleton. Native GA-O-glucosides are
considered a potential source of free GAs. Cultured callus cells synthesize their own GA and can metabolize added GA (Lance et al.,
1976a). Because anti-GAs (see below) alter callus growth rate, these
endogenous GAs may normally influence growth of cultured cells.
There is good evidence of a structure/function relationship for GAs
in floral induction versus effects of GAs on stem elongation. A highly
florigenic GA is one which has 19 carbon atoms; C-7 is a carboxyl
group, ring A is A ~ or A2,3 and ring C is hydroxylated (C-13), or
ideally rings C/D are polyhydroxylated (C-12, 13 and/or 15). Conversely, dihydroxylation (C-313, C-13) with ring A saturated gives a
GA which greatly promotes stem elongation but has low florigenicity
(Pharis et al., 1992). No such relationship has been established for
the effects of GAs in vitro, which may depend upon the plant species/
family in question. The sometimes observed antiflowering effect of
applied GAa and GA7 has been attributed to their long biological
half-life.
GAs have numerous interactions with other hormones. GA-induced a-amylase activity is antagonized by ABA. Ethylene blocks
(or in rice, promotes) the ability of stems to respond to GAs. GA
antagonizes the senescence promoting effects of ABA and ethylene
in leaves and petals. Light conditions (wavelength and photoperiod)
affect GA metabolism; however, the precise mechanism still requires
much study (Reid et al., 1991).
Anti-gibberellins and growth retardants. There are many chemicals
that effectively inhibit GA biosynthesis. Some can have promotory
effects on cultures in which supraoptimal endogenous levels are inhibitory to growth and development but conversely can slow culture
growth in cultures without added GA. Anti-GAs may sometimes promote root formation. Although chlormequat (CCC), AMO 1618, ancymidol, and related compounds are effective inhibitors of GA synthesis, their action is not absolutely specific, and they have toxic side
effects related to GA biosynthesis (Fabijan et al., 1981). If such side
effects have not occurred, one can reverse the effects of the inhibitor
with an exogenous supply of GA. Inability to obtain reversal indicates
that the observed effects may not be solely due to reduced GA synthesis. The mode of action of other compounds (e.g., diaminozide) is
less clear. The triazole growth retardants (placlobutrazol, triadimefon, tetcyclacis, triadimenol) also are all potent inhibitors of GA synthesis but can also interfere with sterol and abscisic acid production
(Rademaeher, 1992). Ancymidol and triazoles protect plants from
some stresses (drought, extreme temperatures, and gaseous pollutants). The problem of hyperhydricity can be overcome often by reduction or prevention of leaf development, coupled with the forma-

