in vitro direct rhizogenesis from gerbera jamesonii bolus leaf
TRANSCRIPT
SHORT COMMUNICATION
In vitro direct rhizogenesis from Gerbera jamesonii Bolus leaf
Saikat Gantait • Uma Rani Sinniah
Received: 18 January 2014 / Revised: 18 July 2014 / Accepted: 20 July 2014
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014
Abstract The present report describes an original proto-
col for in vitro direct induction of roots from leaf explants
of gerbera for the first time. Since gerbera has immense
potential as a premium cut-flower, the major attempts were
made on in vitro mass propagation chiefly through in vitro
multiple shoot proliferation or callus regeneration. Never-
theless, rhizogenesis could be impending an unattempted
method with its yet-to-be known advantages. In our study,
the optimum conditions for direct root induction from leaf
explants were assessed employing tissue culture technique.
Leaves were inoculated to MS medium containing no or
variable auxin sources and concentrations namely, 2,4-
dichlorophenoxyacetic acid, indole-3-acetic acid (IAA),
indole-3-butyric acid or a-naphthaleneacetic acid for root
induction. It was evident that the maximum root induction
(with a frequency of 92.6 %) occurred on MS media for-
tified with 1.5 mg l-1 IAA, wherein root induction was
observed as early as 11 days of culture and an average of
*19 roots with *13 mm length was obtained from 4 cm2
leaf segment after 45 days of culture. Stereo microscopic
observation revealed the induction of roots and gradual
developmental stages of rhizogenesis. The efficiency of
direct root induction without any interim growth stages
(such as, callus or shoots) in our study offers a reproducible
system that could provide a model protocol for more
comprehensive developmental studies on root growth.
Keywords Adventitious rooting � Auxins � Coumarin �Developmental stages � Leaf explant
Abbreviation
2,4-D 2,4-Dichlorophenoxyacetic acid
IAA Indole-3-acetic acid
IBA Indole-3-butyric acid
MS Murashige and Skoog medium
NAA a-Naphthaleneacetic acid
PGR Plant growth regulator
PPFD Photosynthetic photon flux density
Introduction
Gerbera (Gerbera jamesonii Bolus), belonging to the
family Asteraceae, is considered as one of the most popular
cut-flowers, as well as potted floriculture plant. Owing to
its array of colourful inflorescences it has attained out-
standing commercial importance as cut-flowers (Gantait
et al. 2011). The gerbera plant is also used in the prepa-
ration of traditional Chinese medicine (Singh 2006). It
contains naturally occurring three new coumarin deriva-
tives 1–3 and a known coumarin 4 (Inoue et al. 1989; Liu
et al. 2010; Qiang et al. 2011). Coumarins from Gerbera
were found to have broad spectrum anti-tumor and anti-
bacterial activities in vitro and in vivo and are considered
to be potential new cancer chemoprevention agents (He
et al. 2014). It is a clonally propagated plant and shows
restricted growth of apical meristem which restrains the
Communicated by B. Borkowska.
S. Gantait � U. R. Sinniah
Department of Crop Science, Faculty of Agriculture, Universiti
Putra Malaysia, 43400 Serdang, Selangor, Malaysia
S. Gantait (&)
Sasya Shyamala Krishi Vigyan Kendra, Ramakrishna Mission
Vivekananda University, Arapanch, Sonarpur,
Kolkata 700150, India
e-mail: [email protected]
123
Acta Physiol Plant
DOI 10.1007/s11738-014-1643-4
axillary buds from developing offshoots. Consequently, a
low rate of multiplication is evident during conventional
propagation. Tissue culture systems for mass propagation
of gerbera, as a substitute, have been effectively com-
menced by several researchers. Shoot tip, young capitulum
and axillary bud culture are the most frequently used
methods in gerbera for direct organogenesis such as mul-
tiple shoot, somatic embryo (Gantait et al. 2010).
