RESEARCH ARTICLE
Analyses of phylogenetics, starch granule morphology and consumer preference of Canna
indica L. grown in Sri Lanka
R.W.K.M. Senevirathna, L.T. Ranaweera, N.D.U.S. Nakandala, H.M.T.N. Senavirathna, W.M.D.A. Wijesundara,
H.S.M. Jayarathne, C.K. Weebadde and S.D.S.S. Sooriyapathirana
Highlights
• The sequence polymorphism of the atpB gene differentiates Canna indica (Arrowroot/Buthsarana) from
ornamental Canna spp.
• Canna indica has the significantly large starch granules compared to those of Xanthosoma sagittifolium,
Manihot esculenta, Solanum tuberosum, and Ipomoea batatas.
• Canna indica has unique scallop-seashell shaped starch granules.
• The cooked Canna indica tubers are accepted, better than those of Xanthosoma sagittifolium, and rated equally
to Solanum tuberosum, and Manihot esculenta.
Ceylon Journal of Science 49(3) 2020: 261-273
DOI: http://doi.org/10.4038/cjs.v49i3.7777
RESEARCH ARTICLE
Analyses of phylogenetics, starch granule morphology and consumer preference of Canna
indica L. grown in Sri Lanka
R.W.K.M. Senevirathna1, L.T. Ranaweera1, N.D.U.S. Nakandala1, H.M.T.N. Senavirathna1,
W.M.D.A. Wijesundara1, H.S.M. Jayarathne1, C.K. Weebadde2 and S.D.S.S. Sooriyapathirana1,3,*
1
Department of Molecular Biology and Biotechnology, Faculty of Science, University of Peradeniya, Peradeniya, Sri
Lanka.
2
Department of Plant, Soil and Microbial Sciences, College of Agriculture and Natural Resources, Michigan State
University, East Lansing, 48824, USA.
3
Postgraduate Institute of Science, University of Peradeniya, Peradeniya, Sri Lanka.
Received: 22/05/2019; Accepted: 29/06/2020
Abstract: Canna indica is a tuber crop which has many
medicinal values. In Sri Lanka, C. indica tubers are consumed
in rural areas and mainly available in street-markets of NuwaraEliya and Kandy Districts. In the present study, we assessed
the phylogenetics of C. indica, starch granule morphology and
consumer preference of C. indica tubers in comparison to the
popular tuber crops. The phylogenetic analysis was conducted
based on the sequence polymorphism at rbcL, atpB gene, trnLtrnF and trnH-psbA marker-loci with respect to the ornamental
Canna spp. in Sri Lanka and the previously published sequences
of Canna spp. The starch granules were isolated and observed
under optical and scanning electron microscopes. The diameter
and the surface area of the starch granules were measured under
the optical microscope and subjected to analysis of variance.
As C. indica tubers are consumed as boiled tuber pieces in Sri
Lanka, the consumer preference analysis was conducted using the
boiled tuber pieces C. indica, Xanthosoma sagittifolium, Manihot
esculenta, Solanum tuberosum, and Ipomoea batatas. The
phylogenetic tree based on rbcL marker revealed that C. indica
in Sri Lanka is slightly divergent from the other Canna spp. Only
the polymorphism of the atpB gene can be used to differentiate
C. indica from the ornamental Canna sp. in Sri Lanka. The
morphological analysis of starch granules revealed that C. indica
has the biggest scallop-seashell shaped starch granules compared
to other tuber species. The boiled C. indica tubers were accepted
better than that of X. sagittifolium, rated equally to the tubers of
S. tuberosum and M. esculenta, and rated less than I. batatas. The
hardy and fibrous nature of C. indica tubers must be the major
limiting factors for achieving the highest consumer preference
highlighting the need of breeding for better texture in tubers.
Keywords: Devkali, Indian-shot, Large starch granules in plants,
Ornamental Canna spp., Underutilized tuber crops.
INTRODUCTION
Canna indica of family Cannaceae is considered as one
of the traditionally used tuber crops in the world (Prince,
2010). The vernacular names of C. indica are Indian-Shot
in English, Arrowroot in Africa, Achira in Latin America,
Devkali in India, Buthsarana in Sinhala, and Ampurut Alai in
Tamil (Bachheti et al., 2013). It is believed that C. indica was
originated in Latin America (Andrade-Mahecha et al., 2012)
and later naturalized in many countries including Sri Lanka
(Olango et al., 2013; Gunarathna et al., 2016). The rhizome
is the edible part of the plant and all parts of the plant contain
medicinal values including antioxidant property (Mahesh
et al., 2014; Joshi et al., 2009a), HIV reverse transcription
inhibitory activity (Woradulayapinij et al., 2005), antidiarrheal, hemostatic, hepatoprotective, antibacterial and antiworming effects (Rahmatullah et al., 2010; Josephine et al.,
2013; Lin et al., 2011; Joshi et al., 2009b; Anisuzzaman et
al., 2007; Indrayan et al., 2011; Thepouyporn et al., 2012;
Abdullah et al., 2012; Nirmal et al., 2007).
