Indian Forester, 142 (5) : 459-470, 2016 ISSN No.

0019-4816 (Print)
http://www.indianforester.co.in ISSN No. 2321-094X (Online)
RECENT ADVANCES IN UNDERSTANDING THE ROLE OF GROWTH REGULATORS IN PLANT GROWTH AND
DEVELOPMENT IN VITRO - I. CONVENTIONAL GROWTH REGULATORS

SURESH KUMAR1, ROHTAS SINGH2, SANJAY KALIA3, S. K. SHARMA4 AND RAJWANT K. KALIA

Central Arid Zone Research Institute, Jodhpur 342003, India

Email - rajwantkalia@yahoo.com, rajwantkalia@cazri.res.in
ABSTRACT
Growth regulators, a diverse array of organic compounds, are critical components in determining developmental
pathways in plants. They interact at the cellular level to produce physiological and morphological effects. Our
understanding about transport, metabolism and mode of action of growth regulators in plants has considerably
increased in the recent years. Discovery of the chemicals that interfere with synthesis, transport and action of
endogenous growth regulators have further improved our knowledge regarding the role of plant growth regulators
(PGRs) in plant's growth and development. A number of PGRs are being used in plant cell, tissue and organ cultures for
decades, while many of them have recently been discovered and tested for their effects in vitro. In this review, we
attempted to summarize the remarkable progress that has been made over the past decades towards understanding
PGRs. The progress is further improving our knowledge of the molecular mechanisms of their action, and beginning to
explain how PGRs not only have direct influence on cellular growth, but also control various aspects of plant's growth
in vivo as well as in vitro.
Key words: Abscisic acid, Auxin, Cytokinin, Ethylene, Gibberellins, Plant growth regulators.

Introduction requirements of the definition of a hormone, as was

Plant growth regulators (PGRs), also known as defined for the mammalian system. Localized synthesis
plant hormones or phytohormones, include a group of of some of the growth regulators may be observed in
naturally occurring, organic substances having unique plants (as in case of animal hormones), but cell or tissue
metabolism and regulatory properties. They influence specific synthesis of growth regulators also observed. It
physiological processes at a concentration (usually 10
-5 may not be always true that growth regulators in plants
M) far lower than that of nutrients or vitamins required are transported away from their site of synthesis and
to affect these processes. The term 'hormone' was have their action at a distance. While auxins and
initially used in medical science for such a stimulatory cytokinins produce growth responses at a distance from
factor. The mammalian hormone concept defines their site of synthesis, thus fit the definition of
hormone as extracellular signaling molecule, which act transported chemical messengers, ethylene acts within
on target cells distant from its localized site of synthesis the same cell or tissue where it was synthesized. Thus,
(Kendrew, 1994). This involves a localized site of transport is not an essential property of a growth
synthesis, followed by transport to target tissue, and regulator in plants. Moreover, cell-to-cell polar transport
control of a physiological response in the target tissue. of auxins contrasts with the passive allocation of
The concept of hormone in plant sciences came from the hormones in animals through blood (Friml, 2003).
observations on morphogenic and developmental Growth regulators in plants differ from most of those in
correlations by Julius von Sachs at the end of the 19 th animals in having pleiotropic effects; they are involved in
Century, who suggested that morphological differences controlling a wide range of plant developmental
between plant organs are due to differences in their processes (Fig. 1). Therefore, plant growth regulator is a
material composition and postulated the existence of more precise term for such stimulatory factors and will be
root and flower forming substances in plants (Teale et al., used to denote natural, endogenous and synthetic
2006). It is now clear that all the PGRs do not fulfill the growth substances in this and subsequent reviews.

A number of PGRs are being used in plant cell, tissue and organ cultures for decades, while many of them have
recently been discovered and tested for their effects in vitro. In this review, we attempted to summarize the
remarkable progress that has been made over the past decades towards understanding PGRs.

