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Organogenesis and Ultra-structural Features of in vitro-grown Canna indica L. BioMed Research International 2016, Article ID 2820454, 9 Pages,

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Research Article
Organogenesis and Ultrastructural Features of In Vitro Grown
Canna indica L.
Sharifah Nurashikin Wafa, Rosna Mat Taha, Sadegh Mohajer,
Noraini Mahmad, and Bakrudeen Ali Ahmed Abdul
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
Correspondence should be addressed to Rosna Mat Taha; rosna@um.edu.my
Received  October ; Revised  December ; Accepted  December 
Academic Editor: Denise Freire
Copyright ©  Sharifah Nurashikin Wafa et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
An ecient protocol for micropropagation of Canna indica L., an economically and pharmaceutically important plant, was
standardized using rhizome explants, excised from two-month-old aseptic seedlings. Complete plant regeneration was induced
on MS medium supplemented with . mg/L BAP plus .mg/L NAA, which produced the highest number of shoots (. ±.%)
and roots (. ±.%) aer  weeks. Furthermore, the optimum media for multiple shoots regeneration were recorded on MS
enriched with .mg/L BAP (. ±.%). Plantlets obtained were transplanted to pots aer two months and acclimatized in
the greenhouse, with % survival. In addition, ultrastructural studies showed that rhizomes of in vitro grown specimens were
underdeveloped compared to the in vivo specimens, possibly due to the presence of wide spaces. Meanwhile, the leaves of in vivo
specimens had more open stomata compared to in vitro specimens, yet their paracytic stomata structures were similar. Hence, there
were no abnormalities or major dierences between in vitro regenerants and mother plants.
1. Introduction
Canna indica L. belongs to the family Cannaceae and is a
tropical herb grown from rhizomes and seeds, with banana-
like leaves and multicolour owers. e plant can reach a
height of  meters and their habitat includes shady areas,
wet places in forest and savannah, and swampy areas, along
rivers or roadsides.ough C. indica are grown primarily as
an ornamental, a recent study shows that proteins contained
in the water extract from C. indica fresh rhizome have a
potent ability to inhibit human immunodeciency reverse
transcriptase (HIV-RT) virus in vitro (% at 𝜇g/mL) [].
In a dierent study, leaf samples of C. indica exhibited the
strongest anti-HIV  activity compared to roots and rhizome
due to the presence of plastocyanin []. Furthermore, many
parts of C. indica were used in traditional medicine as
diaphoretic and diuretic in fevers and dropsy, as a demulcent,
to stimulate menstruation, treat suppuration, and rheuma-
tism and to regain energy []. It has also been studied for
its antitumor, cytotoxic activity and antibacterial properties
[, ]. Apart from its profound medicinal values, rhizomes
of C. indica, which are rich in starch, have traditionally
been consumed as boiled rhizome and noodles [] and used
to make alcoholic beverages and our. Meanwhile, the leaf
extracts of C. indica showed great potential as botanical
molluscicide [] and the ower extract exhibited abilities as
a natural indicator in acid-base titration [].
Despite its many medicinal values and uses, traditional
propagation of C. indica is hindered by its extremely hard
seed coat [] and slow vegetative propagation of rhizome
which is also susceptible to viral infection during multipli-
cation [–]. Furthermore, common practice of asexual
propagation through rhizome produces genetic stagnancy
and genetic variation limitations in Canna []. Plant tissue
culture provides a solution whereby the method rapidly
micropropagates plants of superior qualities and free from
microorganisms in a relatively short time and minimal space
using few starting materials. At the same time, having a reli-
able micropropagation protocol is a preliminary requirement
for any genetic manipulation and protoplast fusion in order to
improve Canna variety. e rst attempt in this regard using
intact rhizome of C. indica did not show high morphogenetic
Hindawi Publishing Corporation
BioMed Research International
Volume 2016, Article ID 2820454, 9 pages
http://dx.doi.org/10.1155/2016/2820454
BioMed Research International
potential and the plant produced insucient endogenous
cytokinin to induce buds proliferation []. Later, the same
author tried to stimulate shoot development or bud for mation
from meristem tips with little success []. Recently, a study
managed to obtain complete plantlets from leaves explant of
C. indica via indirect regeneration through in vitro callus and
somatic embryogenesis [].
