Adventitious Root Formation in Chloranthus sp.

Stems via Waterlogging and

Auxin Treatment using Hydroponically-grown Seedlings and Stem Cuttings
Cumigad, I.D., Eduardo, A.J., and Gloria, P.C.,
Institute of Biology, University of the Philippines Diliman, Quezon City

ABSTRACT

Adventitious roots are roots that develop from any organ of the plant except the embryonic root.
Although part of the normal development, formation can be a response to stress conditions. In this
experiment, we studied the formation of adventitious roots in Chloranthus sp. seeds and stem cuttings by
hypoxia and phytohormone treatment. Chloranthus seeds were germinated in nutrient solution, acclimatized,
before they were transferred to polystyrene rafts. Hypoxia was induced by deoxygenizing and flushing of
nitrogen until oxygen concentration were 0.4-1.0 mg/L. For the auxin treatment set-up, the nutrient solution
was flushed with NAA and ethylene. Induction of adventitious roots from cuttings was induced by dipping 0.5
cm of the cutting the into powder ontaining 8 g/kg IBA. Cross sections were viewed under the microscope at
72, 96, 144, 192, and 240 hours post-excision. The formation of the meristemoid cells were observed after 72
hours while globular meristem formation was observed 96 hours after. The dome-shaped meristem and
primordia were already present after 144 hours and vascularization at 192 hours. The root emerged 240
hours post-excision. Both set ups yielded the same expected results and successfully induced formation of
adventitious roots. Further research could look into the effects of varying light intensities, moisture, and
temperature in Chloranthus adventitious root formations.

