South African Journal of Botany 108 (2017) 294302

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Assessment of genetic stability amongst micropropagated Ansellia africana,

a vulnerable medicinal orchid species of Africa using SCoT markers
Paromik Bhattacharyya, Vijay Kumar, Johannes Van Staden
Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Micropropagation is an important tool for the conservation of threatened and commercially important plant
Received 18 July 2016 species of which orchids deserve special attention. Ansellia africana is one such medicinally important orchid
Received in revised form 30 September 2016 species having much commercial signicance. However, for large-scale micropropagation to become not only
Accepted 2 November 2016
successful but also acceptable by end-users, somaclonal variations occurring in the plantlets need to be eliminat-
Available online 9 December 2016
ed. In the present study, the clonal integrity of micropropagated A. africana plants derived from two different
Edited by JS Boatwright pathways was assessed using a gene-targeted marker system, i.e. Start Codon Targeted polymorphism (SCoT).
Our studies recorded a signicantly higher gene ow value (Nm = 1.596) amongst the generations with an in-
Keywords: crement in clonal variability. The developed protocol helps to understand the main start point of clonal variability
Ansellia africana in a model tissue culture system and the role played by the various plant growth regulators (PGRs). The protocol
Genetic stability also documents a fast and cost-effective regeneration pathway for commercially important medicinal orchids
Gene targeted molecular markers with reproducible molecular detection system to monitor and detect clonal variability for obtaining clonally
Medicinal orchid micropropagation stable true-to-type plantlets for sustainable commercial use.
SCoT-PCR
2016 SAAB. Published by Elsevier B.V. All rights reserved.
Somaclonal variation

1. Introduction
Amongst horticultural and oral crops, the orchids are inarguably
one of the most charismatic, having captivated the attention of con-
servationists, growers, and enthusiasts worldwide. They are one of
the most pampered plants and occupy a top position amongst all
the owering plants valued for cut ower and potted plants, fetching
a very high price in the international market. The world consump-
tion of orchids was reported to be valued at more than $500 million
in 2000 (Hew and Yong, 2004; Wang, 2004). Apart from their orna-
mental value, orchids are also known for their medicinal importance,
especially in the traditional systems of medicine. It is believed that
the Chinese were the rst to cultivate and describe the orchids, and
they were almost certainly the rst to describe orchids for medicinal
uses (Bulpitt, 2005). As in the world's other traditional medicinal
systems, African pharmacopeia orchids also form an important part
of which Ansellia africana gures prominently, primarily because of
its broad spectrum of medicinal properties, especially affecting the
central nervous system (CNS) (Hossain, 2011; Chinsamy et al.,
2014; Bhattacharyya and Van Staden, 2016). A thorough survey of
literature revealed the usage of stem infusions of A. africana as anti-
dotes to bad dreams (Hutchings, 1996). The smoke from burning
roots is also used for the same purpose (Hutchings, 1996;
Corresponding author.
E-mail address: Rcpgd@ukzn.ac.za (J. Van Staden).
Chinsamy et al., 2014). Recent studies have shown that A. africana
has potent acetylcholinesterase inhibitory activity and can be used
as an important source of various biomolecules for the treatment of
Alzheimer's disease which might be the reason behind the usage of
the leaves and stems for the treatment of madness by the Mpika
tribes of Zambia (Gelfand, 1985). Apart from these, this orchid has
also been used for various ethnobotanical purposes and has signi-
cant horticultural usage in the cut ower industry. Thus having
such a wide array of usage, its natural populations are under tremen-
dous anthropogenic pressure. Due to indiscriminate collection from
the wild and heavy deforestation, the natural populations of
A. africana are severely threatened and presently they are catego-
rized as vulnerable in the IUCN Red Data Book (http://www.
iucnredlist.org/details/44392142/0). In South Africa, the status of
A. africana is declining according to the recent Red List of South
African Plants published by South African National Biodiversity
Institute (SANBI; http://redlist.sanbi.org/).