1
Division of Biochemistry, Indian Agricultural Research Institute, Delhi - 110012, India.
2
School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India
3
Department of Biotechnology, CGO Complex, Lodhi Road, New Delhi 110053, India
4
Directorate of Education, Indian Council of Forestry Research and Education, Dehradun, India
460 The Indian Forester
An important feature distinguishing plants from
animals is their sessile nature; therefore, they have
evolved diverse mechanisms to circumvent unfavorable
growth conditions with an ability to initiate cell division
from almost any tissue of the plant. Body plan of a plant is
determined continuously throughout its life cycle,
allowing it to be exquisitely environment responsive.
Thus, the environmentally regulated development of
new growth axes is analogous to environmentally
regulated animal behaviors (Leyser, 2009). Many
differentiated cells maintain their potential to elongate
to show various tropisms. Thus, plants can change their
shape and size to adjust in a changing environment
(Nicotra et al., 2010). The genetic potential of a cell to
regenerate a whole plant has been termed as
'totipotency', and perhaps was the driving force which
led German physiologist G. Haberlandt to conceive the
idea of plant tissue culture. Plant tissues when cultured in
vitro exhibit a high degree of plasticity, allowing one type
of tissue to initiate another. However, the extent of
plasticity varies depending on the specific medium
components used. The culture medium used to
modulate plant development in vitro contains mainly five
classes of substances (macro- and micro-elements,
vitamins, sugar and growth regulators). The type and
concentration of PGRs included in the medium is the
most critical component determining the morphogenetic
potential of the cultured cells or tissues.
In this review we present a brief history of the
discovery of growth regulators, followed by the
remarkable progress that has been made over the past
decades towards understanding the effects of PGRs in
plant growth and development.
Discovery of growth regulators
Charles Darwin in 1880s made the original
observations on phototropism of grass coleoptiles which
led him to postulate the existence of signal that gets
transported from the tip of the coleoptile to the bending
regions lower down. After extensive characterization of
the ways in which such signals moves, Went (1926) was
able to diffuse the chemical from coleoptile tips into agar
blocks, which, when replaced on the tips of the
decapitated coleoptiles, resulted into the stimulation of
growth of the decapitated coleoptiles. This
demonstrated the existence of a growth promoting
substance that moves in different directions in plant
(reviewed by Teale et al., 2006). This substance was
originally named Wuchsstoff by Went, later it was
named as auxin, and subsequently the substance was
identified to be indole acetic acid (IAA) (Wildman, 1997).
Subsequent investigations led to the discovery of
many other growth regulators. For example, research in
[May
plant pathogenesis led to the discovery of gibberellins
(GA). Similarly, the efforts to culture plant tissues led to
the discovery of cytokinin; attempts to control abscission
and dormancy led to abscisic acid (ABA); and studies on
the effects of illuminating gas and smoke led to the
discovery of ethylene. Auxins, cytokinins, gibberellins,
abscisic acid and ethylene are the five classical, naturally
occurring PGRs. More recently, several other compounds
have been discovered with biological activity which
equals to or exceeds that of the equivalent endogenous
PGRs (Gross and Parthier, 1994). These include:
polyamines, brassinosteroids, jasmonates, salicylic acid,
signal peptides, oligosaccharides, sterols, and systemins.
Interestingly, among all the classical naturally occurring
PGRs, chemical identification of only abscisic acid was
made from plant tissue. Identification of most others
came from the extracts that produced hormone-like
effects in plants. For example, auxin was identified from
urine and the fungal cultures of Rhizopus, gibberellins
from culture filtrates of the fungus Gibberella, cytokinins
from autoclaved herring sperm DNA, and ethylene from
illuminating gas (Davies, 2010). With the available
scientific tools and techniques for purification and
characterization of compounds, only a few milligrams of
tissues would be sufficient for complete analysis of the
sample, while earlier investigators might have used even
kilograms of tissues to purify the desirable compound
using the conventional techniques. The technical
advancements have now made it possible to characterize
growth regulator levels in individual leaves, buds, or even
from tissues within the organs. Therefore, a number of
recently discovered PGRs with more specific effects on
plant tissues are available now. While determining the
exact level and location of the growth regulators within
the tissue and cells is still largely elusive, considerable
progress has been made in analyzing and localizing
expression of the genes responsible for biosynthesis of
the growth regulators using techniques like polymerase
chain reaction, mutant plants, and transgene expression
(Davies, 2010).
Though Haberlandt failed, the subsequent
investigators (Gautheret, White and Nobecourt
independently in 1930s) successfully established tissue
culture technique for a number of plants. Credit for their
success mainly goes to the discoveries of auxin by Went
(1926) and Kogl and Haagen-Smit (1931). Later, the
discovery of cytokinins by Miller et al. (1955) provided
impetus to the advances in plant tissue culture. Role of
PGRs and their uses in plant tissue culture started
becoming evident from the observations made in the
1950s. However, Skoog and Miller (1957) were the first to
point out that balance between the concentrations of
2016] Recent advances in understanding the role of growth regulators in plant growth and development in vitro ...
auxin and cytokinin plays important role in initiation of
shoot and root organogenesis. In plant tissue and organ
culture, auxin and cytokinin play the most important
roles of regulating growth and morphogenesis. A number
of synthetic auxins and cytokinins with even better
biological activity than that of the equivalent natural
growth regulators have been discovered and are being
used in plant tissue culture.
The capacity of cultured plant cells to undergo
morphogenesis, resulting in the formation of discrete
organs or whole plant, has provided tremendous
opportunities for application of plant tissue culture in
basic botanical studies, biochemistry, breeding,
vegetative propagation and transgenic studies. However,
variable response among genotypes of same species has
been explicitly demonstrated in tissue culture of many
species including Dalbergia sissoo (Kalia et al., 2004) and
Garcinia indica (Malik et al., 2005). Usually combination
of two or more growth regulators is recommended;
either applied simultaneously or sequentially (Tefera,
2006). Exogenously applied PGRs not only exert direct
effects on cellular mechanisms, but they can also modify
synthesis, activation, sequestration, transport and
destruction of the same or different types of growth
regulators (Beale and Sponsel, 1993; Davies, 1995).
Auxins
Auxin was the first-identified plant growth
regulator. It is synthesized in the apex of plant and
inhibits the growth of lateral branches, a phenomenon
known as apical dominance. Highest level of auxins
occurs in or near the apex and it is transported
basipetally, particularly in monocotyledons. In
dicotyledons, higher concentration of auxin is found in
the sub-apical zone which also grows rapidly. Auxins are
also abundant in young leaves, floral organs, developing
fruits and seeds (George et al., 2008).
Auxins are virtually involved in every aspect of
plant growth and development. They play an important
role in different activities such as cell enlargement, cell
division, vascular tissue differentiation, root initiation,
tropistic (bending) response of roots and shoots to
gravity and light, apical dominance, delaying leaf
senescence, leaf and fruit abscission (inhibit or promote
via ethylene), delaying fruit ripening, and promoting
femaleness in dioecious flowers (via ethylene). Auxin
controls growth of roots by modulating cellular
responses to gibberellin (GA). Fu and Harberd (2003)
demonstrated that auxin plays important role in GA-
mediated control of root growth. They also found that
any decrease in auxin transport or signaling delays the
GA-induced disappearance of repressor of GA from root
cell nuclei. Through the effect of auxin on GA-mediated
461
DELLA protein destabilization, shoot apex exerts long-
distance control on the growth of plant tissues and
organs. Chung et al. (2011) suggested that auxin
regulates brassinosteroid biosynthesis, and it relies on
synthesized brassinosteroids for some of its growth-
promoting effects in Arabidopsis.
Complexity of IAA biosynthesis, transport and
signaling pathways indicates the diverse functions of
auxins. Auxin being critical in plant's growth and
development, its biosynthesis is highly regulated. IAA can
be synthesized from tryptophan via two pathways: (1)
tryptamine and (2) indole-3-pyruvic acid pathways
(Strader and Bartel, 2008). In Arabidopsis these pathways
have overlapping functions because plants deficient in
either of these pathways show similar abnormalities.
However, in maize IAA can also be synthesized from
indole, bypassing tryptophan (Woodward and Bartel,
2005). Once synthesized, auxin is distributed throughout
the plant via cell-to-cell transport system (Vieten et al.,
2007). The cellular auxin influx and efflux carriers create
local auxin maxima and gradients which signal diverse
growth and developmental processes. Direction of auxin
transport is determined by asymmetric localization of
transporters in the cells. Plant perceives stimuli and
changes the sub-cellular position of auxin-transport
components accordingly. In organized tissues, auxins are
involved in the establishment and maintenance of
polarity, while in whole plant the most marked effect is
the maintenance of tropisms (Friml, 2003).
The commonly used auxins are IAA, IBA (indole-3-
butyric acid), 2,4-D (2,4-dichlorophenoxy acetic acid),
and NAA (-napthaleneacetic acid). In spite of being the
only naturally occurring auxin in most of the plants, IAA is
not frequently used in plant tissue culture because it is
sensitive to light and heat. There are several other
synthetic auxins that have replaced it, they include PCPA
(p-chlorophenoxyacetic acid), 2,4,5-T (2,4,5-
trichlorophenoxyacetic acid), 3,6-dichloro-2-
methoxybenzoic acid (Dicamba), and 4-amino-3,5,6-
trichloropicolinic acid (Picloram). Different auxins differ
in their physiological activities and the extent to which
they move through tissues, remains bound inside the
cells, or get metabolized. Moreover, the naturally
occurring IAA shows less physiological activity compared
to the synthetic auxins. Based on stem curvature assays,
2, 4-D has 8 - 12 times more activity, 2,4,5-T has 4 times,
PCPA and Picloram have 2 - 4 times more activity, while