INTRODUCTION form of 1-Naphthaleneacetic acid (NAA) and

Adventitious roots are plant roots that form
from any non-root tissue and are produced both
during normal development and in response to
stress conditions, such as flooding, nutrient
deprivation, and wounding. In horticulture,
agriculture and forestry, adventitious root (AR)
formation is a prerequisite for the vegetative
propagation of many important crops and it is also
a common practice to maintain genetic identity in
progeny (Hartmann et al., 2011).
The de novo formation of ARs is a complex
developmental process during which the stem
base of cuttings undergoes a series of anatomical
and physiological transformations with the use of
phytohormones. Phytohormones are chemicals
that regulate plant growth, in which they affect the
gene expression and transcription levels, cellular
division, and growth of the plant (Srivastava,
2002). Plant hormones are not nutrients, but
chemicals that in small amounts promote and
influence the growth, development, and
differentiation of cells and tissues. There are five
major hormone classes which are based on their
structural similarities and on their effects on plant
physiology, namely: Abscisic Acid, Auxins,
Cytokinins, Ethylene and Gibberellins. (Weier et
al., 1979). To stimulate root growth from cuttings of
plants for vegetative propagation, auxins in the
Indole-3-butyric acid (IBA) are commonly used
(Osborne and McManus, 2005).
Traditionally, phases of AR formation are
classified into (i) induction, (ii) initiation of ARs,
and (iii) expression (Kevers et al., 1997; Li et al.,
2009). Induction of founder cells occurs before
any histological changes become apparent and
is associated with the physical separation of the
cutting from the stock plant. Disruption of polar
auxin transport caused by severance of the
cutting leads to the accumulation of indole-3-
acetic acid in its stem base, which is considered
to be a prerequisite for induction of the founder
cells (De Klerk et al., 1999; Garrido et al., 2002;
Pop et al., 2011; Ahkami et al., 2013).
Furthermore, disturbance of existing cell
connections and transport of assimilates causes
a depletion of assimilates that in turn leads to the
establishment of a new carbohydrate sink in the
stem base (Ahkami et al., 2009).
Chloranthus is a type genus of the family
Chloranthaceae, which consists of fragrant
shrubs or herbaceous plants that are commonly
occurring in Southeast Asia, the Pacific,
Madagascar, Central and South America, and
the West Indies. The family consists of four
extant genera (Hedyosmum, Ascarina,
Sarcandra, and Chloranthus), totalling about 77
known species (Christenhusz and Byng, 2016). It
is a plant of high importance and significance,
since it is among the early-diverging lineages in
the angiosperms with unknown closely-related
families among other families of angiosperms
(Angiosperm Phylogeny Group, 2009). In
particular, these plants are neither monocot nor
dicot and fossils assigned to the family
Chloranthaceae are among the oldest
angiosperms known (Angiosperm Phylogeny
Group, 2009). Chloranthus plants are used in
traditional medicine, in which aromatic oils may be
extracted from the leaves. Given that the entire
plant has anti-stress, antioxidant, anti-
inflammatory, detoxifying, blood activating, and
antibacterial effects, it has not yet been cultivated
by artificial introduction with the use of stem
cuttings (He et al., 2009).
In this study, Chloranthus sp. seeds and
stem cuttings were used to induce adventitious
root formation. Formation of adventitious roots
were induced by hypoxia (waterlogging) and
phytohormone (auxin and ethylene) treatment in
Chloranthus sp. seeds. Meanwhile, IBA, a form of
auxin was used to induce adventitious root
formation in Chloranthus sp. stem cuttings.
MATERIALS AND METHODS
Adventitious root induction via hypoxia and
ethylene treatment with growth starting from
the seed (adapted from Visser et al., 1996)
Plant Growth
Chloranthus sp. Seeds were collected and
sown in trays filled with polyethylene grains and
nutrient solution (Table IA). After germination,
seeds were subjected to a 16-hour day cycle under
PFFD light (27°C) and an 8-hour night cycle
(10°C). After 1 week, seeds were exposed in a
climate room with 16-hour day cycle (higher PFFD
intensity) and 8-hour night cycle (22°C).
Uniform seedlings were then transferred to
polystyrene rafts that floated on nutrient solution in
20-L containers (containing about 6-8 plants per
raft). Aeration was accomplished by flushing air
through bubble stones.
Hypoxia Treatment
This treatment was simulated by
transferring grown plants from nutrient solution to
unstirred agar solution (0.1% w/v with nutrient
concentration same as before). The agar solution