In order to conserve orchids, plant tissue culture techniques have
been successfully applied for their clonal propagation and conservation
(Tandon and Kumaria, 1998). However, for large-scale propagation,
efciency of propagation methods along with genetic stability of the
regenerated plants is of paramount importance (Haisel et al., 2001).
Reports have shown that the regenerated plants might not always
be clonal copies of their mother plant when passed through
micropropagation pathways (Devi et al., 2014). The presence of cryptic
genetic defects occurring due to somaclonal variations can deregulate
the broader utility of the in vitro propagation system (Salvi et al.,

http://dx.doi.org/10.1016/j.sajb.2016.11.007
0254-6299/ 2016 SAAB. Published by Elsevier B.V. All rights reserved.
 P. Bhattacharyya et al. / South African Journal of Botany 108 (2017) 294302
2001). The occurrence of clonal variability is due to various causes of
which explant source and types of plant growth regulators (PGRs)
used plays a pivotal role (Devi et al., 2014).
Keeping a perspective view of the various advantages, molecular
markers are considered much more efcient tools in detection of clonal
variability, primarily as they are not inuenced by any environmental
factors and also because of their high reproducibility. Traditionally,
various conventional molecular markers like random amplied poly-
morphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple
sequence repeats (SSR) are used extensively in the assessment of clonal
delity (Bhattacharyya et al., 2014, 2015; Devi et al., 2015). However, as
the conventional molecular markers target a specic region of the ge-
nome, they have various limitations which have been largely resolved
by the evolution of gene-targeted molecular markers such as start
codon targeted polymorphism (SCoT) (Collard and Mackill, 2009;
Bhattacharyya et al., 2016a, 2016b). SCoT is an extremely reliable and
consistent molecular marker in which the primers have been designed
in accordance with the short conserved region surrounding the ATG
translation start (or initiation) codon (or translational start site, TSS).
More specically, it is a type of targeted molecular marker technique
with the ATG context as one part of a functional gene; markers generat-
ed from SCoT may be mostly correlated to functional genes and their
corresponding traits (Collard and Mackill, 2009; Bhattacharyya et al.,
2013; Singh et al., 2014). In the recent past, SCoT marker has been
widely used in the assertion of clonal delity in various medicinal plants
including orchids (Bhattacharyya et al., 2016a, 2016b). The present
study was therefore aimed at developing a genetically stable, sustain-
able regeneration protocol for A. africana and to assess how explant
source and plant growth regulators (PGRs) inuence the clonal delity
of the micropropagated plants.
2. Materials and methods
2.1. Chemicals
Agar bacteriological powder was purchased from Du Pont de
Nemours Int., South Africa, and Oxoid Ltd., Basingstoke, Hampshire,
England, respectively. N6-benzyladenine (BA), naphthalene acetic acid
(NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA),
phloroglucinol (PG), myo-inositol, vitamins (thiamine HCl, nicotinic
acid, pyridoxine HCl), and glycine were obtained from SigmaAldrich
Co., Steinheim, Germany. The topolins, meta-Topolin (mT), were pre-
pared as described previously (Doleal et al., 2006, 2007). All chemicals
used in the assays were of analytical grade.
2.2. In vitro seed germination and explants preparation
Mature plants of A. africana were collected from the wild and
were maintained in the greenhouse of the University of KwaZulu-
Natal (UKZN), Pietermaritzburg, South Africa (Fig. 1A). From these
greenhouse-maintained plants, mature capsules were collected and
were germinated asymbiotically in accordance to the protocol
described by Vasudevan and Van Staden (2010). Cultures were
maintained in a culture room at 25 2 C under 12 h light and
12 h dark cycles with 40 mol m 2 s 1 of light intensity provided
by cool-white uorescent tubes. Nodal segments containing axillary
buds were excised aseptically from 1-month-old seedlings and were
used as explants for the micropropagation experiments. The seedlings
which were used as explant source were labeled and maintained as
mother plants.