NAA has 2 times more activity than that of IAA (Gaspar et
al., 1996). Although 2,4-D, 2,4,5-T, PCPA, and Picloram
are often used to induce rapid cell proliferation, exposure
to high levels or prolonged exposure to these auxins,
particularly 2,4-D, results in suppressed morphogenic
462 The Indian Forester
activities. In the culture medium auxins are added to
initiate cell multiplication and callus formation, root and
shoot organogenesis, somatic embryogenesis and to
stimulate growth on shoot apices in shoot tip cultures.
While 2,4-D is commonly used for callus induction and
suspension cultures, NAA is used to initiate
organogenesis or in maintaining suspension cultures
(Hagen et al., 1991). Synthetic IAA and IBA are rapidly
metabolized in plant tissues. This attribute is useful when
developmental phases in tissue culture require less auxin
e.g. in rooting or when change in the auxin to cytokinin
ratio is desirable to induce organogenesis from calli
(Gaspar et al., 1994). BSAA (Benzo(b)selenienyl-3 acetic
acid) is another synthetic growth regulator having auxin-
like effects (Gaspar, 1995). The responsiveness of the
cultured tissues to auxins can also be modified by adding
other classes of PGRs such as cytokinin and ethylene. The
level of endogenous free auxin considerably affects the
tissue culture response, and it depends on the rate of
their anabolism, catabolism, transport, and conjugation
processes (Bandurski et al., 1995). Sometimes, e.g.
during root growth, auxins at high concentrations have
been found to have inhibitory effects invariably mediated
by the auxin-produced ethylene. Thus, the choice of
auxins and the concentration used depends on: (1) the
type of growth/development required, (2) the rate of
uptake and transport of the applied auxin, (3) the level of
endogenous auxin in the explant, (4) sensitivity of the
plant tissue to the auxin used, (5) stability of the auxin in
the medium and within the explant, and (6) interaction
between the applied auxin and the endogenous PGRs, if
any (George et al., 2008).
Cytokinins
Cytokinins are adenine derivatives characterized
by their ability to induce cell division either in vivo, when
they are added exogenously to plant organs or in vitro,
when added in tissue culture medium, particularly in the
presence of auxin. Cytokinins were first discovered in the
herring sperm DNA extract in Skoog's laboratory at
University of Wisconsin. During the experiments to
promote continuous growth of tobacco calli induced
from stem sections kinetin was identified as a cell division
factor (Miller et al., 1955). However, kinetin is not yet
accepted as a naturally occurring cytokinin (George et al.,
2008). Cytokinins have been found to be involved in a
broad range of developmental processes such as seed
germination, root and shoot meristem function and leaf
senescence (To and Kieber, 2008). They also play an
important role in the formation of nitrogen-fixing
nodules and other plant-microbe interactions (Murray et
al., 2007; Frugier et al., 2008).
Cytokinins regulate different activities in plants
[May
such as cell division, morphogenesis, induction of
adventitious budding, leaf expansion, delayed leaf
senescence, enhanced stomatal opening in some
species, and conversion of etioplasts into chloroplasts
(Parthier, 2004). They are important regulators of
cambium development, and the cytokinins either
produced in root or shoot can regulate normal
development of the plant (Matsumoto-Kitano et al.,
2008). Although both auxin and cytokinin are usually
required for growth and morphogenesis, auxin may
inhibit cytokinin accumulation, whereas cytokinins may
inhibit at least some of the actions of auxin (Gaspar et al.,
1996). Determination of cytokinin and auxin
concentrations in the individual plant parts, expression
patterns of genes involved in biosynthesis and
quantification of their transport in plant indicated that
the apical meristem of the shoot produces mainly auxin
and the meristem of the roots synthesize cytokinin. Both
of these are exported from their site of synthesis to the
other parts (Friml et al., 2002; Miyawaki et al., 2004).
Though both the meristems are capable of synthesizing
the other type of growth regulator, they do not do so
without external stimulation by the respective PGR.
Thus, shoot meristems depend on the cytokinin from
roots, and the growing root tips depend on auxin from
shoots which suggest differences in the PGR's regulation
of the meristematic activity in shoot and root. The auxin
and cytokinin concentrations are as important as their
ratio. While auxins affect DNA replication, cytokinins
seem to exert some control over the events leading to
mitosis and cytokinesis (Vesely et al., 1994). Bartrina et
al. (2011) demonstrated that cytokinin regulates the
activity of reproductive meristems, flower organs, ovule
formation, and seed yield in Arabidopsis. Effects of
cytokinins on plant metabolism, physiology, and
development are generally antagonized by ABA and GA.
Fleishon et al. (2011) demontrated mutual antagonistic
effects of GA and cytokinin on various developmental
and molecular processes during tomato plant growth.
The ratio of the two PGRs, rather than their
concentration, determines the final response. In many
tissues, including cultured tissues, cytokinins often
promote ethylene biosynthesis (Abeles et al., 1992).
Generally cytokinins are considered as anti-senescence;
but in some cases they may accelerate the senescence
process. A high level of cytokinins induces programmed
cell death in cell cultures, yellowing of leaves and
reduced root mass in intact plant (Carimi et al., 2003).
The first step in isoprenoid cytokinin biosynthesis
pathway is N-prenylation of adenosine 5'-phosphates
(AMP, ADP, or ATP) at the N6-terminus by the enzyme
adenosine phosphate-isopentenyltransferase
2016] Recent advances in understanding the role of growth regulators in plant growth and development in vitro ...
(Sakakibara, 2006). Regulation of cytokinin levels is
generally complex and involves changes in both synthesis
and metabolism. Cytokinin signaling is remarkably
similar to the two-component signaling systems in
bacteria (To and Kieber, 2008). Cytokinin is perceived by a
membrane-associated hybrid kinase that transfers a
phosphate to AHP (Arabidopsis His Phosphotransfer)
proteins (Santner et al., 2009). AHPs are then
translocated into the nucleus where they phosphorylate
ARR (Arabidopsis Response Regulator) proteins which
may act as negative or positive effectors of cytokinin
signaling. A large number of genes are regulated by
cytokinins, including a family of transcription factor
genes called cytokinin response factors (Peng et al.,
2009). Mutants deficient in members of this family
exhibit defects in cytokinin-regulated transcription
(Rashotte et al., 2003).
The naturally occurring cytokinins include a large
group of structurally related purine derivatives, e.g.
zeatin and N6-(2-isopentenyl) adenine (2iP). The
cytokinins commonly used in tissue culture include 6-
benzylaminopurine (BAP) or 6- benzyladenine (BA),
kinetin, 2iP, and zeatin. Two main properties of cytokinins
that are useful in tissue culture are: stimulation of cell
division and release of lateral bud dormancy. They can
also induce adventitious bud formation in cuttings and
shoot tip cultures (Krikorian, 1995; Kumar et al., 2008). In
most of the tissue culture experiments, particularly for
shoot proliferation, adenine-type cytokinins are used. A
naturally occurring enzyme, cytokinin oxidase, degrades
cytokinins such as zeatin and 2iP by cleaving the side
chain; hence these cytokinins are less effective on plants.
The synthetic cytokinins (e.g. BAP, kinetin) are less
susceptible to oxidative degradation but are generally
less active than natural ones (Hare and van Staden,
1994). Catterou et al. (2002) reported a cytokinin-
overproducing Arabidopsis mutant (hoc) to have high
shoot organogenic capacity. The mutant accumulated
cytokinins at 2 to 7 fold higher concentration compared
to wild type plants in its shoot and roots; thereby the
mutant was capable of auto-regenerating shoots without
exogenous application of growth regulators in the
medium.
Adenine, a naturally occurring compound, has a
base structure similar to that of the cytokinins and shows
cytokinin-like activity (Gasper et al., 1996). Adenosine
and adenylic acid may also have cytokinin-like activity.
Many substituted urea also show cytokinin-like activities
(Krikorian, 1995). Some plant tissues show an absolute
requirement for a specific cytokinin for morphogenesis,
whereas other tissues are considered to be cytokinin
independent. Many aspects of cell growth,
463
differentiation, and organogenesis have been found to
be controlled by interaction between cytokinins and
auxins. The required concentrations vary greatly
according to the plant species, the culture conditions,
and the form of the PGRs used. Higher concentration ( 5
-1
mg L ) of cytokinin generally inhibits or delay root
formation and growth. This is why cytokinins are usually
omitted from shoot culture medium when shoots are to
be rooted. Sometimes subculturing the regenerated
shoots on cytokinin-free medium may be required to
reduce the level of cytokinin in the tissues before
culturing on rooting medium (Kalia et al., 2007).
Gibberellins
Gibberellin (GA) was originally isolated in 1938 as a
metabolite from the rice fungal pathogen Gibberella
fujikuroi (Yamaguchi, 2008). The green revolution
associated dwarf varieties have been molecularly
characterized to be affected in GA signaling pathways
(Peng et al., 1999). GAs are a large family of tetracyclic
diterpenoids. Like other PGRs, GAs play major role in
diverse growth processes including seed development,
organ elongation and the control of flowering time
(Yamaguchi, 2008). While GA1 is the most important
gibberellin in plants which is primarily responsible for
stem elongation, the most widely available compound
GA3 (gibberellic acid) is a fungal product. However, GA3 is
only occasionally used in tissue culture medium (Fry and
Street, 1980; Kumar et al., 2008; Kumar et al., 2009). It is
generally used to promote flowering (particularly for the
species that require long days and/or low temperature),
to break dormancy (Kumar and Bhat, 2012), stem
elongation (by increasing cell division and elongation),
and to induce maleness in dioecious flowers. Their ability
to promote bolting and stem elongation can be exploited
for elongation of shoots before rooting in woody species.
In specific cases, GA3 is added to culture medium to
promote growth of low-density cell cultures, to enhance
callus growth, and to elongate dwarf or stunted plantlets
(Gaspar et al., 1996; Kumar et al., 2008). However,
addition of GA3 in tissue culture medium often prevents
formation of roots, shoots, or somatic embryos, although
the opposite has also been reported (George et al.,
2008). Endogenous GA may be required for callus growth
(Lance et al., 1976), and inhibition in GA biosynthesis can
affect development of cell in liquid culture (Ziv and Ariel,
1991). GA may also influence the availability of
endogenous auxin. Shoot growth in meristem and shoot
cultures may be enhanced by addition of GA3 in the