was deoxygenized by vigorous flushing of
nitrogen gas. Oxygen concentration was
estimated to be at 0.4-1.0 mg/L.
Auxin Treatment
Plants were transferred from containers
to nutrient solution flushed with NAA and
ethylene. 5% ethylene and NAA concentration
was maintained all throughout the experiment.
Adventitious root induction using
Chloranthus sp. Cuttings subjected to Indole-
3-butyric acid (IBA) (adapted from Bryant &
Trueman, 2015)
Seedlings were grown in 2.8L pots and
maintained with day-night cycle similar to the
previous method. When considerable height
was reached, cuttings were made from the plant.
The cuttings were dipped 0.5cm into
powder that contains 8 g/kg IBA for 3 sec, and
placed 1 cm deep into a 70mL micropropagation
tube. Cuttings were harvested 7, 14, 21, and 28
days.
Microscopy
Thin cross-sections were made from the
cuttings and examined under high power
objective (HPO) of a bright field microscope.
RESULTS
In the figure, adventitious root (AR)
formation can be seen in response to application
of auxin in the form of indole-3-butyric acid on
Chloranthus sp. stem bases. Five developmental
stages can be seen from the figure (indicated in
roman numerals), namely: meristemoid formation
(I) which occurred 72 hours post-excision,
globular meristem formation (II) which occurred
96 hours post-excision, formation of dome-
shaped meristems and AR primordia (III) which
occurred 144 hours post-excision, AR primordia
with developing vascularization and elongation of
cells (IV) which occurred 192 hours post-
excision, and emergence of AR (V) which
occurred 240 hours post-excision.
 Figure 1. Anatomy of adventitious root (AR) formation from the vascular ring of Chloranthus sp. stem
bases (cross-section). co, cortex; pp, pith parenchyma; vr, vascular ring.
DISCUSSION
In this study, adventitious roots were
induced in response to two stresses, namely: (1)
hypoxia (waterlogging of grown plants) and (2) in
response to wounding of the stem (cuttings).
Under aerated conditions, gaseous ethylene
escapes from plant tissues. However, during
hypoxic conditions, water acts as a physical
barrier, which traps ethylene in the plant
(Musgrave et al., 1972). Gibberellins enhance the
ethylene-promoted adventitious root growth, while
abscisic acid reduces the ethylene-promoted
adventitious growth (Steffens et al. 2006).
Ethylene triggers reactive oxygen species
production, and together they trigger epidermal
programmed cell death for root emergence and
cortical programmed cell death for lysigenous
aerenchyma formation. In dicots, auxin, in
addition to ethylene signaling is the main
requirement for de novo adventitious root initiation
(Steffens et al., 2012).
Wound-induced adventitious roots are
central to the propagation of forestry and
horticultural species. In intact plants, cytokinin and
strigolactones are predominantly produced in the
root, while auxin is predominantly produced in the
shoot. On wounding, jasmonic acid peaks within
30 minutes and is required for successful root
development (Ahkami et al., 2009). Reactive
oxygen species, polyphenols, and hydrogen
sulfide also increase and promote adventitious
rooting via reducing auxin degradation. Auxin
builds up in the base of the cutting, acting
upstream of nitric oxide to promote adventitious
root initiation (Zhang et al., 2009). Auxin, nitric
oxide, and hydrogen peroxide (H2O2) increase
soluble sugars, which can be used for root
development (Li et al., 2009). Furthermore, levels
of root initiation inhibitors such as cytokinin and
strigolactones are reduced with the removal of the
original root system (Rasmussen et al., 2012). At
later stages, auxin inhibits primordia elongation
while ethylene promotes adventitious root
emergence. As the new root system establishes,
the production of cytokinin and strigolactones is
restored (Gomez-Roldan et al., 2008).
The formation of adventitious roots occurs
naturally and is part of the development of most
plants. Adventitious root formation has three
major stages namely: induction, inititation and
expression. Induction is when the cells are
introduced to stimuli that promote adventitious
root growth, this is usually characterized by the
dedifferentiation of the cells. Initiation on the other
hand occurs when the cell is determined to form
an adventitious root, marked by the formation of
root initials and root primordia, and expression
refers to the maturation and further elongation of
the primordia leading to root emergence
(Beeckman, 2009).
The cell’s competence will determine
whether differentiation will occur. Competence
refers to the ability of specific, differentiated cells
to respond to stimuli which induces root-formation
(Altamura,1996). The molecular basis for cell
competence have not been determined yet;