The tubers of C. indica are a common commodity in
ayurvedic herbal medical preparations (Kankanamalage
et al., 2014). Although C. indica possesses significant
medicinal and food values, it is currently an underutilized
tuber crop in Sri Lanka (Malkanthi, 2017). The tubers of
C. indica rarely appear in the country-wide market. The
younger generation who generally appreciate fast food and
limited array of choices, even do not know that C. indica
tuber is a human food. However, C. indica is fast growing
(Zhang et al., 2008) with an excellent ornamental value
(Zhang et al., 2008) and recently being identified as one
of the best species for the phytoremediation of the polluted
water released from reverse osmosis process of the water
purification units in Sri Lanka (Gunarathna et al., 2016).
It is believed that C. indica contains the largest starch
granules ever known (Hermann et al., 1997) thus, can be
used as a candidate species to broaden our knowledge on
starch science and technology.
In Sri Lanka, C. indica is grown in lowland plains (30 –
200 m above sea level) of Sri Lanka without much attention.
However, C. indica plants thrive well in the central highlands
of Sri Lanka (500 m above mean sea level, 2000 mm annual
rainfall and 23-26 0C of mean temperature) and grow as
large dense bushes where suckers keep coming from the
mother plants. The tubers can be harvested for human
*Corresponding Author’s Email: sunethuop@gmail.com
http://orcid.org/0000-0002-5592-1742
This article is published under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
262
Ceylon Journal of Science 49(3) 2020: 261-273
consumption after six months of planting. There are
slight variations in leaf size, leaf margin and plant height
exist among the C. indica populations found in different
habitats. However, when suckers from one location is
planted in a different habitat, these variations get changed
based on the soil fertility and moisture content. However,
the genetic diversity or the exact species delimits of the C.
indica have not been studied in Sri Lanka. In addition to the
edible C. indica, there are ornamental Canna plants which
can produce attractive flowers and foliage grown in many
places of the country. The species differences between
edible C. indica (i.e. Arrowroot) and the ornamental
Canna spp. have not been studied to date. The extensive
multidisciplinary research attempts are needed to uplift C.
indica from its current underutilized status. Also, the taste
and other sensory attributes of C. indica in comparison to
the popular tuber crops must be studied. Therefore, the
present study was conducted to assess the species delimits
and phylogenetic relationships, starch granule morphology,
and consumer preference of C. indica in Sri Lanka.
MATERIALS AND METHODS
DNA extraction, PCR and DNA sequencing
The DNA was extracted from the immature leaves of
C. indica, plants collected from Kundasale, Matale,
Nuwara-Eliya, Peradeniya (7°16’42.2”N 80°40’33.7”E,
7°27’29.7”N 80°36’31.3”E, 6°52’53.7”N 80°48’55.5”E,
7°16’01.1”N 80°36’23.2”E) and ornamental Canna spp.
plants in Kandy, (7°15’26.8”N 80°35’08.9”E) using
modified cetyltrimethylammonium bromide (CTAB)
method (Porebski et al., 1997). The extracted DNA
samples were stored at -20 °C. The PCR was performed
in a Thermal Cycler (TP600: Takara, Otsu Shiga, Japan)
using four DNA barcoding markers given in Table 1. Each
PCR was performed in a 30 µl reaction mixture with 2× Go
Taq Green Master Mix (15 µl), 10µM forward and reverse
primers (1 µl each) and 10 µM spermidine (7 µl). The PCR
products were resolved in 2.5% agarose gel electrophoresis
(Sambrook and Russell, 2001). The PCR amplicons of the
four DNA markers were purified using QIAquick® PCR
purification kit (Catalog No.: 28104, Qiagen, Hilden,
Germany) and sequenced using ABI 3500 Automated
Phylogenetic analysis
The initial and end noises of the sequencing data obtained
from four DNA markers (Table 1) were observed
independently using MEGA 7 (Kumar et al., 2016). Then
the identities of the trimmed sequences were confirmed
independently by subjecting to a search in Basic Local
Alignment Search Tool (BLAST). We created separate
alignments for each marker in MEGA 7. In order to relax the
family Cannaceae topology, we did not use phylogenetically
distant outgroup, or a too closely related one. Thus, we
used two species from family Commelinaceae as outgroups
which can also be found in the Commelinids clade where
family Cannaceae is located. To compare the Canna spp. Sri
Lanka, the sequences generated were aligned for the marker,
rbcL, with the dataset generated in Prince, (2010) (Table 2).