culture medium (Fry and Street, 1980).
Brian and Hemming (1958) found that for normal
response to GA intact shoot apex is necessary in pea.
When shoot apex is taken off, internodes of GA-deficient
464 The Indian Forester
dwarf mutant pea plants fail to elongate in response to
the GA treatment. Fu and Harberd (2003) investigated
the role of shoot apex in regulating GA-responsive root
growth in Arabidopsis. They found that the GA-deficient
mutants (ga1-3) had shorter primary roots than the wild
type Arabidopsis, clearly indicating that GA regulates
root growth. Removal of the shoot apex inhibited the
growth of primary roots of wild type Arabidopsis
seedlings. Application of IAA on the decapitation site
restored the root growth of the seedling. Thus, shoot
apex delivers auxin to roots via a polar transport stream
to promote root growth. They also reported that roots of
decapitated ga1-3 mutant plants were less responsive to
GA than were the roots of intact ga1-3 mutant plants.
Once again, application of IAA to the decapitated shoot
resulted in recovery of the GA response of the roots. This
suggested that the auxin produced at shoot apex
promotes root growth by facilitating the response of root
cells to GA. Studies have also uncovered certain new
roles of GA in leaf differentiation, photomorphogenesis
and pollen-tube growth (Fleet and Sun, 2005).
GAs are synthesized from geranylgeranyl
diphosphate by a multi-enzyme pathway. While GA
represses the genes involved in its biosynthesis, it
promotes the genes involved in GA inactivation (Fleet
and Sun, 2005). GA signaling shows similarity with auxin
and jasmonic acid (JA) signaling pathway. Mutants for GA
biosynthesis and GA response are becoming important
tools in understanding the molecular mechanism of GA
action. Mutants impaired in GA biosynthesis or GA
response were dwarf and possessed small, dark green
leaves. Some of them were defective in seed germination
and floral development. Identification of the upstream
regulators of GA biosynthesis, elucidation of the
functions of GA signaling components, and isolation of
downstream targets have contributed significantly in our
understanding of GA-regulated morphogenesis in plants.
Abscisic acid
Abscisic acid (ABA) was initially thought to be
associated with abscission and dormancy; therefore, it
was regarded as an inhibitor. Accordingly, it was named
as "abscisin II" because it was then found to control
abscission of cotton bolls. Another group of workers
named it "dormin" for its purported role in bud
dormancy. Later it was named as abscisic acid. However,
it is now clear that ABA has little role in either abscission
(which is regulated by ethylene) or bud dormancy, but
the name continues (Rai et al., 2011). While exogenous
application of ABA can inhibit growth of the plant, it has
been reported to act as a promoter of storage protein
synthesis in seeds (Chen and Zhou, 1998). ABA plays key
role in closing of stomatal apertures, control of water and
[May
ion uptake by roots (in part by increasing hydraulic
conductivity) and promoting leaf abscission and
senescence. It acts antagonistically to GAs in many
systems. ABA, along with ethylene and JA, helps in
defense against insect pests. ABA also plays important
role in dormancy in embryos, buds and bulbs and is
associated with seed dormancy, drought responses and
other growth processes (Nambara and Marion-Poll,
2005). ABA was reported to increase freezing tolerance
of axenically grown plants and cell cultures (Robertson et
al., 1994). It has recently been reported to play a key role
in modulating diverse plantpathogen interactions (Fan
et al., 2009).
The ABA biosynthetic pathway was defined
through genetic studies of mutants with seed dormancy
defects, primarily in maize and Arabidopsis. ABA is
synthesized from glyceraldehyde-3-phosphate via
isopentenyl diphosphate and carotenoids in roots and
mature leaves, particularly in response to water stress.
ABA levels are regulated by a variety of environmental
conditions, particularly during drought. ABA regulation is
mediated by changes in biosynthesis and catabolism.
Though several candidate ABA receptors have been
reported, their roles are still controversial. Considering
diverse ABA responses, it is quite likely that there must be
multiple sites of ABA perception (McCourt and Creelman,
2008).
ABA signal transduction pathways are complex
and involve a variety of small molecules and proteins.
Two protein phosphatases called ABI1 (ABA Insensitive 1)
and ABI2 play a central role in ABA response, as
mutations in either gene affect all ABA responses. In
addition, a variety of kinases, RNA-modifying enzymes
and transcription factors have been proposed to function
in ABA signaling (Hirayama and Shinozaki 2007;
2+
Finkelstein et al., 2008). In the guard cells, Ca , ROS and
NO have been implicated in ABA promotion of stomatal
closing. Ubiquitin-proteasome pathway has been
reported to play important role in ABA signaling. (Zhang
et al., 2005; Stone et al., 2006).
ABA has usually been found to inhibit callus
growth; however, it has also been reported to stimulate
callus growth (Blumenfeld and Gazit, 1970). Lower
concentration (0.1 mg L-1) of ABA could increase haploid
tobacco callus, while a higher concentration (10 mg L-1)
acted as inhibitor (Kochhar, 1980). When ABA was added
in the culture medium it promoted callus induction and
proliferation in carrot (Jimenez and Bangerth, 2001). It
has also been observed to influence morphogenesis in a
number of plants. Shepard (1980) found that adding ABA
to the culture medium (0.05 - 0.2 mg L-1) enhanced the
rate of morphogenesis. Hooker and Thorpe (1998)
2016] Recent advances in understanding the role of growth regulators in plant growth and development in vitro ...
observed that ABA at concentrations more than 0.2 mg L-1
inhibited lateral root emergence in tomato, whereas its
putative biosynthesis inhibitor fluridone enhanced
formation of lateral roots. ABA levels have been reported
to increase the frequency of embryos reaching maturity
and can assist in handling large population of somatic
embryos which can be required for mass propagation
(Ammirato, 1988). The role of ABA in decreasing
proportion of abnormal embryos and promoting
synchronized embryo maturation has been confirmed in
several species (Dunstan et al., 1988; Capuana and
Debergh, 1997; Fernando and Gamage, 2000).
Application of ABA for maturation and normal growth of
somatic embryos is also well documented (Roberts et
al.,1990; Label and Lelu, 1994; Rock and Quatrano, 1995;
Arnold et al., 2002). Moreover, exogenous application of
ABA has been reported to prevent precocious
germination of somatic embryos (Rai et al., 2008) and can
induce a quiescent state in somatic embryos similar to
the dormancy of zygotic embryos (Zimmerman, 1993).
Zhang et al. (2010) reported promoting affects of ABA on
somatic embryos in Larix leptolepis. Though exogenous
application of ABA alone suppresses shoot regeneration,
when added into the culture medium in combination of
other growth regulators it shows promoting effects. The
use of ABA for in vitro conservation of many plant species
has also been reported ( Rai et al., 2011).
Ethylene
Ethylene is a simple hydrocarbon (C2H4) and
naturally occurring PGR. It is primarily associated with
fruit ripening in climacteric fruits, hence also known as
fruit-ripening hormone. Ethylene plays important role in
many other developmental processes (Kendrick and
Chang, 2008).
Ethylene has been reported to stimulate auxin
biosynthesis and its basipetal transport to the elongation
zone, where it activates auxin response leading to
inhibition of cell elongation (Ruzicka et al., 2007). It also
activates auxin signaling pathway and regulates root
growth by stimulating auxin biosynthesis and affecting
the auxin transport machinery. Ethylene does not appear
to be essential for normal vegetative growth and
development of plant, as ethylene deficient transgenic
plants grow normally (Kumar et al., 1998). Studies on
transgenic plants with impaired ethylene biosynthesis
have advanced our understanding of developmental
processes influenced by ethylene such as fruit ripening
and leaf senescence. Reduced ethylene synthesis in
transgenic tomato plants expressing ACC deaminase
(Klee et al., 1991), antisense genes of ACC synthase
(Oeller et al., 1991), or ACC oxidase (Picton et al., 1993)
resulted in delayed fruit ripening and leaf senescence.
465
Pua and Lee (1995) used transgenic mustard plants
expressing antisense ACC oxidase gene to elucidate
regulatory role of ethylene on plant morphogenesis in
vitro. While shoot regeneration in wild type cultivar
required use of ethylene inhibitors in the culture
medium, the transgenic plants showed normal shoot bud
regeneration on medium lacking ethylene inhibitors.
Ethylene is synthesized from methionine via
intermediate 1-aminocyclopropane-1-carboxylic acid
(ACC) in a pathway called the Yang cycle (Adams and
Yang, 1979). Ethylene can be synthesized in all plant
organs; however the pathway is regulated by both
environmental factors and developmental stage such as
the onset of fruit ripening. ACC synthase, one of the key
enzymes in the ethylene biosynthesis, is focus of this
regulation. Auxin induces ethylene synthesis by directly
promoting transcription of ACS (ACC synthase) genes.
Various signals modulate ethylene levels by regulating
the levels of ACS proteins (Christians et al., 2009).
Auxins usually stimulate ethylene production, and
cytokinins may block ethylene action; however, a
synergism between auxin and cytokinin in ethylene
production has been reported (Gaspar et al., 1989). At
higher concentrations, ethylene alters microtubule and
microfibril orientation which results into decreased cell
elongation but increased cell expansion (Steen and
Chadwick, 1981). Like cytokinin, ethylene is perceived by
a two-component protein kinase receptor. However, the
receptor is localized to the endoplasmic reticulum
membrane. The ubiquitin pathway has an important role
in ethylene signaling (Kendrick and Chang, 2008).
Ethylene produced by the in vitro grown plant
tissues may accumulate in the culture vessels,
particularly from the rapidly growing callus or suspension
cultures, hence may influence growth of the plants
(Biddington, 1992). Some cultures produce more
ethylene, which may inhibit further growth and
development of the plant. The type of culture vessel used
and its closure affect the gaseous exchange between the
culture vessel and the outside atmosphere resulting into
the build-up of ethylene concentration. Because tissue
culture is carried out within closed containers, gaseous
products such as ethylene, from the plant tissues gets
accumulated inside the vessel. Therefore, ethylene has
been considered as an unwanted gas in tissue culture.
The research on ethylene and tissue culture has been
centered on understanding how ethylene accumulation
might affect organogenesis and how its effect can be the
controlled. In such studies Ethephon (Ethrel or 2-
chloroethylphosphonic acid) is used as ethylene-
releasing chemical, which releases ethylene below pH
5.0 (Abeles et al., 1992).
466 The Indian Forester [May