however, it is theorized that cell receptors to the
root-inducing stimuli play a huge factor in the
cell’s competence (Mohnen, 1994). When a
competent cell is stimulated by root-inducing
factors, the cell becomes determined to initiate
root formation. Complete determination is
achieved when the inducer is removed and the
cell continues to develop into a root. Once a
competent cell is determined by inducers, it
causes the differentiated cell to dedifferentiate,
forming adventitious root initials (Altamura et
al.,1991). There are no cells specified to be
adventitious root initials, however, in some plants
like willow (Salix), adventitious root initials are
present but remain dormant until the stem has
been cut and placed in the water (Hartmann and
Kester, 1993).
The key stage in adventitious root
formation is the formation of the root initials. The
root initials are small meristematic cells with
chromophil nuclei and nucleoli and no starch
(Altamura, 1996). Root initials usually develop
near the vascular tissues and their continuous
divisions cause the formation of a root dome or
the root primordia. Once the root primordia have
been formed, the continuous divisions of the
meristematic cells lead to the further elongation of
the primordia leading to its emergence and
maturation; emergence and maturation can be
distinguished with the formed connection to the
vascular ring (Beeckman, 2009).
Hypoxia refers to a state of lack of oxygen
which usually occurs in plants after floods since
gas exchange is 10,000 times slower in water
than in air (Armstrong et al., 1991). Well adapted
plants such as those thriving in wetlands, develop
adventitious roots with aerenchyma - air channels
that allow gas diffusion to the shoot (Dawood et
al., 2013). Flooding alone does not induce
formation of adventitious roots, increased
ethylene production caused by the flooding results
to various responses depending on the plant
species. Ethylene responses may vary from
epinasty, hyponastic growth, shoot elongation,
aerenchyma formation and adventitious root
development, all of which aids the plant escape
the hypoxic environment, or improve oxygen
uptake (Jackson, 2002; Visser and Voesenek,
2004; Voesenek et al., 2006; Bailey-Serres and
Voesenek, 2008).
Ethylene and auxin signals are intertwined
in producing adventitious roots. Hypoxia leads to
an increased biosynthesis of ethylene which are
then perceived by the NR receptor, triggering the
transport of auxin towards the flooded part of the
plant. The accumulation of auxin in the stem
stimulates further ethylene production, causing a
flux of auxin in the base of the stem. This
accumulation of auxin at the plant’s base lead to
the formation of adventitious roots (Vidoz et al.,
2013).
Aside from auxin, other hormones are
involved in adventitious root formation namely:
cytokinins, gibberellins, abscisic acid, and
ethylene. Cytokinin promotes cytokinesis,
vascular cambium sensitivity, vascular
differentiation, and root apical dominance (Aloni et
al., 2006). Root tips are the major sites of
cytokinin synthesis (Aloni et al., 2004). There are
reports that exogenous cytokinin application
inhibits adventitious root formation (Bollmark and
Eliasson, 1986). Also, endogenous expression of
zeatin (a type of cytokinin) in pea plants showed
strong inhibition of adventitious roots in the
hypocotyl (Kurohah & Satoh, 2007). Since
cytokinins are transported in the root, they travel
using the transpiration system or the xylem sap.
In the context of zeatin and pea plant, zeatin is
first converted into trans-zeatin riboside before
translocation into the shoot. And the trans-
configuration of zeatin is responsible for
adventitious root inhibition (Kurohah & Satoh,
2007).
Gibberellic acid (GA) is a tetracyclic
diterpenoid compound that stimulates plant
growth and development (Gupta & Chakrabarty,
2013). GA was shown to work synergistically with
ethylene to induce adventitious roots in rice
(Steffens et al., 2006). They claim that GA activity
controls root emergence in rice and GA
concentration controls root growth. They also
claim that endogenous GA promotes growth
induced by ethylene. In Arabidopsis, GA signalling
which results to AR growth is allowed via the
degradation of DELLA proteins (Fu et al., 2002).
Hence, there is a close relationship between
ethylene and GA.
Ethylene, together with GA, positively
regulates AR formation (Steffens et al., 2006). It
plays a central role in regulating AR root growth
because ethylene-insensitive transgenic plants
showed no or reduced AR growth. This hormone
accumulates in flooded plant parts because its
gaseous form traps it in the intercellular spaces
(Musgrave et al., 1972). Endogenous
accumulation causes nodal adventitious root
primordia to exert mechanical force on overlying