283

tion of compact bud aggregates through the use of growth retardants

(Ziv, 1992).
ABSCISICACID
(Walton and Li, 1995 on metabolism; Davies and Jones, 1991,
general review).
Abscisic acid (ABA) and other structurally related natural compounds with similar activity (Fig. 6) are most likely produced by the
cleavage of xanthophyll. ABA is often regarded as being an inhibitor,
as it maintains bud and seed dormancy, inhibits auxin-promoted cell
wall acidification loosening, and slows cell elongation. ABA plays a
key role in closing of stomatal apertures (reducing transpiration),
control of water and ion uptake by roots (in part by increasing hydraulic conductivity) and, with other phytohormones, promoting leaf
abscission and senescence. ABA is important in seed maturation, as
it induces the synthesis of storage proteins in developing seeds. It
acts antagonistically to GAs in many systems. ABA, along with ethylene and jasmonic acid, aids in defense against insect wounding.
With ethylene, ABA is intimately involved with plant responses to a
wide range of environmental stresses.
In tissue cultures, exogenously applied ABA can affect (generally
positively at low concentrations, while high concentrations inhibit)
callus growth and organogenesis (buds, roots, embryos). Some ABA
is essential for the maturation and normal growth of somatic embryos
and only in its presence do they closely resemble zygotic embryos in
their morphological and biochemical development (e.g., Roberts et
al., 1990; Rock and Quatrano, 1995). Manipulation of endogenous
and/or exogenous ABA levels increases the frequency of embryos
reaching maturity (Label and Lelu, 1994 and references therein).
ABA increases freezing tolerance of axenically grown plants and cell
cultures.
Occurrence, activity, and antagonists. In nature, the most common
form of ABA is (S)-(+)-abscisic acid (Fig. 6, formula 2). This is
often called the c/s isomer or simply ABA. The so-called trans isomer
has the carboxyl group on the end of the side chain oriented in the
other direction. The cheaper ABA often sold commercially is a 1:1
mixture of the c/s- and the trans-ABA optical isomers. For rapid
responses, such as stomatal closure, only cis-ABA is active, but for
slower responses, both isomers are effective. Exposure of UV light
converts some of the c/s-ABA to the trans form.
Some effects of ABA on cultured plant tissues suggest that it may
modify cytokinin synthesis or activity. In other instances (where it
opposes the effects of phenols), it might enhance the IAA oxidation
which many phenols appear to prevent.
Fluridone and norfurazon, inhibit ABA synthesis, but unfortunately their action is not specific to ABA, as they act as inhibitors
of carotenoid biosynthesis (Henson, 1984; Gamble and Mullet,
1986). Recently, Wilen et al. (1993) have found that stereoisomeric
analogs of ABA are effective specific antagonists of ABA action.
ETHYLENE
(Abeles et al., 1992; Kende, 1993).
In conjunction with other phytohormones, this gas promotes fruit
ripening, senescence, and leaf abscission. There is often a self-adjusting balance between natural auxin and ethylene levels. Some of
the responses of plants to auxins may be caused by increased ethylene synthesis in response to auxin treatment. At higher concentra-

284

GASPARET AL.

tions the gas appears to alter microtubule and microfibril orientation

which results in decreased cell elongation but increased cell expansion (Apelbanm and Burg, 1971; Steen and Chadwick, 1981). Depending upon the time after subculture, ethylene can stimulate or
inhibit growth and organogenesis in in vitro cultures (Huxter et al.,
1981). Ethylene can specifically affect growth of callus and suspension cultures, stem and root elongation, axillary and adventitious bud
formation, rooting, and embryogenesis. The role of ethylene can be
difficult to understand because it effects vary with developmental
stage and because low concentrations can promote (or sometimes
inhibit) a process, whereas higher levels have the opposite effect.
Also, it may be difficult to interpret the literature because some
researchers use only one concentration of the phytohormone. Full
dose-response curves should always be developed. Another complication is that ethylene can interact with other gases present in the
head space of the culture such as O2 and C02 (Kumar et al., 1987).
However, regulating the levels of the head space gases can increase
beth shoot formation and shoot multiplication (Kevers et al., 1992;
Nour and Thorpe, 1994). Low oxygen promotes the synthesis of the
ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC)
but inhibits conversion of ACC to ethylene. High 02 stimulates the
conversion of ACC to ethylene. Very high COx blocks ethylene action.
Chemicals that promote synthesis and releaseof ethylene. Ethephon
(Ethrel; 2-chloroethylphosphonic acid; or 2-CEPA) can be used as
an ethylene-releasing chemical in tissue cultures. It releases ethylene below pH 5.0 (Abeles et al., 1992); however, once exposed to
cytoplasmic pH, ethylene is evolved. Thus, we have found if it is
added to culture media at pH values above 5.0, the ethylene is
quickly dissipated.
Ethylene-absorbing chemicals. Ethylene in the culture vessel atmosphere can be absorbed by solutions of potassium permanganate
or mercuric perchlorate. Purafil, a bead-like material which contains
potassium permanganate and other similar agents, is a good scrubber
of ethylene in gas streams that are delivered to the cultures (Abeles
et al., 1992, p. 292).
Regulation of ethylene production and activity. As ethylene is produced from all plant cultures (and sometimes from recently autoclaved agar medium) it accumulates in the head space in sealed
culture vessels. Cultured tissues can be affected by this gas, the
generation of which depends upon the type and weight of tissue being
grown, volume of the culture vessel, and the manner in which it is
sealed, and culture conditions (Kumar et al., 1987; Kevers et al.,
1992). Auxins usually stimulate ethylene production, and cytokinins
may block ethylene action; however, a synergism between auxin and
cytokinin in ethylene production has been observed (Gaspar et al.,
1989).
Ethylene is synthesized from methionine (Fig. 7) which in turn is
converted to S-adenosylmethionine (AdoMet) and then ACC. Adding
ACC to plant cultures usually increases ethylene production because
ACC often is the limiting factor. Adding methionine may have the
same effect, and as methionine can be produced in plants from aspartic acid, asparagine, and cysteine, their presence may have a
similar effect as well. Because the genes for ACC synthase and ACC
oxidase have been cloned, it is possible to modulate ethylene synthesis through the use of mRNA antisense and similar techniques
(McKeon et al., 1995).
Useful inhibitors of specific steps in ethylene biosynthesis include: (a) aminoethoxyvinylglycine (AVG) which inhibits ACC-syn-