A model selection analysis in Jmodel test (Posada, 2008)
was implemented to infer the best nucleotide substitution
process of the dataset. A phylogeny was constructed in
Maximum Likelihood framework in RAxML (Stamatakis,
2006). We implemented rapid+boostrap algorithm with
1000 iterations and GTRGAMMA as the nucleotide
substitution model. The analysis was implemented in the
CIPRES supercomputer (Miller et al., 2010). We also
carried out a tree search in the Bayesian framework in
MrBays (Huelsenbeck and Ronquist, 2001) in CIPRES
science gateway. Two hot and cold chains of Markov Chain
Monte Carlo (MCMC) running for 30 million generations
were used to probe trees in the tree-space. The 10% of the
initial trees were removed as burn-in, and the rest of the
probed trees were used to draw the final 50% majority rule
consensus tree. The Effective Sample Size (ESS) of all the
priors used were checked in TRACER v1. 4 (Rambaut and
Drummond, 2007). To check the intra species variability of
the Sri Lankan Canna spp., UPGMA trees were constructed
for the markers, using uncorrected pairwise genetic distance
matrices. The gaps (INDELS) were considered as pairwise
deletions and considered uniformed rates across all the
states. The trees were constructed in MEGA7. Finally,
all the drawn trees were modified using FigTree v1.4.3
(Rambaut, 2014).
Sequencer (Catalog No.: 622-0010, Applied Biosystem). All
the sequences generated were submitted to the Genbank under
the accession numbers: MK952702-MK952733.
Table 1: DNA barcoding markers used in the present study.
DNA marker
Primer sequence
trnH-psbA
F: CGCGCATGGTGGATTCACAATCC
rbcL
trnL-trnF
atpB gene
R: GTTATGCATGAACGTAATGCTC
F: ATGTCACCACAAACAGAGACTAAAGC
R: GTAAAATCAAGTCCACCRCG
F: CGA AAT CGG TAG ACG CTA CG
R: ATT TGA ACT GGT GAC ACG AG
F: TATGAGAATCAATCCTACTACTTCT
R: TCAGTACACAAAGATTTAAGGTCAT
Annealing
Reference
temperature (ºC)
64
Tate and Simpson, (2003)
50
Sang et al., (1997)
Levin et al., (2003)
Kress et al., (2009)
Taberlet et al., (1991)
55
Hoot et al., (1995)
55
263
R.W.K.M. Senevirathna et al.
Table 2: Metadata of the sequences used in the phylogenetic analysis.
Species
trnH-psbA
GenBank accession numbers
atpB gene
rbcL
trnL-trnF
Voucher No.
C. indica (Kundasale: arrowroot)
C. indica (Matale: arrowroot)
DMB115
DMB116
MK952726
MK952727
MK952710
MK952711
MK952702
MK952703
MK952718
MK952719
C. indica (Ambewela: arrowroot)
DMB117
MK952728
MK952712
MK952704
MK952720
C. indica (Nuwara-Eliya: arrowroot)
DMB118
MK952729
MK952713
MK952705
MK952721
C. indica (Peradeniya: arrowroot)
DMB119
MK952730
MK952714
MK952706
MK952722
C. indica (Kandy: ornamental red)
DMB120
MK952731
MK952715
MK952707
MK952723
C. indica (Kandy: ornamental yellow)
DMB121
MK952732
MK952716
MK952708
MK952724
C. indica (Kandy:ornamental orange)
DMB122
MK952733
MK952717
MK952709
MK952725
C. flaccida
Siebert_1403
FJ861136
C. glauca
Prince_1995 239
AF378774
C. indica
Kress_89 2849
AF378763
C. indica
Godfrey_60501
FJ861135
C. indica
Duke_88 124
FJ861130
C. indica
Duke_66 314
FJ861131
C. indica
Prince_1995 210
FJ861132
C. iridiflora
Plowman Davis_4753
FJ861134
C. jaegeriana
Kress_89 2884
FJ861133
C. paniculata
Plowman Kennedy 5700
AY656132
C. tuerckheimii
Kress_89 2853
AF378764
C. tuerckheimii
Duke_85 034
FJ861129
Costus pulverulentus
Duke_GH_86 043
AY656108
Dimerocostus strobilaceus
Kress_94 3601
AF243838
Tapeinochilos ananassae
Kress_90 2984
AF243840
Heliconia irrasa
Kress_76 519
AF378778
Heliconia rostrata
Duke_GH_81 030
AF378767
Orchidantha fimbriata
Kress_87 2159
AF243841
Orchidantha siamensis
Kress_94 3718
AF378771
Haumania sp
DJ_Harris_6672
AY656119
Sarcophrynium brachystachys
Kress_01 7007
AY656126
Ensete ventricosum
Kress_96 5372
AF243843
Musa ornata
Duke_GH_88 110
AF378779
Musella lasiocarpa