Direct induction of only roots exclusive of any inter-
mittent organs such as callus or shoots or somatic embryos
could be a simple yet efficient way to mass produce root-
specific secondary metabolites escaping transgenic inter-
vention (Tennant 2010). Direct root induction from leaf
explant as a method for the rapid root regeneration has
been reported earliest by Tran Thanh Van et al. (1974) on
Begonia rex Putz., later on several researchers reported this
system was employed for several plants viz, Lycopersicon
esculentum Mill. (Coleman and Greyson 1976), Nicotiana
tabacum Linn. (Attfield and Evans 1991), Helianthus oc-
cidentalis Riddell (Samaj et al. 1999), Eustoma grandiflo-
rum Shinn. (Tuan et al. 2006), and Ophiorrhiza prostrata
D. Don (Martin et al. 2008).
Compositions of plant growth regulator (PGR) have
been reported to encompass species-specific or even cul-
tivar-specific effect on in vitro root induction, with auxins
(both in types and concentrations), showing the most
imperative effect on direct root induction (Gantait et al.
2009; Teixeira da Silva 2013). It is noteworthy to mention
that auxin-induced rooting through the application of single
doses of auxins, usually singly, was effective in Chrysan-
themum (Dendranthema 9 grandiflora Ramat. Kitamura)
(Teixeira da Silva 2003), Anthurium andreanum Lind., and
Gerbera jamesonii Bolus (Gantait et al. 2008, 2010). The
influence of auxins on direct rhizogenesis from leaf seg-
ments has been reported on Boerhaavia diffusa Linn.
(Shrivastava and Padhya 1995), Rauwolfia serpentina (L.)
Benth (Pandey et al. 2010), Costus igneus Linn. (Nagarajan
et al. 2011), and Carica papaya Linn. (Teixeira da Silva
2013).
In our knowledge, to the current date, no study has been
done on direct induction of root in gerbera. Hence, con-
sidering the competence of leaf explants, the establishment
of a protocol for direct induction of roots from leaf and
their developmental study will be of practical importance.
Counting Gerbera jamesonii Bolus, there are several
ornamental plants species of Asteraceae such as Calendula
officinallis Linn., Chrysanthemum indicum (L.) Desr.,
Spilanthes acmella Auct. Plur., Tagetes erecta Linn.,
Wedelia calendulacea (L.) Less. those are also well known
for their pharmaceutical values due to the presence of
secondary metabolites (Rothe et al. 2011) which can be
accumulated in root system (Qiang et al. 2011). A repro-
ducible rhizogenic system thus would be if practical
importance to several other species of Gerbera carrying
secondary metabolites. Hence, in this article we propose an
accelerated rhizogenesis system of gerbera, a renowned
cut-flower with pharmaceutical values, via direct root
induction from leaf segments by adjusting types and con-
centrations of auxins, followed by the developmental study
at morpho-anatomical level.
Materials and methods
Plant material
One-year old gerbera plants (Gerbera jamesonii Bolus cv.
Sciella) were collected from commercial nursery and
kept growing under polyhouse, with a maximum
200 lmol m-2 s-1 photosynthetic photon flux density
(PPFD) at Universiti Putra Malaysia, Selangor, Malaysia.
Young fully opened leaves of 4–5 cm length were taken
from the source plant 20 days following their emergence
and used for in vitro study.
Surface disinfection and explant preparation
Collected leaves were rinsed for 3 min in tap water; next
submerged in 1 % (v/v) Tween-20 (dissolved in sterile-
distilled water) in a glass beaker for 5 min, 70 % (v/v)
ethanol for 30 s, 3 % (v/v) hydrogen peroxide for 5 min,
and lastly in 20 % Clorox� (Clorox Co., Broadway, Oak-
land, CA, USA) (1.2 % sodium hypochlorite) solution for
15 min, with thorough shaking under a laminar air flow.
Instant sonication in an ultrasonic cleaner was done with
the leaves at full power for 10 min. Subsequently; the
leaves were thoroughly rinsed three times with sterile-
distilled water, every time after decanting the surface dis-
infectant. Then these were shifted to a sterile Petri dish
(15 9 150 mm) for brief drying for 5 min. Each leaf was
cut perpendicularly to its axis into 4 cm2 sections by a
sharp sterile scalpel.