2.3. Micropropagation of A. africana
The explants were cultured in Murashige and Skoog (1962) medium
containing 3% sucrose (Merck, Germany), 0.8% agar (Oxoid, United
Kingdom), and different plant growth regulators (PGRs) such as
295
N6-benzyladenine (BA), meta-topolin (mT) in a range of 520 M and
indole acetic acid (IAA) at 5 M concentration for shoot induction. In-
dole butyric acid (IBA) and indole acetic acid (IAA) in a range of 5
20 M were used to promote root induction along with phenolic elici-
tors such as phloroglucinol (PG) ranging in concentrations ranging
from 10 to 40 M. The pH of the medium was adjusted to 5.8 before
autoclaving at 121 C for 15 min. For each treatment, 5 replicates were
taken and the experiments were repeated in triplicate. All cultures
were maintained at 25 2 C, 80% RH, and 12 h photoperiod of
40 mol m2 s1 irradiance provided by cool-white uorescent tubes
(OSRAM, Germany). Data on number of explants and number of devel-
oping shoots per explant were recorded.
The regenerated plants with well-developed roots were removed
from culture tubes, washed to remove the solidifying agar, and trans-
ferred to plastic pots containing different potting mixtures constituted
of vermiculite, sand, and decaying litter in a 1:1:1 ratio. Plants were ac-
climatized for 2 weeks at 25 2 C under a 12 h photoperiod
(40 mol m 2 s 1 of light intensity), fed with 1/10th strength MS
medium without sucrose, and transferred to mist house conditions for
acclimatization. Survival rate (%) was recorded after 60 days of transfer
to glass house conditions. All the data were subjected to analysis of var-
iance using SAS and means were compared using Duncan's multiple
range test.
2.4. Assessment of clonal delity of the micropropagated plants
2.4.1. DNA extraction and PCR amplication with SCoT primers
Total DNA from young leaves of 4-month-old A. africana plants were
extracted using the Qiagen Plant DNA extraction kit. The isolated DNA
was checked for quantity and purity in a UV spectrophotometer (Kary
50, Agilant Technologies, USA). Comparison of the ratio of absorbance
at two wavelengths (A260 and A280) with the standard ratio for pure
DNA was done. Initially, 45 SCoT primers were screened and 16 most re-
producible primers were selected for the nal amplication (Table S1).
The PCR amplications were carried out in a Veritti Thermal Cycler
(Applied Biosystems, USA) following the protocol described by
Bhattacharyya et al. (2016a).
2.4.2. Gel electrophoresis
Amplication products were separated by electrophoresis at 1.5%
agarose gel in 1X TAE buffer stained with ethidium bromide under
80 V constant power supply for 3 h and documented under Syngene
Gel DOC, Syngene, Synoptic Ltd., UK.
2.4.3. Data and statistical analysis
Only the clear and unambiguous amplicons were scored across all
samples. Based on the presence (1) or absence (0) of the selected
band, proles generated by SCoT primers were compiled into a data
binary matrix. Dendograms were generated by cluster analysis using
the UPGMA method based on Jaccard's coefcient. To estimate genetic
variation level within the micropropagated plants, genetic diversity pa-
rameters including the percentage of polymorphic loci (Pp), Nei's gene
diversity (h), and Shannon index (I) were calculated with POPGENE
version 1.31 (Yeh et al., 1997, 1999). Gene ow (Nm) was estimated
using the expression Nm = 0.25 (1 Gst)/Gst. Analysis of molecular
variance (AMOVA) was performed using Arlequin version 3.01
(Excofer et al., 2005) at two hierarchical levels to examine differences
among and within populations. The Fixation index or F statistics (FST)
was also calculated with Arlequin v. 3.01. The signicance of this analog
was evaluated by 1000 random permutations of sequences among
populations (Miller, 1998). Pairwise similarity matrices were generated
by Jaccard's coefcient of similarity, using the SIMQUAL format of
NTSYS-pc (Rohlf, 1998).