Kress_94 3709
AF243844
Phenakospermum guyannense
Kress_86 2099D
AF243845
Ravenala madagascariensis
Kress_92 3504
FJ861128
Strelitzia nicolai
Duke_GH_78 044D
AF243846
Globba curtissii
Kress_99 6247
AF243847
Siphonochilus kirkii
Kress_94 3692
FJ861127
Anigozanthos sp
Prince_2003 499
FJ861123
Cartonema philydroides
-
FJ861121
Hanguana sp
Kress_99 6325
FJ861125
Helmholtzia glaberrima
Prince_2003 007
FJ861124
Palisota ambigua
Faden_86/55
FJ861122
Dyckia marnier-lapostollei
T._World_97686
FJ861120
264
Assessment of the starch granules
The starch granule morphology of C. indica was assessed
in comparison to the starch granules of well-known tuber
crops in Sri Lanka; Xanthosoma sagittifolium, Manihot
esculenta, Solanum tuberosum, Ipomoea batatas purpletuber, and I. batatas yellow-tuber. The healthy tubers at the
right maturity stage for harvesting were collected from all
six species.
Isolation of starch
Ceylon Journal of Science 49(3) 2020: 261-273
staining purpose. The observations and the analysis of
tissues were done by using the Zeiss scanning electron
microscope (SEM) (Jeol SEM 6400, Tokyo, Japan). The
images were taken using same magnification, ×1000, as in
optical microscope to illustrate the arrangement of starch
granules inside the cells. The same procedure was carried
out to capture the images of extracted starch granules.
Here, instead of tissues, extracted starch granules of all of
the tuber types were used to capture the images and, the
magnification was adjusted to ×2000 for better resolution.
The starch was isolated from each species using the
modified protocols explained in Alves et al., (1999) and De
Pater et al., (2006). The tubers were thoroughly washed,
peeled and chopped into small pieces. The chopped pieces
were homogenized using a solution containing 0.075%
sodium bisulfate to avoid the browning. The homogenateslurry was then filtered to eliminate the impurities and
fiber. Then the filtrate was allowed to settle for three hours.
The supernatant was discarded and the sediment was resuspended in about five volumes of distilled water for
further purification. The starch was allowed to settle for 30
minutes. The procedure of decanting the supernatant and
re-suspending the sediment in water was repeated for three
to four more times to make sure that the extracted starch
was completely free of impurities. Finally the starch was
allowed to settle for one hour followed by treating with a
solution containing 0.1% NaOH. This step was carried out
to reduce the effects of non-starch polysaccharides which
may greatly interfere with starch isolation procedure by
retaining the smaller granules in the remaining fibrous
matrix. A magnetic stirrer was used to stir the viscous
solution. The extracted starch was then washed with
distilled water to remove the excess NaOH and allowed
to dry overnight at room temperature. The resulting starch
was stored at -20 °C.
Assessment of the consumer preference on boiled
tubers
Starch granule observation under optical microscopy
(OM)
RESULTS AND DISCUSSION
A few drops of starch granule suspension of each type
of tuber were treated with iodine and observed under
the optical microscope at ×1000 magnification. As a
control, the same observations were made by using starch
granule suspensions without treating with iodine. An
optical microscope (Carl Zeiss Microscopy GmbH, SN
3150000610) equipped with the camera Zeiss AxioCam
ICc 5 was used for the observations, and the captured
photos were analyzed using the image analysis software,
Zen lite 2.1. The diameter and the cross-sectional area of
20 starch granules were measured for each type of tuber
assessed. The data were subjected to ANOVA procedure
using the statistical package SAS 9.4 (SAS Institute, NC,
Cary, USA).