Culture conditions
The basal MS (Murashige and Skoog 1962), containing
3 % (w/v) sucrose and 100 mg l-1 myo-inositol, gelled
with agar was used. Auxins (Sigma-Aldrich, St. Louis,
USA) were added to the medium as specified below. The
pH of the medium was adjusted to 5.8 using 1 N NaOH or
HCl, prior to addition of solidifying agent. After the
addition of 7 % (w/v) agar, media were sterilized in an
autoclave (Hirayama, Japan) at 121 �C and 1 kg cm-2 for
20 min. Auxin solutions were filter-sterilized (membrane
filtration 0.22 lm) and added to autoclaved basal medium
when the temperature fallen to 50 �C. Medium (20 ml) was
Acta Physiol Plant
123
dispensed into each of the culture tubes (50 mm
height 9 25 mm inner diameter with 1 mm thick) and kept
those slant. Polypropylene autoclavable lids (24 mm
height 9 26 mm inner diameter with 1 mm thick) were
used as closures for culture tubes. The cultures were
maintained at 25 ± 1 �C under a 12 h photoperiod, which
was provided by cool, white fluorescent light (Phillips Life
Max, Thailand) with an irradiance of 60 lmol m-2 s-1
PPFD (measured with LI-COR, Lincoln, USA).
Optimization of auxins in culture medium for root
induction
Sterilized explants were placed adaxial side down in culture
tube containing MS medium supplemented with 0.5, 1.0, 1.5,
and 2.0 mg l-1 of either 2,4-dichlorophenoxyacetic acid
(2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid,
(IBA) or a-naphthaleneacetic acid (NAA), to assess the
effects of the auxin types and concentrations on root induc-
tion and development. MS medium without any PGR was
treated as control. All cultures were grown under environ-
mental conditions as mentioned above. The cultures were
sustained till initiation and development of roots occurred.
Observation was carried out frequently in order to record the
time (in days) taken to root induction. Data collection on
frequency of root induction from explants, and mean number
of root induced per explant was made at 45 days after the
inoculation of explants. In addition, the lengths of the indi-
vidual root were measured. Microscopic observations were
carried out at 109 magnification using a Leica stereozoom
microscope (Leica, Germany) at every week of culture. The
frequency of root induction was determined after 30 days,
using the formula: F = [(E/E0) 9 100] %; wherein: F fre-
quency of root induction, E number of explants inducing
roots, and E0 number of explants inoculated.
Morpho-anatomical observation
Detailed morphological and anatomical examination and
photography of the cultures were carried out using a digital
camera (Panasonic LX5, Japan) and stereozoom micro-
scope (Leica, Germany), correspondingly. Once the
spherules (a protruding structure induced from the undif-
ferentiated tissue prior to root formation) were formed,
observations were recorded weekly to trace stage-wise root
development. For stereo microscopic examination, about
10 roots at each developmental stage were indiscriminately
collected from culture tubes.
Experimental design and statistical analysis
The experiments were arranged in completely randomized
design (CRD). All the aforementioned experiments were
replicated three times, using 20 samples per replicate. Each
inoculated leaf segments were considered as single exper-
imental unit. Visual observations were made every single
day. Data were statistically analysed by means of SAS�-
vers. 6.12 (SAS Institute, Cary, NC, USA) (SAS Institute
1999) by ANOVA. Statistical significance between
mean ± SE were compared based on Duncan’s multiple
range test (DMRT) (Duncan 1955) at a P value = 0.05.
Data expressed as percentage were transformed using arc
sine before ANOVA and converted back to the initial scale
(Compton 1994).