Considering the norm of vegetative propagation for this
plant, current study focuses on establishing direct organogen-
esis and ecient protocol for in vitro regeneration system of
C. indica from rhizome explants. Furthermore, plan for future
development and commercialization of this plant requires
optimum media for induction of multiple shoots. Lastly, as
an early detection system for occurrence of somaclonal vari-
ation, ultrastructural features of in vivo and in vitro samples
from leaves and rhizomes of this species were examined using
Field Emission Scanning Electron Microscope (FESEM).
2. Materials and Methods
2.1. Seeds Scarication and Establishment of Sterile Culture.
Initially, we attempted culture with leaf and rhizome explants
from in vivo conditions; however, we encountered high
problems of contamination and browning. To overcome
these, we grew plantlets aseptically in the laboratory and used
dierent seed scarication methods to increase the germina-
tion rate. Seeds of red owered C. indica were purchased from
TROPILAB, St. Petersburg, Florida, and germinated under
in vitro conditionsandongardensoilinthegreenhouse
as control. Aseptic seedlings were obtained by subjecting
C. indica seeds to chemical scarication of sulphuric acid
(H2SO4) []. e process begins with soaking the seeds in
distilled water for  day to dulcify or soen the seeds. Next,
theseedsweresoakedin%sulphuricacid(H
2SO4)for
hours,thenrinsedseveraltimes,andfurthersoakedin
distilled water for one day on a shaker table at  rpm.
Prior to inoculation in the laminar ow hood, seeds were
surface-sterilized with % alcohol for  min followed by
repeated washing with sterile distilled water. Seeds were then
germinated on basal Murashige and Skoog (MS) [] medium
supplemented with %(w/v) sucrose and .% gellan gum
(Gelzan) as solidication agents []. Each ask of mL
media was inoculated with one seed and germinated seeds
were allowed to grow profusely for two months in culture
room at 25 ± 1C under a -hour photoperiod, before the
rhizomes were excised as explants.
2.2. Complete Plantlet Regeneration and Shoot Multiplication.
For organogenesis study, the nutrient medium was pre-
pared by combining commercial MS salts (Sigma), % (w/v)
sucrose, and .% Gelzan as gelling agent. Plant growth
regulators (PGRs) were added to the medium priorto adjust-
ment of pH (.) and sterilization (autoclaving at Cfor
 min). About  mL of the media was evenly dispensed into
sterilized tubes where one explant was inoculated per tube.
Approximately  rhizome explants (. cm2) per aseptic
seedlings were excised and cultured in a horizontal position
with the cutting side facing the MS medium augmented with
combinations of .–. mg/L -benzylaminopurine (BAP)
with .–. mg/L ,-dichlorophenoxyacetic acid (,-D),
.–. mg/L BAP with .–. mg/L 𝛼-naphthalene acetic
acid (NAA), and .mg/L kinetin (KN) with .–. mg/L
NAA to observe direct organogenesis into complete plantlet.
Meanwhile, multiple shoots were tried to induce using
single hormone of BAP (.–. mg/L), KN (.–. mg/L),
NAA (.–. mg/L), and Triiodobenzoic acid (TIBA; .,
. mg/L). All chemicals used were of analytical grade (Sigma
Chemical Co., USA). Each treatment consists of  repli-
cates. e cultures were maintained in the culture room
at temperature of 25 ± 1C under a -hour photoperiod
with a photosynthetic photon ux density of  𝜇mol m−2 s−1
provided by cool white uorescent lamps.
e cultures were observed daily for any contamina-
tion and explants suering from minor contamination will
quickly be transferred into new media. Tubes containing
fast growing explants will require frequent subculturing into
new media or container in order for growing process to
continue. Plant responses were observed and recorded for
each replicate aer  weeks. Multiple shoots induced were
later excised and cultured on optimum media for complete
plant regeneration.
2.3. Acclimatization of Plantlet. Aer  months, complete
plantlets with well-developed shoots and roots from media
of direct organogenesis were removed from the culture
medium, washed gently under running tap water, and trans-
ferred to plastic pots containing black (peat) soils under
diuse light conditions []. Plantlets were then covered with
clear plastic bags (with holes) and placed in the culture room
at temperature of 25 ± 1C for  days prior to eld transfer
to the greenhouse. Watering with tap water was done twice
a day with maximum volume of  mL per session, at early
morning and late evening, for the period of  months with
constant monitoring.