Fig. 1 : Growth regulators control various aspects of plant's growth and development. Some of the classical and newly discovered PGRs
with their major functions (structure of the representative compound is depicted).

Although its use in plant tissue culture is not
common, it has been reported to have considerable
(adverse) effects on plant growth and development.
Ethylene can specifically affect growth of calli and
suspension cultures, stem and root elongation,
adventitious root formation, axillary and adventitious
bud formation, leaf and fruit abscission, induction of
femaleness in dioecious flowers, flower opening, flower
and leaf senescence, and embryogenesis. High
concentrations of ethylene in culture have been reported
to be responsible for regeneration recalcitrancy in many
species (Pua and Lee, 1995). Callus proliferation in
Brassica oleracea hypocotyl cultures was increased by
the ethylene precursors such as S-adenosylmithionone
and ACC whereas shoot initiation was increased by
ethylene inhibitors like aminoethoxyvinylglycine (AVG),
CoCl2, and AgNO3 (Sethi et al., 1990). Likewise, ACC
increased callus production in maize but inhibited
subsequent shoot formation (Songstad et al., 1988).
AgNO3, promoted shoot regeneration in wheat callus
2016] Recent advances in understanding the role of growth regulators in plant growth and development in vitro ... 467

culture, and reversed the inhibitory effect of ethylene
and 2,4-D on morphogenesis (Radojevic, 1988). AgNO3
also promoted somatic embryogenesis in carrot (Roustan
et al., 1990). Though, ethylene influences growth,
differentiation, and senescence in plants at very low
-1
concentrations (0.01 m l L ), mode of action of ethylene in
tissue culture is still unclear.
Concluding remarks
In the last two decades, our understanding of
molecular mechanisms of growth regulator biosynthesis,
perception and response has improved considerably. It
has been demonstrated that change in the degree of
interaction between PGRs induces changes in plant
morphology and adaptability to the environment. This
suggests that morphology and environmental
adaptability of plants can be modulated by growth
regulators in vivo. Although a balance of action of each
PGR is adjusted to produce a normal plant by controlling
its transport, distribution, and signal transduction, it is
more directly regulated by controlling their biosynthesis
and metabolism. Though plant cell posses a remarkable
potential for cellular totipotency, its behavior during in
vitro culture is largely not predictable. Therefore,
thorough understanding of cell differentiation is
essential to get desirable results in plant cell and tissue
cultures. Despite the considerable progress that has
been made in elucidating some of the pathways of signal
transduction, there is still a lot to know before we fully
understand their complex interactions.