Starch granule observation under scanning electron
microscopy (SEM)
The freshly harvested tubers from each of the six species
were used to obtain tissue samples. The tissues were
subjected to the vacuum for about 15 min. The vacuumdried tissue samples were mounted on stubs with carbon
tabs. The tissue samples were exposed to gold particles for
The fresh and healthy tubers at the right maturity stage
were collected from all six species. The tubers were
peeled, washed thoroughly, cut into approximately 3
cm x 3 cm pieces, and boiled in water for 20 mins. The
water was filtered out, and the salt was added to ascertain
the generally preferred taste. A taste panel of 40 human
subjects was employed representing randomly selected
equal proportions of males and females in the age range
of 18 - 65 years. The required instructions were provided
to the panelists before the assessment. Each panelist was
given 40 g of boiled tuber pieces of each tuber type for
tasting. The panelists were requested to taste and rank
the tubers for seven sensory attributes; color, aroma,
texture, bitterness, fibrous nature, hardness, and overall
taste according to a three-tier scoring system. Score “1”
was assigned for the least preferred level. Score “2” was
assigned for medium preferred level and Score “3” was
assigned for the highest preferred level. The care was
taken to refresh the palates of each panelist by providing
water in between the tasting each tuber dish (Leighton et
al., 2010). The ranked data generated by the taste panel
were subjected to association analysis using FREQ
procedure in SAS.
Morphology of C. indica
Canna indica is a perennial herb that grows up to 1-3 m in
height at flowering stage (Figure 1). The newly emerged
rhizome (Figure 1A) is elliptical in shape and covered with
whitish scale leaves. The interior of the newly emerged
rhizome is cream-white to ivory in color (Figuers1B
and 1C). The fully-grown rhizome is irregular shaped,
monopodial or sympodial, stoloniferous or tuberous,
branched and also covered with scale leaves where the
distal part of the scale leaves are blackish-brown, and
proximal parts are whitish-yellow (Figure 1D). However,
at maturity, the external color of the rhizome is brown.
The root system of C. indica consists of an excessively
branched network of fibrous roots. The roots do not go
deep into the soil; however, forms a mat within 30 cm of
soil depth (Figure 1E). The roots are cylindrical and thick
with numerous hairs. The color of the roots is creamy
white (Figure 1E). As C. indica has a rhizome, the roots
are adventitious in nature. When rhizomes are separated
from the mother plant or in case where mother plant is
dead, sprouting of rhizomes happen immediately upon
touching with moist soils (Figure 1F). After two weeks,
265
R.W.K.M. Senevirathna et al.
the shoot gets elongated up to 5 - 15 cm. At this stage, no
roots are apparent. In the next three to four weeks, the shoot
is extending further, and roots start to grow (Figure 1G).
Within another week or so, the first leaf is unfolding and
continuing to produce new leaves sequentially (Figure 1H).
The plant remains in the vegetative stage for four months
and then starts flowering. A potted C. indica plant at its fully
grown vegetative stage is shown in Figure 1I. The stem is a
pseudo stem covered with sheathing leaves. They are erect,
herbaceous and cylindrical (Figure 1I). Appearance of the
leaves is somewhat similar to the banana leaves; however,
the size is less as C. indica leaves are 15 – 20 cm wide
and 20 – 50 cm long. The leaf shape is more or less ovate.
The leaf lamina is dark green with purplish brown margins.
Moreover, the leaf lamina is parallel veined and has
smooth and wavy margins. The leaf apex is acute (Figures
1J and 1K). Red, solitary flowers are normally 1 cm to 2
cm long and sepals are also having the same length range.
In flowers, the reddish-brown coral tube is a prominent
character. The flowers are hermaphrodite (Figure 1L). The
fruits are spiny, 2 cm to 3 cm long structures consisting of
green capsules (Figure 1L). The seeds are 5 – 6 mm long,
2 – 3 mm wide and white color at the initial stage, and
black color at the maturity stage (Rao and Donde, 1955). A
cultivated C. indica which appears as a bush at five months
of age is shown in Figure 1M. Figures 1N and 1O show two
C. indica plants grown in a natural habitat and an infertile
land, respectively.