296 P. Bhattacharyya et al. / South African Journal of Botany 108 (2017) 294302

Fig. 1. Micropropagation of A. africana. (A) Mother plant. (B) Initiation of shoot buds in MS + 15 M BA (Bar = 1 cm). (C) Initiation of shoot buds in MS + 10 M mT (Bar = 1 cm) (Bar =
1 cm). (D) Development of multiple shoots after 15 days of initiation in MS medium + 15 M BA (Bar = 1 cm). (E) Development of multiple shoots after 15 days of initiation MS
medium + 10 M mT (Bar = 1 cm). (F) Proliferation of multiple shoots after 30 days when maintained in 20 M BA + 5 M NAA (Bar = 1 cm). (G) Proliferation of multiple shoots
after 30 days when maintained in 10 M mT + 5 M NAA (Bar = 1 cm). (H) Root induction from shoots at 15 M IBA (Bar = 1 cm). (I) Root induction from shoots at 30 M PG
(Bar = 1 cm). (J) Rooted shoots in MS medium + 15 M IBA + 30 M PG(Bar = 1 cm) (K) Inset view of rooted plants (Bar = 1 cm). (L) Full-grown plants (Bar = 1 cm)
(M) Greenhouse hardened plantlets after 4 months of transfer.

3. Results and discussions

3.1. Multiple shoot induction and proliferation of A. africana plants Table 1

Effects of various concentrations of mT and BA alone or in combination with NAA in MS
Formulation of an effective micropropagation strategy of any rare, medium on formation and shoot multiplication of A. africana.
endangered threatened (RET) category plant species, including orchids, Plant growth Response frequency (%) Shoots/explant PLB formation (%)
is of utmost importance for their sustainable utilization and conserva- regulators (M)
tion. Development of successful clonal propagation protocols is largely
mT BA NAA
inuenced by usage of proper PGR combinations. In achieving desired 0.0 0.0 0.0
rates of shoot proliferation, cytokinins play a signicant role. Although 5 56 0.4d 3.1 0.1 g 68 0.2c
cytokinins have vital roles in various plant metabolic pathways, their 10 74 0.2b 6.2 0.2d 74 0.5b
regulatory role on various cellular physiological and development pro- 15 71 0.3b 6.1 0.4d 72 0.4b
20 69 0.2c 5.3 0.5e 67 0.3c
cesses in plants are well reported (Werner et al., 2001). Furthermore, 5 32 0.4f 1.3 0.6i 31 0.4f
regeneration and proliferation of multiple shoots is closely correlated 10 47 0.3e 2.2 0.4 h 42 0.2e
with the type and concentrations of cytokinin used (Amoo et al., 15 54 0.4d 3.2 0.3 g 51 0.3d
2014). In orchids, formation and development of protocorm-like bodies 20 51 0.1d 2.1 0.4 h 48 0.2e
5 5 73 0.5b 7.2 0.2c 76 0.5b
(PLB)s from explants such as shoot-tips, axillary buds, stem segments is
10 5 81 0.3a 9.3 0.4a 83 0.6a
one of the accepted methods of in vitro propagation in orchids (Dohling 15 5 80 0.3a 8.3 0.3b 79 0.5b
et al., 2012). In the present study, we tested the effects of a conventional 20 5 77 0.2b 7.6 0.2c 77 0.4b
cytokinin (BA) as well as an aromatic cytokinin (mT) on the in vitro re- 5 5 39 0.3f 2.2 0.2 h 34 0.3f
generation pathways of A. africana. The explants responded differential- 10 5 46 0.2e 3.3 0.3 g 45 0.2e
15 5 55 0.4d 5.4 0.2e 57 0.6d
ly to BA and mT; however, both PGRs showed response within 15 days 20 5 57 0.2d 4.3 0.5f 51 0.5d
of culture. When the explants were cultured in MS medium supple-
Mean values within a column followed by the same letter are not signicantly different by
mented with BA, formation of direct shoots and PLBs were also
Duncan's multiple range test (P 0.05).
observed. However, the frequency of PLB formation and number of Values correspond to means (SE) of three independent experiments. Five replicates
shoots was less in comparison to that of mT (Table 1; Fig. 1B). However, were used for each experiment.