Ikkni o`f esa o`f fu;a=kdksa dh Hkwfedk le>us esa gky dh mUufr;ka ,oa ik=ks&I ikjEifjd o`f fu;a=kdksa dk fodkl
lqjs'k dqekj] jksgrkl flag] lat; dkfy;k] ,l-ds- 'kekZ ,oa jktoar ds- dkfy;k
lkjka'k
vkxsZfud ;kSfxdksa ds ,d fofo/ foU;kl o`f fu;a=kd ikniksa esa fodklkRed ekxksZa ds fu/kZj.k esa vge ?kVd gSaA ;s 'kkjhfjdh; ,oa
vkdkfjdh; izHkkoksa dks mRiUu djus ds fy, dksf'kdk Lrj ij ikjEifjd f;k djrs gSaA ikniksa esa ifjogu] p;kip; vkSj o`f fu;a=kdksa dh f;k
dh iz.kkyh ds ckjs esa gekjh le> gky ds o"kksZa esa dkiQh c<+h gSA la'ys"k.k] ifjogu vkSj vUrtkZr o`f fu;a=kdksa dh f;k ds lkFk gLr{ksi djus
okys jlk;uksa dh [kkst us ikni dh o`f vkSj fodkl esa ikni o`f fu;a=kdksa dh Hkwfedk ds laca/ esa gekjh tkudkjh esa vkxs lq/kj fd;k gSA n'kdksa
ls ikni dksf'kdk] rd vkSj vax lao/Zuksa esa vusdksa ikni o`f fu;a=kdksa dk mi;ksx gks jgk gS] tcfd buesa ls dbZ dh gky esa [kkst dh xbZ gS vkSj
ik=ks buds izHkkoksa dh tkap dh xbZ gSA bl iqujh{k.k esa] geus ikni o`f fu;a=kdksa dks le>us dh fn'kk esa xr n'kdksa esa dh xbZ mYys[kuh; izxfr dh
leh{kk djus dk iz;kl fd;k gSA izxfr budh f;k dh vkf.od f;kfof/ dh gekjh tkudkjh esa vkxs lq/kj dj jgh gS vkSj ;g Li"V djuk
'kq: fd;k gS fd dSls ikni o`f fu;a=kdksa dk u dsoy dksf'kdk o`f ij izR;{k izHkko gS cfYd thos lkFk gh lkFk ik=ks ikni dh o`f ds fofHkUu
igyqvksa dks Hkh fu;af=kr djrk gSA
References
Abeles F.B., Morgan O.W. and Sahveit M.E. (1992). Ethylene in plant biology, 2nd ed. Academic Press, San Diego
Adams D.O. and Yang S.F. (1979). Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the
conversion of methionine to ethylene. Proc Natl Acad Sci USA, 76:170174
Ammirato P.V. (1988). Role of ABA in regulation of somatic embryogenesis. Hort Sci., 23:520
Arnold S.V., Sabala I., Bozhkov P., Dyachok J. and Filonova L. (2002). Developmental pathways of somatic embryogenesis. Plant Cell Tiss Org
Cult., 69:233-249
Bandurski R.S., Cohen J.D. and Slovin J. (1995). Auxin biosynthesis and metabolism. In: Plant hormones (Davies PJ ed). Dordrecht: Kluwer
Academic Publishers, pp 39-65
Bartrina I., Otto E., Strnad M., Werner T. and Schmlling T. (2011). Cytokinin regulates the activity of reproductive meristems, flower organ
size, ovule formation, and thus seed yield in Arabidopsis thaliana. The Plant Cell : 10.1105/tpc.110.079079.
Beale M.H. and Sponsel V.M. (1993). Future directions in plant hormone research. J Plant Growth Regul., 12:227-235
Biddington N.L. (1992). The influence of ethylene in plant tissue culture. Plant Growth Regul., 11:173-187
Blumenfeld A. and Gazit S. (1970). Interaction of kinetin and abscisic acid in the growth of soybean callus. Plant Physiol., 45:535-536
Brian P.W. and Hemming H.G. (1958). Complementary action of gibberellic acid and auxins in pea internode extension. Ann Bot., 22:117
Capuana M. and Debergh P.C. (1997). Improvement of the maturation and germination of horse chestnut somatic embryos. Plant Cell Tiss
Org Cult 48:23-29
Carimi F., Zottini M., Formentin E., Terzi M. and Lo Schiavo F. (2003). Cytokinins: new apoptotic inducers in plants. Planta., 216:413421
Catterou M., Dubois F., Smets R., Vaniet S. and Kichey T. (2002). hoc: an Arabidopsis mutant overproducing cytokinins and expressing high in
vitro organogenic capacity. Plant J., 30:273-287
Chen J.G. and Zhou X. (1998). Involvement of abscisic acid in mesocotyl growth in etiolated seedlings of a Foxtail millet dwarf mutant. J Plant
Growth Regul., 17:147151
468 The Indian Forester [May