Results and discussion
Gerbera, employed in the present study to establish a direct
root induction protocol using leaf segments, gave a number
of significant results. It was found that leaf segments could
be used to induce roots even in PGR-free media. Never-
theless, the competence of leaf explants inducing roots
significantly varied with the auxin types and levels tested,
in terms of percentage induction, the number of roots and
time taken for root induction. Interestingly, auxin-free
medium induced in vitro roots but with very low frequency
and less number. In addition, the time taken to induce roots
was much longer as compared to the media supplemented
with auxins (Table 1). Fortification of three different aux-
ins (IAA, IBA and NAA) at four levels of 0.5–2 mg l-1 in
MS medium was indispensable for enhanced root induction
and well developed root system within a brief culture
period, whilst 2,4-D was not beneficial like control and was
able to result in analogous response (Table 1). All treat-
ments inclusive of control but excluding 2,4-D with higher
concentrations, resulted in root induction with varying
frequencies (from 17.3 to 92.6 %). There was a significant
difference (P = 0.05) among the effects of four auxin
sources according to Duncan’s multiple range test. Root
induction in the presence of 2,4-D was very low in com-
parison to other auxins used, to the point no root induction
was observed when concentration was more than 1 mg l-1.
Amid IBA and NAA, the latter showed improved effect on
root induction, whereby very low concentration of
0.5 mg l-1 triggered 74.3 % root induction from the
wounded leaf surface. Rising the concentration of these
two PGRs, appeared to have inhibitory influence on rhi-
zogenesis. Among the auxins employed, induction of root
was most proficient in the presence of IAA where
*81–93 % induction was recorded with IAA supplemen-
tation ranging from 0.5 to 2 mg l-1. The superlative
response was recorded for the medium fortified with
1.5 mg l-1 IAA, on which 92.6 % of the explants induced
on an average of 19 roots per 4 cm2 leaf segment, within
45 days of culture. The experiment was evaluated at
Acta Physiol Plant
123
45 days of culture since, at the initial stage roots were
appeared as a mass of structures, making them unfeasible
to count, even with a stereomicroscope.
The morphogenetic phases from direct induction to
development of roots are reviewed in Fig. 1. The initial
symptom of morphogenesis was observed in the form of
swelling from the cut portions (Fig. 1a). As reviewed by
Blakesley et al. (1991) the first visible sign of a new
meristematic locus was the expansion of a single cell, with
simultaneous nuclear swelling. The cytoplasm of such cells
was much denser than that of adjacent cells. However, this
morphogenesis turned either into callus mass (Fig. 1b) with
the influence of higher 2,4-D levels or induced roots
directly. Initial morphological signal of root induction
appeared as swollen, small nodule-like meristemoids on the
wound abaxial surface of leaves, within 11 days after
inoculation of explants (Fig. 1c). Subsequently the nodules
advanced into slender cylindrical bodies with globular root
primordial tip, rough surfaces and were pale pinkish in
color (Fig. 1d). The basal part of the protruding structures
appeared to have tiny root hairs. The whole leaf segment
disorganized with subsequent rhizogenesis. This formula-
tion also resulted in the highest mean length (*13 mm) of
roots. The progressive morphological change during
growth and development of the root system appearance
during the maturity stage is shown in Fig. 1e. Roots started
to disintegrate and became prominent from each other as its
growth progressed to a more advanced developmental
stages. However, with excess auxin concentration, the
development of rhizogenesis was affected with reduced
root length and profuse root hairs formation (Fig. 1f).
Although little research has been carried out on in vitro
direct root induction or organogenesis from leaf explants in
ornamentals, there are a few describing structural details
(Blakesley et al. 1991; Gantait et al. 2012). However, this
is the first study, which traced the various developmental
stages of induction of roots through direct organogenesis
with naked eye with corroboration to the details derived
from micromorphological observations in gerbera. The
formation of initial swelling, induction of primordial
structure and complete rootlets with root cap has been
revealed to be imperative events in the development of
in vitro rhizogenesis. Direct in vitro induction of roots from
leaf explants was first documented by Tran Thanh Van
et al. (1974). Since then a number of studies on direct root
induction using leaf explants have been reported (Coleman
and Greyson 1976; Attfield and Evans 1991; Samaj et al.