Phylogenetics of C. indica
The best substitution model for combined datasets of Prince,
(2010) and the Canna spp. in Sri Lanka is TPM3uf+I+G (A/
C=2.100; A/G=8.260; A/T=1.000; C/G=2.100; C/T=8.260;
G/T=1.000). The maxmum convergence for the MCMC
tree is at 20 millon chain runs and the ESS value for each
prior is above 200. Almost similer branching pattern is
obtained for the trees constructed in the baysin and ML
frameworks. However, the ML tree resolves the best, thus
presented in this study (Figure 2). The Canna spp. in Sri
Lanka are separately clustered in the Cannaceae clade of
the phylogram (Figure 2A). However, the phylogenetic
relationships of the Cannaceae clade in this phylogeny are
poorly resolved with few polytomic relationships. This
has also been described in the Prince, (2010), where they
concluded that plastid markers failed to resolve the correct
phylogenetic relationships within the Cannaceae family.
The nucleotide variation is recognized in the selected
loci of the chloroplast genome of C. indica and ornamental
Canna spp. (Figures 2B, 2C and 2D). Out of the two coding
and two non-coding markers employed in the present study,
only rbcL, atpB gene and trnH-psbA can be effectively
employed to identify the nucleotide polymorphism and
thereby to designate the species in respective clusters within
a UPGMA tree. The marker trnL-trnF is monomorphic for
all the samples sequenced. The informative substitutions
in the atpB gene are detected; thus an enhanced separation
is obtained in the phylogeny (Figure 2B). Accordingly,
five edible Canna spp. are cladded in the same cluster.
Remarkably, the point mutation (A/C) at the 2nd position in
atpB gene is sufficient to ascertain the variance between the
ornamental Canna spp. from the edible Canna spp. with
a genetic divergence of 0.0008. However, the ornamental
Canna sp. (red flower bearing) is diverged out from the
clade which contains the other two ornamental Canna
spp. (yellow and orange flower bearing). This might be
attributed to the synonymous mutations at 1417th and
1418th positions in the atpB gene.
Furthermore, five transversions (C/G, C/A, T/G) and
one unique transition (A/G) within the trnH-psbA region
are detected. C. indica (Kundasale, Matale and NuwaraEliya) are grouped into one clade with a unique haplotype,
thus clearly differentiating them from ornamental Canna
sp. (yellow) with a genetic divergence of 0.0014. The clade
containing C. indica (Nuwara-Eliya) and ornamental Canna
sp. (red) is distinguishable from C. indica (Peradeniya) with
a genetic divergence of 0.0014. The ornamental Canna
sp. (orange) can be considered as the most evolutionary
diverged group, by relying predominantly on the SNP
variation in trnH-psbA locus (Figure 2D). Moreover,
synonymous mutations are identified in the rbcL region.
Consequently, C. indica (Matale, and Nuwara-Eliya) and
ornamental Canna sp. (red) are nested in the same clade
while C. indica (Nuwara-Eliya), ornamental Canna spp.,
yellow and orange, are cladded together. Similarly, C.
indica (Kundasale and Peradeniya) are fallen within the
same clade with a 0.0018 genetic divergence (Figure 2C).
Based on the results, it can be suggested that the usefulness
of the two loci; trnH-psbA and rbcL, in discriminating the
ornamental and edible Canna species is less, compared with
the informativeness of atpB gene. The ornamental Canna
spp. are also identified as C. iridiflora and C. jaegeriana
in the published literature (Prince, 2010; Tanaka et al.,
2009). However, in the present study for rbcL and trnHpsbA, the ornamental Canna spp. do not show any clear
separation from C. indica (edible Arrow-root) indicating
that ornamental Canna spp. could also come under C.
indica. conflicting with the nomenclature provided in
(Prince, 2010; Tanaka et al., 2009) (Figures 2C and 2D).
The ornamental Canna spp. and C. indica are clearly
separated by the polymorphism at the atpB gene (Figure
2B) highlighting the possible speciation or sub speciation
which requires further studies.
Morphological variability of starch granules
The variability of the size of the starch granules are given
in the Table 3. The results revealed that the granule size
and the shape are highly variable among the tuber crops
studied. C. indica starch granules are significantly larger
than the granules of the other popular starch crops studied
for the comparison purpose. The mean diameter and the
mean cross-sectional area of the starch granules are 53.72
µm of 2180.46 µm2 respectively for C. indica and they
are the largest reported values for the six species studied
(P<0.05). The microscopic analysis of the starch granules
in the present study further verifies the past observations
reporting that C. indica has large starch granules (Hermann
et al., 1997). The smallest size of the starch granules is
observed in I. batatas (purple) (mean diameter: 10.56 µm,
cross sectional area: 91.44 µm2) (P<0.05). The appearance
of starch granules under the OM and SEM are presented in
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Ceylon Journal of Science 49(3) 2020: 261-273
Figure 1: Morphological appearance of the whole plant and the different parts of C. indica plant.