2.4. Statistical Analysis. All data and variables were statisti-
cally analyzed using SPSS statistical package version  (SPSS
Inc., Chicago, III). Values are presented as mean ±SE. One-
way ANOVA and Multiple Range Analysis were done on all
data using Duncans test at 𝑝 = 0.05.
2.5. Ultrastructural Studies Using Field Emission Scanning
Electron Microscope (FESEM). Fresh rhizomes and leaves (
months) of in vivo and in vitro C. indica were collected and
cleaned prior to excise into small pieces (specimen) to exhibit
important parts of the samples to be viewed. e samples
were placed on a stub and examined using a JEOL JSM-F
Field Emission Scanning Electron Microscope (FESEM).
3. Results and Discussion
3.1. Establishment of Aseptic Seedlings. Preliminary exper-
iments showed that C. indica seeds did not germinate
satisfactory in the in vitro condition without scarication.
erefore, the black hard seed coat was scaried mechan-
ically and chemically. e best scarication method was
soaking of seeds for three hours in sulphuric acid (H2SO4)
which produced the highest percentage of successful seeds
BioMed Research International
(a) (b)
(c) (d)
F : Dierent stages of Canna indica L. plantlet development. (a) Complete plant regeneration aer  month. (b) Vigorous plantlet
growth aer  months. (c) Subcultured plantlet in a larger container. (d) Acclimatized plantlet on garden soil.
germination (.% ±.). Similarly, the positive eects of
H2SO4scarication (more than .%) were also reported
by other researches for C. indica seedssowninthein vivo
conditions []. In regard to these studies, the slight variations
in germination percentage between in vivo and in vitro seed
germination could be attributed to the variability of sowing
medium and the vigorous method of seeds sterilization.
Furthermore, dierences in seed viability could be caused
by nonhomogeneous seed lots due to uneven maturation
and dierences in seed dormancy levels []. Scarication
using H2SO4was also favoured by seeds of tamarind [] and
bladder-senna []. e coatless seed commences germina-
tion by breaking the quiescence or dormancy. Process of seed
development begins with the formation of radicle (growing
root) aer day . is is followed by the coleoptile which
encloses the embryonic shoots and then pushes upward
to the surface aer day . Both will resume elongation
and promoting multiple roots, hairy roots, and rhizome
aer day . Once elongation process stops, the rst leaf
emerged. Aer two months, aseptic seedlings with well-
developed roots and leaves started to accumulate in the sterile
tube. Seedlings were then ready to be used as source of
explants.
3.2. Induction of Shoots, Roots, and Multiple Shoots. Rhizome
explants produced new shoots and roots simultaneously aer
two weeks of inoculation on MS medium supplemented
with combinations of BAP plus NAA. However, root and
shoot induction varied in dierent concentrations of the
hormones. High frequency of shoots (.%) and roots
(.%) which resulted in complete plantlet regeneration
(Figure (a)) was observed in MS medium supplemented
with . mg/L BAP plus . mg/L NAA. e plantlets grew
vigorously (Figure (b)) and subsequently required subcul-
turing into larger containers (Figure (c)). Aer  months, the
plantlets were acclimatized on garden soil, rst in the culture
room for  days and later in the greenhouse (Figure (d)).
Table  shows that lower concentration of BAP with higher
concentration of NAA only induced high formation of roots,
while increasing the concentrations of NAA with optimum
concentration of BAP reduced both shoot and root forma-
tion. Meanwhile, the combination of KN and NAA was not
as eective as the other hormone combinations at promoting
shoots and roots development. In contrast, a previous study
[] reported that intact rhizome explants produced shoot
buds and regenerated into complete plantlets eectively in
media containing a combination of  mg/L IAA and  mg/L
BioMed Research International
T : e combined eects of hormones on new shoot and root formation of Canna indica L. aer  weeks.
MS + hormones (mg/L) New shoot formation (%) Number of shoots per explant New root formation (%) Number of roots per explant
. BAP + . ,-D . . 
. BAP + . ,-D . . 
. BAP + . ,-D . . 
. BAP + . ,-D . . 
. BAP + . ,-D . . 