1999; Tuan et al. 2006; Martin et al. 2008). The leaf seg-
ments of gerbera used in our study displayed high regen-
erative competence by inducing direct root formation from
the cut surface of leaves which inflicted wound. Wounded
surface have been often reported to induce callus as an
indicator of repair system. In several ornamentals, instead
of producing callus, roots were induced directly. As a
consequence of the wounding-response phenomenon, dor-
mant unwounded cells near the cut surface become active
and initiate cell proliferation. The commencement of cell
proliferation from intact, unwounded cells have been
Table 1 Effect (growth period
45 days) of variable levels of
auxins on direct root induction
from leaf segment of gerbera
(Gerbera jamesonii Bolus)
Data represent
mean ± standard error of 20
replicate explants per treatment
in three repeated experiments
(C control)
Data expressed as percentage
were transformed using arc sine
prior to ANOVA and converted
back to the original scale for
demonstration in the table
(Compton 1994)
Means within a single column
followed by different letters are
significantly different according
to Duncan’s multiple range test
(Duncan 1955) at P = 0.05
MS ? auxins (mg l-1) Frequency of root
induction (%)
Days to root
induction
No. of roots Root length
(mm)2,4-D IBA IAA NAA
C 33.3 ± 0.3 gh 19.6 ± 0.4 ab 5.2 ± 0.6 fg 5.2 ± 0.1 d
0.5 26.2 ± 0.6 i 21.3 ± 0.4 a 6.6 ± 0.6 f 4.4 ± 0.2 de
1 17.3 ± 1.2 j 19.6 ± 0.3 ab 4.5 ± 0.7 g 4.8 ± 0.0 de
1.5 0.0 ± 0.0 k 0.0 ± 0.0 e 0.0 ± 0.0 h 0.0 ± 0.0 f
2 0.0 ± 0.0 k 0.0 ± 0.0 e 0.0 ± 0.0 h 0.0 ± 0.0 f
0.5 34.6 ± 0.4 gh 17.6 ± 0.4 b 4.3 ± 0.4 g 4.6 ± 0.3 de
1 37.8 ± 0.2 fg 17.1 ± 0.3 b 11.6 ± 0.3 cd 4.8 ± 0.2 de
1.5 41.3 ± 0.5 f 19.8 ± 0.2 ab 8.6 ± 0.2 e 6.2 ± 0.2 cd
2 28.6 ± 0.2 hi 22.6 ± 0.3 a 5.3 ± 0.2 fg 5.9 ± 0.3 cd
0.5 84.6 ± 0.7 c 15.3 ± 0.6 c 12.3 ± 0.6 c 7.6 ± 0.1 c
1 87.4 ± 1.3 b 15.6 ± 0.4 c 15.4 ± 0.3 bc 9.6 ± 0.1 b
1.5 92.6 ± 0.7 a 11.3 ± 0.4 d 19.3 ± 1.1 a 12.8 ± 0.1 a
2 81.3 ± 0.6 cd 13.6 ± 0.3 cd 16.4 ± 0.3 b 9.2 ± 0.3 b
0.5 74.3 ± 0.3 d 16.3 ± 0.3 bc 12.8 ± 0.6 c 4.7 ± 0.3 de
1 71.8 ± 0.6 de 17.1 ± 0.6 b 13.6 ± 0.4 c 5.3 ± 0.1 d
1.5 66.7 ± 0.3 e 17.6 ± 0.3 b 11.4 ± 0.5 cd 6.5 ± 0.3 cd
2 65.3 ± 0.7 e 19.8 ± 0.7 ab 7.3 ± 0.2 ef 3.6 ± 0.1 e
Acta Physiol Plant
123
attributed to a range of biochemical transformation such as
degradation of starch, raise in peroxidase and polyphenol
oxidase which have been activated within the quiescent cell
(Imaseki 1985). Conversely, the relationship between these
metabolic changes and root induction is still indistinct and
leftover unexamined.