A: Newly emerged rhizome; B: Cross section of the newly emerged rhizome; C: Longitudinal section of the newly emerged rhizome;
D: Fully grown rhizome at the harvesting stage (economically important part); E: Root system with sprouting rhizomes; F: Close-up
view of sprouting rhizome; G: Rhizome sprouted fully and ready to produce leaves; H: Sprouted rhizome with the first leaf; I: C. india
plant in a pot (a poly bag); J: Adaxial view of a fully grown leaf; K: Abaxial view of the fully grown leaf; L: Flower and fruit; M: A
cultivated bush of C. indica; N: C. indica plant naturally grown; O: C. indica plant grown in an infertile land.
Figure 3. A pronounced difference in the shape of the starch
granules is observed in C. indica. The shape is like a shell of
a bivalve with curve-like patterns (scallop-seashell shaped)
throughout the surface when viewed under OM (Figure
3A1; 3A2). The granules displayed a disk shape with a
smooth surface under SEM (Figure 3A3) and the similar
results have previously been reported by Jane et al., (1994).
However, within the tissue, the cells contain much smaller
number of starch granules in C. indica in comparison to the
other tuber crops studied (Figure 3A4). According to the
SEM images and the surface area measurements under OM,
the highest surface area is recorded for C. indica. Several
other studies also have reported the same observation
(Wickramasinghe et al., 2009, Piyachomkwan et al., 2002,
Hung and Morita, 2005, Cisneros et al., 2009). Moreover,
the high viscosity and the ability to make a clear paste of
C. indica starch make it a good candidate as a thickening
agent (Andrade-Mahecha et al., 2012).
R.W.K.M. Senevirathna et al.
267
Figure 2: Phylogenetic structure of Canna spp. in Sri Lanka.
A: rbcL phylogram showing the phylogenetic position of Canna spp. inhabited in Sri Lanka; B: UPGMA tree drawn for atpB gene; C:
UPGMA tree drawn for rbcL; D: UPGMA tree drawn for trnH-psbA.
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Ceylon Journal of Science 49(3) 2020: 261-273
Figure 3: Morphological variability of starch granules in root and tuber crops, observed under the optical microscope and scanning
electron microscope.
A: Canna indica; B: Xanthosoma sagittifolium; C: Manihot esculenta; D; Solanum tuberosum; E: Ipomoea batatas-purple; F: Ipomoea
batatas-yellow. 1: Optical microscopic photographs of granules isolated from the tissue and stained with iodine; 2: Optical microscopic
photographs of granules isolated from the tissue and not stained; 3: Scanning electron microscopic photographs of granules isolated from
the tissue (Magnification: ×2K); 4: Scanning electronic microscopic photographs of granules present within the tissue; (Magnification:
×1K).
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R.W.K.M. Senevirathna et al.
Table 3: Variation of the average diameter and the cross sectional area of the starch granules.
Mean diameter
Mean surface
(µm)
Area (µm2)
Canna indica
53.72a
2180.46a
Solanum tuberosum
14.55c
148.26c
Xanthosoma sagittifolium
15.82c
235.28b
Manihot esculenta
19.86b
322.47b
Ipomoea batatas-yellow
20.01b
313.99b
Ipomoea batatas-purple
10.56d
91.44d
Species
Means denoted by the same letters within the column are not significantly different at P<0.05.
Figure 4: The boiled tuber pieces of the six species used for the taste panel analysis. A: Canna indica; B: Xanthosoma
sagittifolium;C: Manihot esculenta; D; Solanum tuberosum; E; Ipomoea batatas (purple); F: Ipomoea batatas (yellow).
In contrast, X. sagittifolium is found to possess a
significantly high number of starch granules within the
tissue with irregular shapes (Figure 3B1; 3B2; 3B3; 3B4).
The granules observed under the OM reveals that those of
the I. batatas (yellow) and I. batatas (purple) are similar
in morphology with a polygonal shape (Figures 3E1;
3E2; 3F1; 3F2). The exception is being M. esculenta and
S. tuberosum which appears to be having a mixture of
shapes (Figures 3C1; 3C2; 3D1; 3D2 respectively). The
OM photographs show that some of the S. tuberosum
granules are oval shaped whereas the others are elliptical
(Figures 3D1; 3D2) while SEM displays disk and oval
shaped S. tuberosum granules (Figure 3D3). The cells of
I. batatas are observed with spherical and irregular shapes
of granules (Figure 3E3; 3F3) and fairly a high number of
granules when viewed under the SEM (Figures 3E4; 3F4).