. BAP + . NAA . ±.a.±.ab
. BAP + . NAA . ±.a . ±.ab
. BAP + . NAA . ±.ab .±.bc
. BAP + . NAA . ±.c . ±.c
. BAP + . NAA . ±.b . ±.ab
. BAP + . NAA . ±.b . ±.abc
. KN + . NAA . . 
. KN + . NAA . ±.a.±.a
. KN + . NAA . . 
Data represent mean ±standard error (SE) from  replicates per treatment. Means with dierent letters in the same column are signicantly dierent at 𝑝=
0.05 according to Duncans multiple range test (DMRT).
T : Eects of single hormone on shoots, multiple shoots, and root formation of Canna indica L. aer  weeks.
MS + hormone (mg/L) Shoot
formation (%)
Multiple shoots
formation (%)
Number of shoots
per explant
Root
formation (%)
Number of roots
per explant
MS (control) . ±.abc . . ±.ab
BAP
. . ±.bcde . . ±.cde
. . ±.def . ±.a.±.de
. . ±.bcde . ±.a . ±.abcd
. . ±.ef . ±.abcd . ±.cde
. . ±.f. ±.cd .±.bcde
.  . ±.ef . ±.d . ±.abc
. . ±.cdef . ±.cd . ±.a
KN
. . ±.bcde . ±.a . ±.cde
. . ±.abcde . . ±.de
. . ±.abcd . ±.a . ±.ef 
. . ±.abcd . ±.a . ±.de
. . ±.def . ±.a.±.de
. . ±.def . ±.bcd . ±.de
NAA
. . ±.abc . . ±.f
. . ±.ab . ±.ab . ±.f
. . ±.ab . ±.a . ±.f
. . ±.a. . ±.f
TIBA . . ±.ef . ±.abc . ±.abcd
. . ±.abcd . ±.a . ±.de 
Data in percentage are mean ±standard error (SE) from  replicates per treatment. Means with dierent letters in the same column are signicantly dierent
at 𝑝 = 0.05 according to Duncan’s multiple range test (DMRT).
KN. A further study by Kromer and Kukulczanka [] was
also in disagreement with the present study, whereby they
preferred combinations of KN, adenine sulphate, and NAA
to combinations of BAP plus NAA. e combinations of
BAP plus NAA are substantially important in propagation of
various ornamental species, whereby at equal concentration,
the combination can yield production of callus while at
other concentrations it can result in direct regeneration and
rhizogenesis [].
e response of rhizome explants to various plant growth
regulators used singly, in terms of shoot, root, and multiple
shoot formation, is depicted in Table . All single hormones
tested induced formation of shoots and roots but at dierent
induction levels. According to the table, the percentage of
BioMed Research International
(a) (b) (c)
F : Multiple shoot induction from Canna indica L. rhizome explants, in (a) . mg/L BAP; (b) . mg/L BAP; and (c) . mg/L BAP
aer  weeks.
shoot formation increased with the increase of BAP up to
the optimum concentration of . mg/L and later gradually
decreased. e pattern was in agreement with previous
researchers [, ] which observed a less eective formation
of shoots with higher concentrations of BAP. e variants
in optimum concentration of BAP for regeneration could
be attributed to the types of explants used. Regeneration of
plantlets from somatic embryos of C. indica only required low
concentration of BAP (. mg/L) [] while, in the present
study, rhizome explants required a high concentration of BAP
(. mg/L) to induce optimum shoot and root formation.
Similarly, rhizome buds of Curcuma manga give the best
response of shoot formation in MS medium containing a high
concentration of BAP (. mg/L BAP) []. In media with
KN only at the concentrations tested, the percentage of shoot
formation recorded was less than %, while inversely, more
than % root formation was promoted with the gradual
increase of its concentration. A low concentration of TIBA
(. mg/L) encouraged formation of shoots, while at higher
concentration, the formation of shoots declined drastically
and only formation of roots was promoted. TIBA is an
antiauxin that promotes lateral shoot initiation in shoot tip
culture of Canna edulis [] and indirect regeneration of
turmeric from callus []. Among the cytokinins used, BAP
wasfoundtobethemostsuitableinpromotingcelldivi-
sion, shoot multiplication, and axillary bud formation, while
inhibiting root development []. However, we observed
profuse rooting in all the cases, which may be a general phe-
nomenon for Canna [] and the members of Zingiberales as
similar phenomena were previously reported in ginger []
and turmeric [].