epidermal cells which result in epidermal
apoptosis (Steffens et al., 2012). The close
relationship between ethylene and GA is
established by experiments showing that ethylene
alone can induce root emergence but GA cannot
however presence of both results in growth. This
is explained via the GA signalling which involves
DELLA proteins. DELLA proteins accumulate in
the nucleus as a response to ethylene while the
response of DELLA to GA is mentioned in the
above paragraph. Hence, a crosstalk is formed
using this protein which links their relationship
(Steffens et al., 2006). While synergistic
relationship exists between GA and ethylene,
abscisic acid exerts an antagonistic effect on
them (Steffens et al., 2006).
Abscisic acid (ABA) is a competitive
inhibitor of GA activity and of ethylene signaling
(Steffens et al., 2006). It is an important signal
molecule for abiotic stress adaptation but also
acts as a developmental signal. It is inhibitory on
seedling root growth (Beaudoin et al., 2000) and
development of lateral roots (De Smet et al.
2003). ABA negatively controls root emergence.
10uM of ABA resulted to a 50% decrease in AR
penetration in rice (Steffens et al., 2006).
Similarly, it inhibits root growth and was shown to
primarily act using the ethylene signaling
pathway. This was established because ABA was
not able to affect ethylene mutants. So far, only
the hormones central to AR formation has been
discussed. Equally important are the genes at
play to induce ARs.
Whole genome transcriptomic analysis
reveals that genes associated with metabolism
(GO Term: biological process) and catalytic
activity (GO Term: molecular process) are highly
upregulated in shoots subjected to AR stimulating
factors (Park et al., 2017). In the context of
hormone-associated genes, auxin genes are both
up- and down-regulated while ethylene and GA-
associated genes are down-regulated. Cytokinin
related genes were found to be the same (Park et
al., 2017).
CYP79B3, a gene encoding cytochrome
P450 enzyme of auxin biosynthesis, was highly
upregulated (Park et al., 2017). This is involved in
the indole-3-acetaldoxamine (IAOx) pathway
among multiple pathways for auxin biosynthesis.
Logically, auxin responsive factor genes are also
significantly upregulated and as a consequence,
transcriptional repressors of auxin elements are
downregulated (Park et al., 2017).
Specific genes responsible for certain
steps in AR were also identified in numerous
studies. ROOT REDIFFERENTIATION (RRD)
were identified in Arabidopsis (Sugiyama, 2003).
RRD1 and RRD2 are for active cell proliferation
while RRD4 is needed in hypocotyl generation
from callus. SCARECROW-LIKE1 and SHORT-
ROOT genes identified from forest species are
found to be involved in the earliest stages of AR
formation as they play a role in auxin-signaling
pathways in rooting-competent cuttings (Sanchez
et al., 2007; Sole et al., 2008; Ricci et al., 2008).
PINHEAD/ZWILLE-like proteins were upregulated
during root initiation phase where they play a role
in cell replication and cell wall weakening (Brinker
et al., 2004). Most importantly, Auxin Response
Factors (ARFs) are transcription factors which
regulate auxin response genes (Guilfoyle and
Hagen, 2007). They play an important role in
allowing auxin to produce its specific effects in
inducing AR. Aside from endogenous factors
which affect AR induction; external factors are of
relevance in determining a response.
When a stem cutting with axillary bud is
used to induce AR, rooting is exhibited. However,
when the buds are removed or the phloem
beneath the node is removed, rooting is not
induced (Kibbler et al., 2004). Presence of leaves
in a cutting help induce rooting because
carbohydrates and auxin from the leaf travel to
the AR initiation sites (Koyuncu & Balta, 2004).
Polyphenol oxidase and IAA oxidase are group of
compounds known as rooting co-factors which act
synergistically with auxin to induce rooting in
selected cuttings to induce AR (Jackson &
Harney, 2011).
Environmental factors such as moisture
and temperature are shown to be important in
adventitious root induction (Briske & Wilson,
1978). Their experiment utilized Bouteloua grama
(Blue Grama) which needed 96% moisture and
15°C to form AR. Light intensity, duration and
oxygen concentration are equally important in AR
induction (Lee, 2011). The physiological status of
a plant should be also considered. This includes
(1) stock plant health and etiolation, (2)
carbohydrate content, (3) mineral nutrition, and
(4) girdling. Factors for the selection of wood to be
used should also be considered and include the
following: (1) lateral versus terminal shoots (2)
proximal versus distal selections (3) flowering
versus vegetative shoots and (4) ‘heel’ versus
‘non-heel’ cuttings (Lee, 2011).