However, M. esculenta granules possess both spherical and
irregular shapes (Figure 3C3). As shown by SEM image,
the number of granules within the tissues of M. esculenta
is higher than that of C. indica, however, less than that
of X. sagittifolium (Figure 3C4). Moreover, S. tuberosum
displays relatively a smaller number of starch granules
within the tissue, nevertheless a high number of granules
compared to C. indica.
Consumer preference
The representative samples of the boiled tubers prepared
for the taste panel are shown in Figure 4. The boiled tubers
of C. indica are brown in color (Figure 4A) compared to
other tubers boiled. All the taste parameters assessed except
bitterness are significantly associated with the type of
tuber (i.e., tuber species) (Figure 5, P<0.05). The strongest
association is detected between the type of tuber and the
preferred color (Figure 5A). The highest preferred color is
recorded for I. batatas (yellow) (88%) and S. tuberosum
(68%). The color of C. indica, M. esculenta and I. batatas
(purple) are equally preferred (15-18%). It is interesting to
note that the color of C. indica is preferred more than that
of X. sagittifolium which is a more popular tuber crop than
C. indica. The highest preferred aroma is observed for I.
batatas (yellow) (28%) followed by S. tuberosum (15%).
The respondents equally rank C. indica and M. esculenta
for the preferred level of aroma (Figure 5B). The highest
preferred texture is reported for the two types of I. batatas;
however, the texture of C. indica is preferred more than X.
sagittifolium (Figure 5C). The bitterness felt is low for all
the tuber types assessed; however, 10 % of the respondents
rank the highest felt bitterness for C. indica tubers (Figure
5D). The highest felt fibrousness is observed for C. indica
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Ceylon Journal of Science 49(3) 2020: 261-273
Figure 5: The associations between the taste parameters and six tube crop species studied. Y-axis represent the percentage (%)
respondents. The % respondents for each category of association is given within the bars.
R.W.K.M. Senevirathna et al.
(73%) followed by I. Batatas (purple) (21%) (Figure 5E).
The highest felt hardness is also reported for C. indica.
The felt hardness is found be the least for S. tuberosum
and I. batatas (yellow). The tubers of X. sagittifolium and
M. esculenta are ranked as the hardest by 25% and 20%
respondents respectively (Figure 5F). In the ranking of
tubers for overall taste, 70% and 50% of the respondents
said they highly prefer the taste of I. batatas (yellow) and
I. batatas (purple) respectively. Interestingly, C. indica and
M. esculenta get equal ranks for the overall taste (Figure
5G).
The association analysis based on the taste panel data
provided important insights on the potentials of C. indica
tubers and the avenues for the crop improvement. It is
evident from the analysis that consumers prefer yellow
color in boiled tubes but the color of C. indica is not entirely
rejected. The color of C. indica accepted better than that of
X. sagittifolium. The highest fibrousness and the hardness
could be the reasons for the less preference for the tubers
of C. indica. However, when considering the overall taste,
C. indica gets equally ranked to the highest levels as the
two of the world famous and established tuber crops, S.
tuberosum and M. esculenta (Figure 5G). Thus, improving
the tuber properties; softness and less fibrousness, through
breeding and selection will undoubtedly uplift C. indica
from its current underutilized status.
CONCLUSIONS
The phylogenetic tree constructed based on rbcL marker
revealed that C. indica is slightly divergent from the other
Canna species in the world. The polymorphism of the atpB
gene can be used to differentiate C. indica from ornamental
Canna spp. The polymorphism of rbcL and trnH-psbA
cannot be used to differentiate C. indica from the rest. The
starch granules morphological analysis revealed that C.
indica has the significantly large (scallop-seashell shaped)
starch granules compared to Xanthosoma sagittifolium,
Manihot esculenta, Solanum tuberosum, and Ipomoea
batatas. The consumer preference analysis indicates that
boiled C. indica tubers are accepted better than that of X.
sagittifolium. The C. indica tubers rate equally to the tubers
of Solanum tuberosum, and M. esculenta. The relatively
hardy fibrous nature of the tubers of C. indica is the
major limiting factor for achieving the highest consumer
preference. The breeding programs on C. indica must be
planned to achieve softer and less fibrous tubers for higher
appetite.
ACKNOWLEDGEMENTS
The authors wish to thank Departments of Geology
and Zoology of the Faculty of Science of University
of Peradeniya, Sri Lanka for kindly providing electron
microscopic and light microscopic facilities.
STATEMENT OF CONFLICT OF INTEREST
The authors declare no conflict of interest.
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