thase (excess AVG causes necrosis and other unwanted side effects
unrelated to ethylene), (b) a-aminoisobutyric acid (AIBA) and cobalt
ions which inhibit ACC oxidase (EFE) and prevent the conversion
of ACC to ethylene, and (c) n-propyl gallate, ioxynil, bromoxynil,
3,4,5-trichlorophenoI, polyamines, and salicylic acid which can, in
some situations, inhibit the conversion of ACC to ethylene. Also,
ethylene production ceases when oxygen supply is reduced and oxidation inhibitors are present, because oxygen is required to convert
ACC to ethylene (Abeles et al., 1992).
Several chemicals can inhibit ethylene action, perhaps by preventing the gas binding with its active site. 2,5-Norbornadiene (NBD)
and silver ions (from silver thiosulfate, STS) or silver nitrate) very
effectively block ethylene action (Abeles et al., 1992). STS is preferred as it produces less toxic symptoms and is more mobile in the
plant (Veen and van de Geijn, 1978; Liu et al., 1990). Other inhibitors of ethylene action are chelating agents, such as i-hydroxyquinoline and diethyldithio-carbamic acid, and high concentrations of
carbon dioxide.
POLYAMINES
(Evans and Malmberg, 1989; Galston and Kaur-Sawhney, 1995).
Less is known about polyamines (PAs) than other phytohormones,
and not all researchers are prepared to classify them as hormones.
They require higher concentrations to produce an effect than do the
more traditional plant hormones. However, PAs seem to be involved
in a wide range of growth and developmental phenomena, as tissues
that are deficient in PAs are abnormal. In particular, aliphatic PAs
like putrescine, spermidine, and spermine have been shown to enhance morphogenesis in many cultured tissues (Bagni et al., 1993;
Turbicio et al., 1993).
At a physiological pH, PAs act as polycations and complexing
agents. They bind strongly to phospholipid groups and other anionic
sites on membranes (thus affecting membrane fluidity), cell wall
polysaccharides, and to RNA and DNA. Nucleic acids are stabilized
by their association with PAs. Free PAs can compensate for ionic
deficiencies or the damaging effects of some stresses on membranes.
Part of their function may be to act as buffers to minimize fluctuations
in cellular pH, to modulate enzyme activities, and enhance DNA
replication and transcription. PAs appear to be involved in cell division, cell elongation, and rooting and in certain circumstances can
be used as a substitute for auxin treatment, which has led some to
consider them as secondary messengers.
Through the analyses of the variations of their endogenous levels
and the use of inhibitors of their biosynthesis in in vitro cultures,
PAs have been found to be involved in pollen maturation, adventitious vegetative shoot formation, adventitious rooting, and somatic
embryogenesis (Bagni et al., 1993; Turbicio et al., 1993; Galston and
Kaur-Sawhney, 1995). There is also good evidence for the role in
flower development (Rastogi and Sawhney, 1990). It has been argued
that PAs might be active, not by themselves, but through their catabolic pathway and the generation of H202 (Hausman et al., 1994).
Naturally occurring PAs, precursors, and conjugates. The most
common of the PAs are putrescine (a diamine), spermidine (a triamine) and spermine (a tetraamine). They are formed from arginine
and ornithine (Fig. 8). PAs conjugate with phenolic acids such as
cinnamic, coumaric, caffeic, and ferulic derivatives (Smith, 1985).
Regulation of PA synthesis and activity. The biosynthesis of PAs
is influenced by the medium used for in vitro culture; biosynthesis