 P. Bhattacharyya et al. / South African Journal of Botany 108 (2017) 294302 297

using mT, formation of both PLBs as well as direct shoot formation cycles of sub-culturing, the proliferated shoots were transferred to
was observed at a signicantly higher rate (Table 1; Fig. 1C). Further- rooting media for root induction.
more, treatment with mT alone resulted in a signicant increment in
the conversion of PLBs into multiple shoot. The explants cultured in 3.2. Rooting and acclimatization
plain basal MS medium did not show any response. Coupled with
that, mT also exhibited a higher response frequency of 74% generat- The multiple shoots generated from both the pathways, i.e. mT- and
ing both shoot buds as well as primary PLBs in comparison to BA BA-mediated were transferred to a medium supplemented with IBA,
where the highest observed regeneration frequency was 54%. After IAA, and PG at varying concentrations (Table 2). As the rooting percent-
3 weeks, multiple shoot buds were initiated along with the forma- age was less in full-strength MS medium (data not shown), the medium
tion of secondary PLBs in which the efcacy of mT was established strength was reduced to half strength. Both IAA and IBA and phenolic
over BA. The rate of formation of multiple shoots was signicantly elicitor PG were found to be conducive to root induction (Table 2). In
lower in case of BA-derived plants with no signs of development of this medium, 35 roots per shoot were obtained after 6 weeks of culture
secondary PLBs (Table 1; Fig. 1D, E). When the explants were using root inducers separately (Table 2; Fig. 1H). Being an epiphyte, de-
grown in cytokinin and auxin (5 M NAA) supplemented medium, velopment of adequate branching is of utmost importance. Thus, in
a higher rate of response frequency of shoot buds and PLBs was order to increase the rooting frequency, varying concentrations of IBA
observed in all PGR combinations. Being potent cytokinins, the were used, along with PG to enhance the rate of root induction and pro-
presence of either BA or mT has been reported to trigger the liferation. The efcacy of PG on in vitro rooting has been reported in var-
meristematic activation in the tissue cells, which ultimately ious plant species including orchids (Bhattacharyya et al., 2015, 2016a;
assists in the formation of multiple shoots (Koir et al., 2004). In Kumar et al., 2016). The conducive effect of PG in the in vitro rooting of
the present study, synergistic effects of cytokinin and auxin the orchids might be due to the auxin-phenol synergism resulting in the
combinations induced an effective multiple shoot induction rate down-regulation of the peroxidase activity in the rooting medium,
(Table 1; Fig. 1F, G). thereby protecting the endogenous auxin from peroxidase-catalyzed
However, a comparative assessment of functional efcacy be- oxidation (De Klerk et al., 1999). Furthermore, being a precursor in
tween BA and mT puts forward the latter as a more superior PGR the lignin biosynthesis pathway, PG also prevents hyperhydricity within
than the former. The induction rate varied with type and concentra- the micropropagated plants by providing precursors which are general-
tion of growth regulators. The combination, concentrations, and the ly at extremely low levels or not synthesized at all in hyperhydric tis-
ratio between the growth regulators are critically important for the sues, and thus by increasing the activity of enzymes involved in lignin
formation of shoots and PLBs in orchids (Dohling et al., 2007; biosynthesis (Phan and Hegedus, 1986; Ross and Grasso, 2010). In the
Bhattacharyya et al., 2016b). The efcacy of the mT over the conven- present study, the synergized effect of IBA and PG promoted a signi-
tional purine-based cytokinins such as BA and KN has been reported cantly higher rate of rooting with highest recorded rooting frequency
by various coworkers for various medicinal plant species including of 76% with an average of 8 roots/shoot within 1 month (Table 2;
orchids (Werbrouck et al., 1995, 1996; Bogaert et al., 2004; Bairu Fig. 1I, J, K). The plants were maintained in the optimum rooting medi-
et al., 2007; Aremu et al., 2012; Bose et al., 2016; Bhattacharyya um for another 2 months and the full-grown plants (Fig. 1L) were trans-
et al., 2016b). Being less toxic in nature, mT accounts for a much ferred to plastic cups in the greenhouse, resulting in 87% survival. There
higher multiple shoot proliferation rate, and simultaneously, it is be- were no visual or morphological abnormalities observed within the
coming a much accepted PGR in comparison to conventional purine- micropropagated plants (Fig. 1M).