Christians M.J., Gingerich D.J., Hansen M., Binder B.M. and Kieber J.J. (2009) The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively
to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels. Plant J., 57:332345
Chung Y., Maharjan P.M., Lee O., Fujioka S. and Jang S. (2011) Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in
Arabidopsis. Plant J., 66:564578
Davies P.J. (1995) Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, Dordrecht, The
Netherlands, Norwell, MA, USA
Davies P.J. (2010) The plant hormones: their nature, occurrence, and functions. In: Davis PJ (ed), The Plant Hormones: Biosynthesis, Signal
Transduction, Action. Springer, USA.
Dunstan D.I., Bekkaoui F., Pilon M., Fowke L.C. and Abrams S.R. (1988) Effects of abscisic acid and analogues on the maturation of white
spruce (Picea glauca) somatic embryos. Plant Sci., 58:77-84
Fan J., Hill L., Crooks C., Doerner P. and Lamb C. (2009) Abscisic acid has a key role in modulating diverse plantpathogen interactions. Plant
Physiol., 150:17501761
Fernando S.C. and Gamage C.K.A. (2000) Abscisic acid induced somatic embryogenesis immature embryo explant of coconut (Cocos nucifera
L.). Plant Sci., 151:193-198
Finkelstein R., Reeves W., Ariizumi T. and Steber C. (2008) Molecular aspects of seed dormancy. Ann Rev Plant Biol., 59:387415
Fleet C.M. and Sun T.P. (2005) A DELLAcate balance: the role of gibberellin in plant morphogenesis. Curr Opin Plant Biol., 8:77-85
Fleishon S., Shani E., Ori N. and Weiss D. (2011) Negative reciprocal interactions between gibberellin and cytokinin in tomato. New Phytol.
DOI: 10.1111/j.1469-8137.2010.03616.x
Friml J. (2003) Auxin transport - shaping the plant. Curr. Opin. Plant Boil., 6:7-12
Friml J., Wisniewska J., Benkova E., Mendgen K. and Palme K. (2002) Lateral relocation of auxin efflux regulator AtPIN3 mediates tropism in
Arabidopsis. Nature, 415:806809
Frugier F., Kosuta S., Murray J.D., Crespi M. and Szczyglowski K. (2008) Cytokinin: secret agent of symbiosis. Trends Plant Sci., 13:115120
Fry S.C. and Street H.E. (1980) Gibberellin-sensitive cultures. Plant Physiol., 65:2- 477
Fu X. and Harberd N.P. (2003) Auxin promotes Arabidopsis root growth by modulating gibberellin response. Nature., 421:740743
Gaspar T. (1995) Selenieted forms of indolylacetic acid: new powerful synthetic auxins. Across Org Acta 1:65-66
Gaspar T., Kevers C. and Hausman J. (1994) Peroxidase activity and endogenous free auxin during adventitious root formation. In:
Lumsdenm PJ, Nicholas JR, Davies WJ (ed), Physiology, growth and development of plants in culture. Dordrecht: Kluwer Academic
Publishers, pp 289-298
Gaspar T., Kevers C., Penel C., Greppin H., Reid D.M. and Thorpe T. (1996) Plant hormones and plant growth regulators in plant tissue culture.
In vitro Cell Dev Biol-Plant, 32:272-289
Gaspar T., Kevers C. and Bouillenne H. (1989) Ethylene production in relation to rose micropropagation. In: Clysters H, De Proft M, Marcelle R
(eds), Biochemical and physiological aspects of ethylene production in lower and higher plants. Dordrecht: Kluwer Academic
Publishers, pp 303-312
George E.F., Hall M.A. and Klerk G.J.D. (2008) Plant Propagation by Tissue Culture, 3rd Edition pp 205-226
Gross D. and Parthier B. (1994) Novel natural substances acting in plant growth regulation. J Plant Growth Regul., 13:93-114
Hagen S.R., Muneta P. and Augustin J. (1991) Stability and utilization of picloram, vitamins and sucrose in a tissue culture medium. Plant Cell
Tiss Org Cult., 25:45-48
Hare P.D. and van Staden J. (1994) Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell
Physiol., 35:1121-1125
Hirayama T. and Shinozaki K. (2007) Perception and transduction of abscisic acid signals: keys to the function of the versatile plant hormone
ABA. Trends Plant Sci., 12:343351
Hooker T.S. and Thorpe T.A. (1998) Effects of fluridone and abscisic acid on lateral root initiation and root elongation of excised tomato roots
cultured in vitro. Plant Cell Tiss Org Cult., 52:199203.
Jimenez V.M. and Bangerth F. (2001) Endogenous hormone levels in explants and in embryogenic and non- embryogenic culture of carrot.
Physiol Plant., 111:389-395
Kalia R.K., Arya S., Kalia S. and Arya I.D. (2007). Plantlet regeneration from fascicular buds on seedling explants of Pinus roxburghii. Biol.
Plant, 51:653-659
Kalia S., Kalia R.K. and Sharma S.K. (2004) Evaluation of clonal variability in shoot coppicing ability and in vitro responses of Dalbergia sissoo
Roxb. Silvae Genet., 53:212-220
Kendrew J. (1994) The encyclopedia of molecular biology. Blackwell Science, Oxford, UK
Kendrick M.D. and Chang C. (2008) Ethylene signaling: new levels of complexity and regulation. Curr. Opin. Plant Biol., 11:479485
Klee H.J., Hayford M.B., Kretzmer K.A., Barry G.F. and Kishore G.M. (1991). Control of ethylene synthesis by expression of a bacterial enzyme
in transgenic tomato plants. Plant Cell, 3:11871193
2016] Recent advances in understanding the role of growth regulators in plant growth and development in vitro ... 469

Kochhar T.S. (1980). Effect of abscisic acid and auxins on the growth of tobacco callus. Z Pflanzenphysiol, 97:1-4
Kogl F. and Haagen-Smit A.J. (1931). Uber die Chemie des Wuchsstoffs K. Akad. Wetenschap. Amsterdam. Proc. Sect. Sci., 34:1411-1416
Krikorian A.D. (1995). Hormones in tissue culture and microprupagation. In: Plant hormones(Davies PJ ed.). Dordrecht: Kluwer Academic
Publishers, pp 774-796
Kumar P.P., Lakshmanan P. and Thorpe T.A. (1998). Regulation of morphogenesis in plant tissue culture by ethylene. In Vitro Cell Dev Biol-
Plant, 34:94 - 103
Kumar S. and Bhat V. (2012). High frequency direct plant regeneration via multiple shoot induction in the apomictic forage grass Cenchrus
ciliaris L. In Vitro Cell Dev Biol-Plant, 41: (In Press)
Kumar S. and Chandra A. (2009). Direct plant regeneration via multiple shoot induction in Stylosanthes seabrana. Cytologia, 74:391-399
Kumar S., Chandra A. and Gupta M.G. (2008). Plantlet regeneration via multiple shoot induction in Indian accessions of lucerne (Medicago
sativa L.). J Plant Biochem Biotech., 17:181-184
Label P. and Lelu M.A. (1994). Influence of exogenous abscisic acid on germination and plantlet conversion frequencies of hybrid larch
somatic embryos (Larix leptoeuropaea). Plant Growth Regul., 15:175-182
Lance B., Reid D.M. and Thorpe T.A. (1976). Endogenous gibberellins and growth of tobacco callus cultures. Physiol. Plant, 36:287-292
Leyser O. (2009). The control of shoot branching: an example of plant information processing. Plant Cell Environ., 32:694-703
Malik S.K., Chaudhury R. and Kalia R.K. (2005). In vitro multiplication and conservation of Garcinia indica: A medicinal tropical tree. Sci. Hort.,
106:539-553
Matsumoto-Kitano M., Kusumoto T., Tarkowski P., Kinoshita-Tsujimura K., Vaclavkova K., Miyawaki K. and Kakimoto T. (2008). Cytokinins are
central regulators of cambial activity. Proc Natl Acad Sci USA 105:2002720031
McCourt P. and Creelman R. (2008). The ABA receptorswe report you decide. Curr Opin Plant Biol., 11:474478
Miller C., Skoog F., Von Saltza M.H. and Strong F.M. (1955). Kinetin, a cell division factor from deoxyribonucleic acid. J. Am Chem. Soc.,
77:1392
Miyawaki K., Matsumoto-Kitano M. and Kakimoto T. (2004). Expression of cytokinin biosynthetic isopentenyltransferase genes in
Arabidopsis: Tissue speci?city and regulation by auxin, cytokinin, and nitrate. Plant J., 37:128138
Murray J.D., Karas B.J., Sato S., Tabata S., Amyot L. and Szczyglowski K. (2007). A cytokinin perception mutant colonized by Rhizobium in the
absence of nodule organogenesis. Science, 315:101104
Nambara E. and Marion-Poll A. (2005). Abscisic acid biosynthesis and catabolism. Ann. Rev. Plant Biol., 56:165185
Nicotra A.B., Atkin O.K., Bonser S.P., Davidson A.M. and Finnegan E.J. (2010). Plant phenotypic plasticity in a changing climate. Trends Plant
Sci., 15:684-692
Oeller P.W., Min-Wong L., Taylor L.P., Pike D.A. and Theologis A. (1991). Reversible inhibition of tomato fruit senescence by antisense RNA.
Science, 254:437-439
Parthier B. (2004). Phytohormones and chloroplast development. Biochem Physiol Pflanz, 174:173-214.
Peng J., Richards D.E., Hartley N.M., Murphy G.P., Devos K.M., Flintham J.E., Beales J., Fish L.J., Worland A.J. and Pelica F. (1999).'Green
Revolution' genes encode mutant gibberellins response modulators. Nature, 400:256261
Peng Z.Y., Zhou X., Li L., Yu X. and Li H. (2009). Arabidopsis Hormone Database: a comprehensive genetic and phenotypic information
database for plant hormone research in Arabidopsis. Nucleic Acids Res., 37:D975D982
Picton S., Barton S.L., Bouzayen M., Hamilton A.J. and Grierson D. (1993). Altered fruit ripening and leaf senescence in tomatoes expressing
an antisense ethylene-forming enzyme transgene. Plant J., 13:469-481
Pua E.C. and Chi G.L. (1993). De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene.
Physiol Plant., 88:467-474
Pua E.C. and Lee J.E.E. (1995). Enhanced de novo shoot morphogenesis in vitro by expression of antisense 1-aminocyclopropane-1-
carboxylate oxidase gene in transgenic mustard plants. Planta, 196:69-76
Radojevic L. (1988). Plant regeneration of Aesculus hippocastanum L. (Horse chestnut) through somatic embryogenesis. J. Plant Physiol.,
132:322-326
Rai M.K., Jaiswal V.S. and Jaiswal U. (2008). Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium
guajava L.). Sci. Hort., 117:302-305
Rai M.K., Shekhawat N.S., Gupta H.A.K., Phulwaria M. and Ram K. (2011). The role of abscisic acid in plant tissue culture: a review of recent
progress. Plant Cell Tiss. Org. Cult., 111(2): 179-190
Rashotte A.M., Carson S.D., To J.P. and Kieber J.J. (2003). Expression profiling of cytokinin action in Arabidopsis. Plant Physiol.,
132:19982011
Roberts D.R., Flinn B.S. and Webb D.T. (1990). Abscisic acid and indole-3- butyric acid regulation of maturation and accumulation of storage
proteins in somatic embryos of interior spruce. Physiol. Plant, 78:355-360
Robertson A.J., Weninger A., Wilen R.W., Fu P. and Gusta L.V. (1994). Comparison of dehydrin gene expression and freezing tolerance in
Bromus inermis and Secale cereale grown in controlled environments, hydroponics, and the field. Plant Physiol., 106:1213-1216
470 The Indian Forester [May