285

GROWTH ACTIVESUBSTANCESIN CULTURE

is greater on ammonium-based medium than when nitrogen is supplied as nitrate ions. Biosynthesis is generally enhanced by the presence of auxins, cytokinins, and GA (Slocum and Flores, 1991).
Known inhibitors of specific stages in the PA biosynthetic pathway
are illustrated in Fig. 8: AG (amino-guanidine), CHA (cyclohexylamine), DFMA (DL-a-difluoromethylarginine), DFMO (adifluoromethylornithine), MGBG (methylglyoxal-bis-guanylhydrazone), and CHA (cyclohexylamine); and these have proven useful in
examining the role of PAs in growth and organized development.
Ethylene can diminish the rate of PA synthesis by reducing the
activity of arginine decarboxylase. PAs themselves inhibit ethylene
biosynthesis by blocking the conversion of ACC to ethylene, possibly
because they can act as free radical scavengers. They may also interfere with ethylene action. This mutual antagonism may in part
have arisen because PAs and ethylene share the common precursor
S-adenosylmethionine. Techniques are also available to study PA
catabolism (Aribaud et al., 1994).
OLIGOSACCHARINS
(Ryan and Farmer, 1991; Darvill et al., 1992).
Oligosaccharins are oligosaccharides (short chains of sugar residues connected by glycosidic linkages) and are cell wall fragments
which, at low concentrations, exert biological effects on plant tissues
other than as carbon or energy sources. Along with the polypeptide
systemin, some oligosaccharins (the oligogalacturonides) initiate
some aspects of a plant's response to attack by pathogens and insects.
However, other oligosaccharins have effects on development and
morphogenesis (Tran Thanh Van et al., 1985) that are not obviously
related to disease resistance.
Some oligosaccharins inhibit endogenous growth and the growth
induced by auxin and H +, GA~, and fusicoccin. Some of them have
been shown to interact with auxin-binding sites at the plasma membrane level. Another set of oligosaccharins can promote elongation
in the absence of auxin in some biological tests. They may be substrates for xyloglucan endotransglycosylases.
Effects observed in tissue cultures include (Tran Thanh Van et
al., 1985; Tran Thanh Van and Trinh, 1990): (a) promotion of callus
proliferation (in the presence of auxin), (b) increase in the number
of adventitious roots in cultured wheat embryos (in the absence of
auxin), (c) control of organogenesis in thin cell layers of tobacco,
(d) inhibition of auxin-stimulated rooting in tobacco leaf explants,
(e) inhibition of auxin-dependent embryogenesis in carrot cultures,
and (f) restoration of the ability of nonembryogenic (mutant) carrot
cells to undergo somatic embryogenesis (the effect is also obtained
by the addition of a specific 32-kDa isoenzyme of chitinase).
Some of these effects again are in agreement with the idea that
oligosaccharins are anti-auxins. This may be justified partly through
the model presented by Aldington and Fry (1993) to explain the
oxidative burst evoked by oligosaccharin elicitors. According to this
model, any component which interferes with receptor-reductase coupling or which consumes electrons, or which blocks the oxidase
would be expected to inhibit both the bleaching and transmembrane
signalling functions of the oxidative burst. Interacting primarily with
the plasma membrane, it also appears plausible that some oligosaccharins secondarily induce a change in intracellular free Ca2 maybe
through the generated H202 (Price et al., 1994). Certain protein kinases are Ca2+-dependent, so the tertiary effect could be on protein
phosphorylation. Ca2 also regulates the phosphatidylinositol phos-

phate signalling system. Also it should be noted that specific oligosaccharins are the "nodulation factors" produced by Rhizobium.