Multiple shoots of C. indica with more than .% induc-
tion were successfully obtained in MS media supplemented
with the single hormone of BAP (., ., ., and . mg/L)
and KN (.mg/L) (Table ). e multiple shoots were
healthy with normal looking leaves and roots. e highest
number of multiple shoots formation (33.0%± 0.48) with
occurrence of ve multiple shoots per explant (Figure (a))
was recorded in MS medium supplemented with . mg/L
BAP. Furthermore, there were also observations of four and
three multiple shoots per explant on MS medium supple-
mented with . mg/L BAP (Figure (b)) and .mg/L BAP
(Figure (c)), respectively. Induction of multiple shoots using
a combination of cytokinin and auxin has been reported by
many researchers [, , ] and is considered as an impor-
tant step for commercial exploitation of a micropropagation
protocol. However, the ability of producing multiple shoots
with roots using a single hormone (cytokinin) in a tissue
culture system is highly desirable, especially in reducing
costs for mass production of any species. However, the
optimum requirement at propagule proliferation stage diers
from species to species. In research involving other species
related to C. indica, shoot multiplication of C. aromatic was
foundtobeoptimalatmg/LBAP[]andforC. zedoary,
mg/L BAP was reported to be suitable for multiplication
[].
Acclimatization of plantlets is the crucial phase where
plantlets are in transition from in vitro phase to in vivo
phase. Plantlets with well-developed shoot and roots were
removed from the culture media and transferred to plastic
pots containing black (peat) soils. ey were maintained for
about  days under plastic covers to avoid desiccation prior
to eld transfer to the greenhouse. e gradual transition
process from culture container to the greenhouse produced
normal plant growth and morphology with plant survival
rate as high as .% by minimizing physiological stress on
the regenerated plantlets []. Similarly, in another study C.
indica acclimatized in clay pots containing an autoclaved
mixture of soil and sand produced .% survival rate of
regenerated plantlets []. On the other hand, a simple
acclimatization method was preferred for Kaempferia galan-
gal, whereby a survival rate of –% was easily achieved
by dismissing the hardening process and simply keeping the
plants in % shade and watering twice a day [].
BioMed Research International
(a) (b)
(c)
Void spaces
(d)
F : Ultrastructural comparison of Canna indica L. rhizome. e outer layer of in vivo rhizome (a) is smoother and well-formed
compared to the in vitro rhizome (b). At x magnication, there are no conspicuous void spaces in the in vivo rhizome(c)whilevoid
spaces were visible in the in vitro rhizome (d).
3.3. Ultrastructural Studies. In a tissue culture system, it is
important to produce plantlets identical to the parent plants
and avoid formation of somaclonal variation. e present
study provides the rst comparison between rhizome spec-
imens from in vivo and in vitro C. indica under eld emission
scanning electron microscope (FESEM). Macromorphologi-
cally, no morphological abnormalities in tissue culture raised
plants were observed when compared to plants grown in eld.
However, going deeper into rhizome ultrastructural obser-
vations, there were minor structural dierences, especially
at the outer layer of the rhizome (Figures (a) and (b)).
e In vivo specimen (Figure (c)) exhibited an absence of
void spaces, while void spaces were very apparent in the
in vitro specimen (Figure (d)). is indicated that the in
vitro rhizome was underdeveloped compared to the in vivo
rhizome specimen. It could be hypothesized that since the
culture medium was providing all the necessary foods and
nutrients to the plant, thus the rhizome’s natural function as
a storage compartment for food materials such as starch was
underutilized. Furthermore, rhizomes of specimens grown
in in vitro conditions required a longer time to reach their
full developmental stage or maturity, even though both
specimens were collected at the same plant age. According
to Mahmad et al. [], in vitro lotus plants (Nelumbo nucifera
Gaertn.) grew vigorously and possessed similar physiological
characteristicsasthemotherplantonlyaermonthsof
acclimatization.
Stomata distribution and structure were compared
between adaxial and abaxial leaf surface of both in vivo
and in vitro specimens of C. indica. Figure (a) shows that
there are an abundance of open stomata detected on the
leaf surfaces of in vivo specimens compared to in vitro
specimens that are rich in closed stomata (Figure (b)).