Rock C.D. and Quatrano R.S. (1995). Hormones during seed development. In: Davies PJ (ed) Plant hormones. Dordrecht: Kluwer Academic
Publishers, pp 671-697
Roustan J.P., Latche A. and Fallot J. (1990). Control of carrot somatic embryogenesis by AgNO3, an inhibitor of ethylene action: effect on
arginine decarboxylase activity. Plant Sci., 67:8995
Ruzicka K., Ljung K., Vanneste S., Podhorska R. and Beeckman T. (2007). Ethylene regulates root growth through effects on auxin biosynthesis
and transport-dependent auxin distribution. Plant Cell, 19:21972212
Sakakibara H. (2006). Cytokinins: activity, biosynthesis, and translocation. Ann. Rev. Plant Biol., 57:431449
Santner A., Calderon-Villalobos L.I.A. and Estelle M. (2009). Plant hormones are versatile chemical regulators of plant growth. Nature Chem.
Biol., 5:301-307
Sethi U., Basu A. and Guha-Mukherjee S. (1990). Control of cell proliferation and differentiation by modulators of ethylene biosynthesis and
action in Brassica hypocotyl explants. Plant Sci., 69:225-229
Shepard J.F. (1980). Abscisic acid-enhanced shoot initiation in protoplast derived calli of potato. Plant Sci. Lett., 18:327-333
Skoog F
andMiller C.
(1957). Chemical
regulation
of
growth
and
organ
formation
in
plant
tissues
cultured
in vitro
Symp.
Soc
Exp
Biol
11 :
118-140.
Songstad D., Duncan D. and Widholm J. (1988). Effect of l-aminocyclopropane-l-carboxylic acid, silver nitrate, and norbornadiene on plant
regeneration from maize callus cultures. Plant Cell Rep., 7:262 - 265
Steen D.A. and Chadwick A.V. (1981). Ethylene effect in pica stems tissue: evidence of microtubule mediation. Plant Physiol., 67:460-466
Stone S.L., Williams L.A., Farmer L.M., Vierstra R.D. and Callis J. (2006). Keep on going, a ring E3 ligase essential for Arabidopsis growth and
development, is involved in abscisic acid signaling. Plant Cell, 18:34153428
Strader L.C. and Bartel B. (2008). A new path to auxin. Nat. Chem. Biol., 4:337339
Teale W.D., Paponov I.A. and Palme K. (2006). Auxin in action: signaling, transport and the control of plant growth and development. Nature
Rev. Mol. Cell Biol., 7 : 847-859
Tefera Wannakrairoj (2006). Synergistic effects of some plant growth regulators on in vitro shoot proliferation of korarima (Aframomum
corrorima (Braun) Jansen). Afr. J. Biotech., 5:1894-1901
To J.P. and Kieber J.J. (2008). Cytokinin signaling: two-components and more. Trends Plant Sci 13: 8592
Vieten A., Sauer M., Brewer P.B. and Friml J. (2007). Molecular and cellular aspects of auxin transport-mediated development. Trends Plant
Sci., 12:160168
Vesely J., Havlicek L. and Strnad M. (1994). Inhibition of cyclin-dependent kinases by purine analogues. Eur J Biochem 224:771-786
Went F.W. (1926). On growth accelerating substances in the coleoptile of Avena sativa. Proc. K. Ned. Akad. Wet. Ser. C., 30:10
Wildman S.G. (1997). The auxin-A, B enigma: Scientific fraud or scientific ineptitude? Plant Growth Regul., 22:37-68
Woodward A.W.and Bartel B. (2005). Auxin: regulation, action, and interaction. Ann. Bot. (Lond), 95:707735
Yamaguchi S. (2008). Gibberellin metabolism and its regulation. Ann. Rev. Plant Biol., 59:225251
Zimmermann J.L. (1993). Somatic embryogenesis: a model for early development in higher plants. Plant Cell, 5:1411-1423
Ziv M. and Ariel T. (1991). Bud proliferation and plant regeneration in liquid-cultured Philodendron treated with ancymidol and
paclobutrazol. J. Plant Growth Regul., 10:53-57
Zhang S., Han S., Yang W., Wei H., Zhang M. and Qi L. (2010). Changes in H2O2 content and antioxidant enzyme gene expression during the
somatic embryogenesis of Larix leptolepis. Plant Cell Tiss. Org. Cult., 100:2129
Zhang X., Garreton V., and Chua N.H. (2005). The AIP2 E3 ligase acts as a novel negative regulator of ABA signaling by promoting ABI3
degradation. Genes Dev., 19:15321543.

Abbreviations
ABA - abscisic acid; ACC - 1-aminocyclopropane-1-carboxylic acid; ACS -ACC synthase; AgNO3 Silver nitrate; AVG aminoethoxyvinylglycine; BA - 6-
benzyladenine; BAP - 6-benzylaminopurine; BSAA - Benzo(b)selenienyl-3 acetic acid; Ethephon - 2-chloroethylphosphonic acid; GA gibberellic acid; 2iP -
N6-(2-isopentenyl) adenine; IAA indole 3-acetic acid; IBA - indole-3-butyric acid; NAA - -napthaleneacetic acid; NPA - N-l-naphthylphthalamic acid; PGRs -
Plant growth regulators; 2,4,5-T - 2,4,5-trichlorophenoxyacetic acid.