SALICYLATES
(Raskin, 1992; Pierpoint, 1994).
Salicylic acid (SA, o-hydroxybenzoic acid) and its derivatives (glycosides of SA, esters of SA, glycosides of SA esters, and other salieyl
alcohol derivatives) (Fig. 9) belong to the large group of plant pbenolics and are probably ubiquitous in higher plants. SA probably is
biosynthesized from cinnamic acid.
The most typical biological responses of SA are: (a) the induction
of flowering (but it is debated whether SA is a specific regulator of
flowering), (b) the induction of tuberization in vitro (although they
are less active than jasmonates), (c) induction or regulation of thermogenesis (heat production during flowering of thermogenic plants
like Arum lilies), (d) systemic signal for the induction of disease
resistance, in particular against necrotrophie pathogens [the action
of SA is likely mediated by elevated amounts of H202, since it specifically inhibits catalase activity in vitro and induces an increase of
H202 concentration in vivo (see Chen et al., 1993)], and inducer of
certain pathogenesis-related (PR) proteins, and (e) inhibition of germination. SA is leached from the leaves and bark of certain trees,
where it restricts regeneration beneath their canopies and therefore
may have an allelopathic effect.
The specific roles and interactions of SA, oligosaccharins, ethylene, and jasmonates in wounding and pathogen attack still need
much research. However, a parallel often is made between the SA
control (inhibition) of the synthesis of the animal hormones prostaglandins (enzymatically derived from fatty acids following injury or
hormonal stimulation) and the generation of jasmonic acid in
wounded plant tissues. This might suggest that the role of SA in
coordinating plant defense gene expression is indirect. It is known,
on the one hand, that SA is not the translocated signal responsible
for inducing systemically acquired resistance, but SA is required in
signal transduction. On the other hand, plant defense genes are synergistically induced by ethylene and methyljasmonates.

JASMONATES
(Sembdner and Parthier, 1993).
Following early detection and chemical identification from Jasminum, Rosarinus and other plants containing fragrant essential oils,
jasmonic acid (JA) and in particular its volatile methylester (MeJA)
have been detected in many species. The two physiologically active
compounds, (-)-jasmonic acid and (+)-7-iso-jasmonic acid, are
stereomerie forms normally found in a molar ratio of 9:1. Both can
be metabolically transformed. The biosynthetic pathway starts with
linolenic acid and is synthesized by the sequential action of several
enzymes starting with lipoxygenase (Fig. 10). Many naturally occurring jasmonates have been identified (Sembdner and Panthier, 1993).
Wounding and pathogens can promote JA synthesis as follows: sytemin and oligouronides act as signals which eventually activate lipases, releasing linolenic acid and leading to JA formation. The jasmonates then activate genes for the formation of stress proteins
(different from the PR proteins) such as proteinase inhibitors. In
some cases JA also promotes ethylene synthesis (Emery and Reid,
1996).

286

GASPARET AL.

Jasmonates are thus involved in the cellular transduction processes between external stress (herbivore, pathogen, desiccation, mechanical, or osmotic stresses) and macromolecular stress responses
involving the expression of "defense genes" and production of JAinduced proteins (JIPS) (Reinbothe et al., 1994). Jasmonates display
a multiplicity of effects in plants (Gross and Parthier, 1994) such as
the promotion of leaf senescence, abscission, fruit ripening, tendril
coiling, and tuber formation (tuberonic acid or 12-hydroxylated jasmonic acid is very effective here). Jasmonates also inhibit seed germination and stem elongation. As inducers of vegetative storage protein gene expression, jasmonates also stimulate bulb formation in
vitro.
Jasmonic acid has been shown to retard callus formation, inhibit
rhizogenesis, and promote bud formation in cultured potato meristems (Ravnikar and Gogala, 1990). In recently completed studies,
we found that exogenous JA (106-10 -4 M) caused fewer and shorter
shoots in cultured radiata pine cotyledons, tobacco callus, and tobacco leaf disks (Tampe, P. A.; Reid, D. M.; Thorpe, T. A., unpubl.).
Interestingly, it also caused non-shoot-forming tobacco callus to form
shoots. JA interfered with the development of radiata pine meristemoids into shoot primordia. In cultured roots of tomato, low concentrations of JA promoted the frequency of lateral root initiation and
elongation, whereas high concentrations (10 -s M) inhibited root
growth and reduced lateral root formation (Tung, P., et al., in press).
JA-treated roots produced more ethylene than controls, but inhibitors
of ethylene action and synthesis did not block the JA effects. Clearly
more studies with JA and in vitro cultures are needed.
Traumatic acid is another product of lipid peroxidation and like
JA, is derived from linolenic acid (Fig. 10). As a wound response,
traumatic acid is thought to modify auxin levels or auxin activity.
MISCELLANEOUSSUBSTANCESWITH
PHYTOHORMONAL-LIKEACTIVITY