Knowledge of AR induction mechanisms
and techniques are valuable because it is usually
used in asexual reproduction of important crops.
Hence, knowledge derived from basic and applied
research regarding AR technologies are
indispensable in agriculture and horticulture
(Bagherabadi, 2016).
SUMMARY AND CONCLUSION
In this experiment, we studied the
feasibility of the formation of adventitious roots by
hypoxia and phytohormone treatments in
Chloranthus sp. seeds and cuttings. Both set-ups
yielded the same expected results and
successfully induced formation of adventitious
roots.
The mechanisms of actions of the two
treatments were also studied. Waterlogging leads
to a hypoxic environment due to the slow diffusion
of gas in water. The hypoxic environment leads to
LITERATURE CITED
Ahkami AH , Lischewski S , Haensch KT et al.
(2009). Molecular physiology of
adventitious root formation in Petunia
hybrida cuttings: involvement of wound
response and primary metabolism. New
Phytologist 181, 613–625.
Ahkami AH , Melzer M , Ghaffari MR , Pollmann S
, Ghorbani Javid M , Shahinnia F ,
Hajirezaei MR , Druege U . (2013)
Distribution of indole-3-acetic acid in
Petunia hybrida shoot tip cuttings and
relationship between auxin transport,
carbohydrate metabolism and adventitious
root formation. Planta 238, 499–517.
Aloni R., Langhans, M., Aloni, E., Ullrich, C.I.
(2004). Role of Cytokinin in the Regulation
of Root Gravitropism. Planta, 220(1), 117-
82.
Aloni,R. ., Aloni, E.., Langhans, S.., & Ullrich, C. I.
(2006). Role of Cytokinin and Auxin in
Shaping Root Architecture: Regulating
Vascular Differentiation, Lateral Root
Initiation, Root Apical Dominance and
Root Gravitropism. Annals of Botany,
97(5), 883–893.
an increased biosynthesis of ethylene which then
triggers the transport of auxin to the flooded part
of the plant. The increased concentration of auxin
leads to the production of more ethylene, forming
a positive feedback loop, further increasing the
auxin concentration which initiates the
adventitious root formation. In line with this,
cuttings with leaves showed more production of
adventitious roots since carbohydrates and auxin
from the leaf travel to the AR initiation sites.
The effects of other hormones such as
gibberellic acid and abscisic acid were also
explored and the roles of genes such as
CYP79B3 and the RRD in adventitious root
formation were identified.
Environmental factors affecting
adventitious root formation in Chloranthus were
not fully explored. Further research could look
into the effects of varying light intensities,
moisture, and temperature in Chloranthus
adventitious root formations.
Altamura MM, Capitani F, Serafini-Fracassini
D,Torrigiani P, Falasca G. (1991). Root
histogenesis from tobacco thin cell layers.
Protoplasma 161, 31-42
Altamura MM. (1996). Root histogenesis in
herbaceous and woody explants cultured
in vitro. A critical review. Agronomie 16,
589-602
Angiosperm Phylogeny Group III (2009). "An
update of the Angiosperm Phylogeny
Group classification for the orders and
families of flowering plants: APG III".
Botanical Journal of the Linnean Society.
161 (2): 105–121.
Armstrong W, Justin SHFW, Beckett PM, Lythe S.
Root adaptation to soil waterlogging.
Aquatic Botany. 1991;39:57–73.
Bagherabadi, M. (2016). Adventitious root
formation in Arabidopsis: underlying
mechanisms and applications (PhD
Dissertation), Laboratory of Plant
Breeding, Wagenigen University,
Netherlands.
Bailey-Serres, J. and Voesenek, L.A.C.J. (2008)
Flooding stress: acclimations and genetic
 diversity. Annu. Rev. Plant Biol. 59, 313–
339.
Beaudoin N, Serizet C, Gosti F, Giraudat J (2000)
Interactions between abscisic acid and
ethylene signaling cascades. Plant Cell
12:1103–1115
Beeckman, T. (2009). Root development.