Open stomata distribution was also much more visible on
the abaxial surface (Figure (c)) compared to the adaxial
surface (Figure (a)) of in vivo leaves. is is a normal
and expected occurrence of leaves from in vivo conditions.
Abundance of closed stomata of in vitro leaves on both
adaxial (Figure (b)) and abaxial (Figure (d)) surfaces
could be the result of nonfunctional stomata, which was
similarly reported in the potato plant []. e stomata were
probably nonfunctional or failed to mature to a normal
functioning state due to prolonged exposure of the plantlets
to high relative humidity and low CO2concentration. e
presence of sucrose in the medium and accumulated ethylene
in the headspace of the vessel might also have played a role
[]. As a consequence, photosynthesis, transpiration, and
uptake of water, nutrients, and CO2couldbesuppressedand
dark respiration enhanced, resulting in poor growth [].
However, functioning closed stomata or plantlet with no fully
open stomata is a great acclimatization condition for in vitro
plantletswhereby,asaconsequence,theplantletwillonly
wilt slightly and the epidermal cells can recover and become
turgid quickly aer transfer to ex vitro conditions, leading to
increased survival rate []. e surface view shows that both
in vivo (Figure (a)) and in vitro (Figure (b)) C. indica leaves
possess paracytic stomata, characterized by the position of
the subsidiary cell which is parallel to the guard cells []. It
BioMed Research International
Open
stomata
(a)
Closed stomata
(b)
(c) (d)
F : Surface view of stomata distribution of Canna indica L.leaves.Moreopenstomataaredetectedonthein vivo leaves ((a) adaxial;
(c) abaxial) compared to in vitro leaves ((b) adaxial; (d) abaxial).
Guard cells
Subsidiary
cells
(a) (b)
19.5 𝜇m
(c)
7.6 𝜇m
(d)
F : Ultrastructural comparison of stomata structure of Canna indica L. leaves. Stomata of in vivo (a) and in vitro (b) leaves are paracytic
in structure and the pores of in vivo stomata (c) are larger in comparison with the stomata of in vitro leaves (d).
BioMed Research International
has typical monocot stomata in which the dumbbell-shape
guard cells with unevenly thickened walls are dwarfed by the
larger subsidiary cells, which are lacking in dicot stomata
[]. Furthermore, the subsidiary cells and the long axes of
thestomatalieparalleltoeachother.eporesofin vivo
stomata (Figure (c)) are almost three times bigger than the
pores of in vitro stomata (Figure (d)).
Electron microscopical observations of rhizome and
leaves belonging to regenerants from in vitro culture of C.
indica presentfrommanypointsofviewasimilarstructure
with those from the native plants, which demonstrates
that our experimental in vitro conditions did not induce
signicant morphological alterations at the ultrastructural
level. Direct complete plant regeneration of C. indica was
possible using sterile rhizome explants cultured in MS
medium supplemented with .mg/L BAP plus . mg/L
NAA. Meanwhile, multiple shoots were successfully induced
in medium with a high concentration of the single hor-
mone BAP (. mg/L BAP). e nding paves the way for
future research on biotechnology manipulations and com-
mercialization, especially in producing virus-free plants and
synthetic seed of these medicinally important ornamental
species. Furthermore, all plantlets were successfully acclima-
tizedtothegreenhousewith%survivalrate.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgment
e authors would like to thank University of Malaya for
the postgraduate PPP grant (PV/) and the facilities
provided.
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A study was carried out to investigate the effects of sulphuric acid and hot water treatments on the germination of Tamarind (Tamarindus indica L). Seeds were placed on moistened filter papers in 28 cm diameter Petri dishes under laboratory condition for germination. 330 seeds of T. indica (10 seeds per Petri dish) with three replicates each were used. The highest germination was recorded in seeds treated with 50 per cent sulphuric acid concentration with 60 min soaking period. Germination was observed to be enhanced by increase in the sulphuric acid concentration, water temperature, and soaking period in all the trials, except with absolute sulphuric acid where poor response was observed. Results of this study may serve as useful information in the production and improvement of the tree species, as knowledge on seed germination requirements is a critical factor in seedlings production.
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