A variety of compounds synthesized by plants and microorganisms

have been shown to possess growth active properties. These include
compounds such as systemin (Ryan, 1992), sterols (Mandava, 1988),
brassinosteroids (Milborrow and Pryce, 1973; Gross and Panthier,
1994), and turgorins (Kallas et al., 1990).
Systemin is an 18 amino acid polypeptide which is synthesized
after wounding. Unlike the oligosaccharins, it is transported away
from the site of wounding. It probably causes the release of linolenic
acid from membranes leading to increased jasmonic acid, which then
promotes production of proteinase inhibitors at sites distant from the
original site of wounding (Ryan, 1992).
Some sterols and brassinosteroids have been shown to have growth
regulatory properties and can be active at low concentrations (Mandava, 1988; Arteca, 1995). Sterols (or steroid alcohols) are triterpenoids with three six-membered rings (A, B and C) and a five-membered ring (D). Brassinosteroids (BRAs) are steroid derivatives with
a structure fairly similar to that of the sterols, although some BRAs
have a seven-membered oxolactone B ring. The sterols, stigmasterol,
and vitamins D2 and Ds have been found to promote the rate of
axillary proliferation and to stimulate root formation. Sterols may
modulate auxin action, alter the sensitivity of cells to auxin, and
influence auxin transport or oxidation.
The brassinosteroids (BRAs) consist of a group of natural products
biologically active at very low concentrations typical of those of hormones (Gross and Panthier, 1994). The steroids, brassinolide, cas-

tasterone, and 6-deoxodolichosterone are the best known among over

30 characterized BRAs. Pollen contains relatively high concentrations (about 100 ~g/kg), whereas the content of other plant tissues
is in the nanogram range. BRAs have many effects on growth and
development such as promotion of stem elongation, inhibition of root
elongation, and promotion of epinasty and ethylene synthesis. BRAs
often, but not always, act synergistically with auxins. BRAs also
protect against disease, cold, and salt stresses.
Dehydrodiconiferyl alcohol glucosides (DCGs) may be derived
from the aromatic alcohol coniferyl alcohol (Orr and Lynn, 1992).
DCGs have been isolated from transformed Vinca rosea tumor cells,
and they can replace the cytokinin requirement for growth of tobacco
pith and callus cells in culture. In tobacco pith cells, cytokinin treatment greatly promotes the concentrations of DCGs. For DCG synthesis, coniferyl alcohol is dimerized by a soluble intracellular peroxidase and then glycosylated. Breakdown of DCGs takes place
slowly. In the cotyledons of Pharbitis nil, DCGs accumulate during
the induction of flowering by low temperature treatment (Hirai et al.,