Chichester, West Sussex, U.K.: Wiley-
Blackwell.
Bollmark M, Eliasson L (1986) Effects of
exogenous cytokinins on root formation in
pea cuttings. Physiol. Plant. 68:662-666.
Brinker M, van Zyl L, Liu WB, Craig D, Sederoff
RR, Clapham DH, von Arnold S (2004).
Microarray analyses of gene expression
during adventitious root development in
Pinus contorta. Plant Physiol 135(3):1526-
1539.
Brisk, D. & Wilson, M. (1978). Moisture and
Temperature Requirements for
Adventitious Root Development in Blue
Grama Seeds. Journal of Range
Management, 31(3), 174-178.
Bryant, P.H., & Trueman, S.J. (2015). Stem
Anatomy and Adventitious Root Formation
in Cuttings of Angophora, Corymbia, and
Eucalyptus. Forests, 6, 1227-1228.
Christenhusz, M. J. M.; Byng, J. W. (2016). "The
number of known plants species in the
world and its annual increase". Phytotaxa.
Magnolia Press. 261 (3): 201–217.
Dawood, T., Rieu, I., Wolters-Arts, M., Derksen,
E. B., Mariani, C., & Visser, E. J. W.
(2014). Rapid flooding-induced
adventitious root development from
preformed primordia in Solanum
dulcamara. AoB Plants, 6, plt058.
http://doi.org/10.1093/aobpla/plt058
De Klerk G-J , Van Der Krieken W , De Jong JC .
(1999). The formation of adventitious
roots: new concepts, new possibilities. In
Vitro Cellular & Developmental Biology
35, 189–199.
De Smet I, Signora L, Beekman T, Inze D, Foyer
CH, Zhang H (2003) An abscisic acid-
sensitive checkpoint in lateral root
development of Arabidopsis. Plant J
3:543–555
Fu, X, Harberd NP (2003) Auxin promotes
Arabidopsis root growth by modulating
gibberellin response. Nature 421:740– 743
Garrido G , Ramón Guerrero J , Angel Cano E ,
Acosta M , Sánchez-Bravo J . (2002).
Origin and basipetal transport of the IAA
responsible for rooting of carnation
cuttings. Physiologia Plantarum 114, 303–
312.
Gomez-Roldan V, Fermas S, Brewer PB, Puech-
Pagès V, Dun EA, Pillot JP, Letisse F,
Matusova R, Danoun S, Portais JC, et al.
(2008) Strigolactone inhibition of shoot
branching. Nature 455: 189–194
Guilfoyle TJ, Hagen G (2007). Auxin response
factors. Current Opin Plant Biol 10(5):453-
460.
Gupta, R., & Chakrabarty, S. K. (2013).
Gibberellic acid in plant: Still a mystery
unresolved. Plant Signaling & Behavior,
8(9), e25504.
Hartmann HT , Kester DE , Davies FT , Geneve
RL . (2011). Hartmann and Kester’s plant
propagation-principles and practices . New
Jersey: Prentice Hall.
He, RR; Yao, XS; Li, HY; Dai, Y; Duan, YH; Li,
YF; Kurihara, H (2009). The anti-stress
effects of Sarcandra glabra extract on
restraint-evoked immunocompromise. Biol.
Pharm. Bull. 32: 247–52.
Jackson, M. & Harney, P. (2011). Rooting
cofactors, indole acetic acid, and
adventitious root initiation in mung bean
cuttings (Phaseotus aureus), Canadian
Journal of Botany, 48(5), 943-946.
Jackson, M.B. (2008) Ethylene-promoted
elongation: an adaptation to submergence
stress. Ann. Bot. 101, 229–248.
Kevers C , Hausman JF , Faivre-Rampant O ,
Evers D , Gaspar T . (1997) Hormonal
control of adventitious rooting: progress
 and questions. Angewandte Botanik 71,
71–79.
Kibbler, H., Johnston, M., Williams, R. (2004).
Adventitious root formation in cuttings
Backhousia citriodoria F. Muell: 1. Plant
Genotype, juvenility, characteristics of
cuttings. Scientia Horticulturae, 102(1),
133-143.
Kurohah, T., Satoh, S. (2006). Involvement of
Cytokinins in Adventitious and Lateral
Root Formation. Plant Root, 1, 27-33I.
Kuyuncu, E. & Balta, F. (2004). Adventitious root
formation in leaf-bud cuttings of tea
(Camellia sinensis L.), Pakistan Journal of
Botany.
Lee, C. (2011). Principles of Propagation by
Cuttings. North Dakota State University.
Li SW , Xue L , Xu S , Feng H , An L . 2009.
Mediators, genes and signaling in
adventitious rooting. Botanical Review 75,
230–247.
Mohnen, D. (1994). Novel experimental systems