The other remarkable observation in our study is the
typical regulatory consequence of auxin concentrations on
direct root induction. It was evident that the presence of
auxin enhanced the incidence of root number as well as
reduced the time taken for its initiation. In consideration of
the fact that the survival of explants was a role of the auxin
present in the medium, it is rational to infer that a passable
endogenous PGR level may have been present in those
explants that survived in the control (Gantait and Sinniah
2012). Consequently, one rational explanation for the
incidence of rhizogenesis in the control medium would be a
potential role of endogenous auxin. This opinion is based
on a preceding proposition that the endogenous auxin
content of initial explants might have an imperative func-
tion in organogenic competence (Pandey et al. 2010). In the
present study, the occurrence of root induction increased
with raising concentrations of auxins, but started declining
following an optimum level. Reduced root induction might
be attributed to the excess cumulative level of accumulated
endogenous and raised exogenous auxins. As reviewed by
Blakesley et al. (1991) the presence of exogenous auxin
was most effective for direct root formation but the con-
centration required for optimum response was dependent
upon the type of auxin used. Significant differences in root
induction rate of gerbera were observed between IAA
treatments when compared to that of the 2,4-D, IBA or
NAA. It has been suggested in several experiments that
IAA proved to be the most effective for root induction in
comparison to other auxins (Gantait et al. 2008, 2010). Its
mode of action may be attributed to its ability to influence
the biosynthesis and accumulation of endogenous auxins
(Reviewed by Blakesley et al. 1991). According to Murch
Fig. 1 Direct induction and development of root system in Gerbera
jamesonii Bolus. a Initial symptom of morphogenesis in form of
swelling from the cut portions due to expansion of a cells (Bar 5 mm),
b initiation of callus from the wounded surface at higher 2,4-D
concentrations (Bar 3 mm), c root induction appeared as swollen,
small nodule-like meristemoids on the wound abaxial surface of
leaves, within 11 days after inoculation (Bar 3 mm), d the nodules
advanced into slender cylindrical bodies with globular root primordial
tip, rough surfaces and were pale pinkish in color (Bar 5 mm),
e growth and development of the disintegrated and prominent
rhizohenesis at advanced stage (Bar 5 mm), f rhizogenesis affected
with reduced root length and profuse root hairs at with excess auxin
concentration (Bar 5 mm)
Acta Physiol Plant
123
et al. (1997) PGR induces stress in plants, under in vitro
condition and induction of roots might be an adaptive
organogenic mechanism of leaf segments to overcome the
combined stress induced by wound and IAA. The modes of
actions of IAA are not absolutely explored. IAA may be
involved in the reprogramming and expression of the
competent cells necessary for them to undergo differenti-
ation and development (Reviewed by Blakesley et al.
1991). Although earlier researches have revealed that the
regenerative response might be due to stimulation of the
endogenous PGR system, it can be suggested based on the
present results that IAA presumably induces an array of
biochemical pathways to generate particular metabolites
essential for root induction procedure. In the present study,
IAA at 1.5 mg l-1 alone induced 92.6 % roots compared to
33.3 % in control. On the contrary, a number of reports
have shown the inadequacy of IAA alone on in vitro root
induction from young leaf explants and leaf segments
(Pandey et al. 2010; Nagarajan et al. 2011).
A competent system for the accelerated organogenesis
in terms of direct root induction from gerbera leaf has been
described in the current study. To our knowledge, such an
effort concerning the complete protocol and developmental
study of direct root induction has been made for the first
time on any ornamental plant.
Author contribution S. Gantait, U.R. Sinniah—Con-
ceived the research idea and designed the experiments;
S. Gantait—Executed the experiments; S. Gantait,
U.R. Sinniah—Wrote the manuscript.
Acknowledgments The authors are indebted to Department of Crop
Science, Faculty of Agriculture, Universiti Putra Malaysia for pro-
viding the research facilities.
Conflict of interest We, the authors of this article, declare that there
is not conflict of interest and we do not have any financial gain from it.
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