1994).
Turgorins (Kallas et al., 1990) are signalling molecules which induce nastic movements in touch-sensitive plants such as Mimosa.
They are 4-O-glycosides of gallic acid (which has close chemical
similarity to salicylic acid), protocatechuic acid and p-hydroxybenzoic acid, with the 6-hydroxy group of the glucosyl moiety being
esterified with sulfuric acid. Turgorins bind to plasma membranes.
We are unaware of any specific effect of these compounds on in vitro
cultures.
Fungal products such as fusicoccin and plant products strigol,
sorgoleon, alectrol, and sorgolactone (sesquiterpene lactones) are
germination stimulants (Bewley and Black, 1982). Strigol and chemically related compounds (Cook et al., 1972) are exuded by host roots
and break the seed dormancy of the root-parasitic plants such as
Striga.
There are of course many other compounds (some synthetic) which
may be of use in investigations of growth and organized development
in plants cultures. For example, the herbicide glyphosate might be
useful as it blocks the shikimate pathway and thus inhibits tryptophan synthesis (Jensen, 1986). Short dipping treatments or addition
of glyphosate to culture media can cause the development of more
axillary and adventitious shoots (George, 1993, p. 469).
Growth of most plant cultures is improved by the addition of myoinositol to the medium, although there are few reports of it having a
regulatory role in morphogenesis (Thompson and Thorpe, 1986). The
majority of exogenous myo-inositol is incorporated into phosphatidylinositol, which is considered to be an important factor in the functioning of membranes. The phosphatidyl cycle controls various cellular responses to physical and chemical, biotic or abiotic signals by
generating secondary messengers such as myo-inositol-l,4,5-triphosphate (Cot6 and Crain, 1993). It is becoming clearer that plant
hormones and regulators (including toxins and elicitors) mediate
their effects through transduction and amplication pathways somewhat similar to those seen in animals (Libbenga and Mennes, 1995).
Finally, there are a large number of naturally occurring chemicals,
many of microbial origin, which have been found to promote or inhibit processes such as seed germination and root initiation at concentrations of 10 -4 M and below. To date, over 300 microbial and
so-called secondary plant products processing bioregulatory activity
in plant systems have been detected and described (Gross and Panthier, 1994). This article describes compounds isolated since 1990.

GROWTH ACTIVESUBSTANCESIN CULTURE

Earlier compounds can be found in other reviews (e.g., Bearder,
1980; Gross, 1991). Compounds such as capillarol (a cinnamic acid
derivative) from Artemisia; sequiterpene lactones from a variety of
plants, including Artemisia and Podocarpus; "G-inhibitors" from
leaves of the Myrtaceae; acremoauxin A (o-arabitol ester of 3-indolylpropionic acid) from the fungus Acremonium; 2-hydroxyl-l'-methylzeatin from Alternaria sp; and 6-acetoxylinoleic acid from the alga
Spatoglossum pacificum, are but a few. We are not aware of any
reports on the effects of these substances on in vitro cultured tissues.
CONCLUSIONS
Many hormone-like growth regulators have now been identified,
but despite the long list of their effects and the progress that has
been made in elucidating some of the pathways of signal transduction, we have still a long way to go before we fully understand the
complex interaction between plant hormones. The growth and development processes in vivo and in vitro are excessively complex and
composed of interdependent physiological phases (competence, induction, determination, initiation, expression) which have different
requirements. In these processes a single phytohormone can, over
time, more than once play the role of inducer/stimulator or inhibitor
as tissues move through phases of changing sensitivity to specific
hormones.
A further complication, making it difficult to determine the specific effects of any particular phytohormone, is that one hormone
appears to be able to modify the synthesis of other classes of hormones. This may be effected via a direct influence of phytohormone
A on the enzymes of synthesis/metabolism of phytohormone B. Alternatively, the effect may be indirect. For example, phytohormone
A may alter the overall growth rate or change the direction of development, which in the long term, feeds back and adjusts the balance
of all other phytohormones. In essence, because the tissues are now
growing at a different rate or are in a different developmental state,
the cells are required to modify the manufacture of all phytohormoRes.
Recently much has been said about "sensitivity" of tissues to plant
hormones (see Davies, 1995) and this is clearly a factor that needs
much more investigation. However, "sensitivity" is difficult to define
and not easy to measure in a meaningful way. For example, an increase in the numbers or receptiveness of phytohormone receptors,
a decrease in phytohormone breakdown, a reduction in metabolism
to conjugated or inactive forms, and a reduction in transport away
from the active site could all be interpreted as an increase in sensitivity to an exogenous phytohormone.
Nevertheless, for cells in culture, an array of growth active substances which may be used to modify their growth and differentiation
(biochemical and/or morphogenic) is available. This allows for systematic empirical studies to be conducted, particularly with recalcitrant tissues that do not respond to the classical phytohormones.
Although auxins and cytokinins will continue to be the major plant
hormones used in tissue culture, it is clear that there are many other
growth active substances that can be used to enhance growth and
differentiation in vitro. As pointed out in this article, the effects of
many of these potential phytohormones have not yet been examined
under in vitro conditions.
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