for determining cellular competence and
determination.In: Biology of Adventitious
Root Formation
Musgrave A, Jackson MB, Ling E (1972)
Callitriche stem elongation is controlled by
ethylene and gibberellin. Nature 238: 93–
96
Osborne, Daphné J.; McManus, Michael T.
(2005). Hormones, signals and target cells
in plant development. Cambridge
University Press. p. 158. ISBN 978-0-521-
33076-3.
Park, S., Elhiti, M., Wang, H., Xu, A., Brown, D.,
Wang, A. (2017). Adventitious root
formation In Vitro peach shoots is
regulated by auxin and ethylene. Scientia
horticulturae, 226, 250-260.
Pop TI , Pamfil D , Bellini C . (2011). Auxin control
in the formation of adventitious roots.
Notulae Botanicae Horti Agrobotanici Cluj-
Napoca 39, 307–316.
Rasmussen A, Beveridge CA, Geelen D (2012)
Inhibition of strigolactones promotes
adventitious root formation. Plant Signal
Behav 7: 694–697.
Ricci A, Rolli E, Dramis L, Diaz-Sala C (2008).
N,N ‘-bis- (2,3-methylenedioxyphenyl)urea
and N,N ‘-bis-(3,4-
methylenedioxyphenyl)urea enhance
adventitious rooting in Pinus radiata and
affect expression of genes induced during
adventitious rooting in the presence of
exogenous auxin. Plant Sci 175(3):356-
363.
Sanchez C, Vielba JM, Ferro E, Covelo G, Sole A,
Abarca D, De Mier BS, Diaz-Sala C
(2007). Two SCARECROW-LIKE genes
are induced in response to exogenous
auxin in rootingcompetent cuttings of
distantly related forest species. Tree
Physiol 27(10):1459-1470.
Sole A, Sanchez C, Vielba JM, Valladares S,
Abarca D, Diaz-Sala C (2008).
Characterization and expression of a
Pinus radiata putative ortholog to the
Arabidopsis SHORT-ROOT gene. Tree
Physiol 28(11):1629-1639.
Srivastava, L. M. (2002). Plant growth and
development: hormones and environment.
Academic Press. p. 140. ISBN 0-12-
660570-X.
Steffens B, Kovalev A, Gorb SN, Sauter M (2012)
Emerging roots alter epidermal cell fate
through mechanical and reactive oxygen
species signaling. Plant Cell 24: 3296–330
Steffens, B., Wang, J., Sauter, M. (2006).
Interactions between ethylene, gibberellin,
and abscisic acid regulate emergence and
growth rate of adventitious roots in
deepwater rice. Planta, 223, 604-612.
Sugiyama M (2003). Isolation and initial
characterization of temperature-sensitive
mutants of Arabidopsis thaliana that are
impaired in root redifferentiation. Plant Cell
Physiol 44(6):588-596.
Vidoz, M. L., Loreti, E., Mensuali, A., Alpi, A. and
Perata, P. (2010), Hormonal interplay
during adventitious root formation in
 flooded tomato plants. The Plant Journal,
63: 551–562. doi:10.1111/j.1365-
313X.2010.04262.x
Visser, E.J.W. and Voesenek, L.A.C.J. (2004)
Acclimation to soil flooding – sensing and
signal-transduction. Plant Soil, 254, 197–
214.
Visser, E.J.W., Cohen, J.D., Gerard, W.M.,
Barendse, G.W., Cornelis, W.P., Blom, M.,
Laurentius, A., Voesenek, C.J. (1996). An
Ethyelene-Mediated Increase in Sensitivity
to Auxin Induces Adventitious Root
Formation in Flooded Rumex palustris
Stem. Plant Physiology, 122, 1687-1692.
Voesenek, L.A.C.J., Colmer, T.D., Pierik, R.,
Millenaar, F.F. and Peeters, J.M. (2006)
How plants cope with complete
submergence. New Phytol. 170, 213–226.
Weier, Thomas Elliot; Rost, Thomas L.; Weier, T.
Elliot (1979). Botany: a brief introduction to
plant biology. New York: Wiley. pp. 155–
170.
Zhang H, Tang J, Liu XP, Wang Y, Yu W, Peng
WY, Fang F, Ma DF, Wei ZJ, Hu LY
(2009) Hydrogen sulfide promotes root
organogenesis in Ipomoea batatas, Salix
matsudana and Glycine max. J Integr
Plant Biol 51: 1086–1094.

APPENDIX

Table IA. List of micronutrients and

macronutrients included
in the nutrient medium

Reagent Concentration

Ca(NO3)2 2mM

K2SO4 1.25mM

MgSo4 0.5mM

K2PO4 0.5mM

Fe-EDTA 90 uM

NaCl 50uM

H3BO3 25uM

MnSO2 2uM

CuSO4 0.5uM

H2MoO4 0.5uM