based cytokinins like BA or Kinetin (Amoo et al., 2011). The primary
reason behind the superiority of mT over BA and other conventional 3.3. Assessment of clonal delity among the micropropagated plants of
cytokinins is the fact that the localized accumulation of the topolins A. africana
in plant tissues is prevented by its fast translocation rate (Kamnek
et al., 1997). Coupled with this, the metabolic end products of mT In spite of having various advantages, a major criticism of the tissue-
are easily degradable and the hydroxyl group in the side chain of culture-raised plants is the development of somaclonal variations.
mT makes possible the formation of O-glucoside metabolites, a
metabolite which is considered to be a cytokinin storage form, stable Table 2
under certain conditions but rapidly getting converted to active Effects of auxins and phenolic elicitor on root formation and proliferation of A. africana.
cytokinin bases when required (Parker et al., 1978; Werbrouck Root inducers Frequency of shoots Shoot/root Root length (cm)
et al., 1996; Bairu et al., 2009). The reversible sequestration of the (Auxins/phenolic with roots
O-glucosides, in turn, allows the continuous availability of cytokinins elicitor)
at a physiologically active level over a prolonged period of time IBA IAA PG
resulting in a high shoot multiplication rate in in vitro cultures 0.0 0.0 0.0
(Strnad, 1997). The present experimental nding also advocates 5 53 0.3c 2.1 0.2 g 1.2 0.5e
10 61 0.2b 3.2 0.3f 1.4 0.3e
the efciency of mT over BA and is in close proximity with the nd-
15 72 0.5a 5.3 0.4d 1.5 0.4e
ings of Bhattacharyya et al. (2016a) in Dendrobium nobile, a medici- 20 65 0.4b 5.1 0.3d 1.3 0.5e
nal orchid species. As previously mentioned, the rate of shoot 5 32 0.3e 2.1 0.5 g 1.1 0.3e
proliferation magnied signicantly in synergy with 5 M NAA, 10 45 0.5d 3.1 0.3f 1.1 0.2e
which acted as an auxin trigger. In the present study, the highest 15 41 0.3d 2.1 0.4 g 1.2 0.3e
20 36 0.2e 1.3 0.3 h 1.3 0.5e
number of multiple shoots were induced when the proliferating
10 42 0.5d 2.1 0.5 g 2.1 0.4d
shoot buds were cultured in MS media supplemented with 10 M 20 47 0.3d 3.5 0.3f 2.4 0.5d
of mT and 5 M NAA (Table. 1). Along with alleviated shoot multipli- 30 56 0.4c 3.2 0.5f 3.2 0.6c
cation rates, formation of secondary PLBs was also observed facilitat- 40 43 0.3d 3.5 0.3f 2.0 0.5d
5 10 53 0.2c 4.2 0.4e 4.2 0.4b
ing a higher clonal propagation rate. The present nding is also
10 20 62 0.4b 6.1 0.2c 4.6 0.3b
supported by the ndings of Bairu et al. (2008) and Amoo et al. 15 30 76 0.3a 8.1 0.6a 5.7 0.5a
(2011), where a signicant increment in callus yield as well as ad- 20 40 69 0.4b 7.1 0.5b 5.1 0.6a
ventitious shoot proliferation was recorded using mT. The multiple Mean values within a column followed by the same letter are not signicantly different by
shoots generated from both the pathways, i.e. mT-derived as well Duncan's multiple range test (P 0.05).
as BA-derived were sub-cultured after 1 month interval. After two The values represent the means (SE) of three independent experiments.
298 P. Bhattacharyya et al. / South African Journal of Botany 108 (2017) 294302
Table 3
Comparison of genetic variations in all three consecutive regenerations of A. africana as revealed by SCoT markers.
Regenerations SPAR methods No of primers used Total no of bands amplied
1st mT pathway 16 56
BAP pathway 62
2nd mT pathway 72
BAP pathway 56
3rd Acclimatized 70
*PP = percentage polymorphism; PB = polymorphic bands.
Reports suggest that synthetic PGRs used during the course of
micropropagation create stress in the culture environment due to the
release of cytotoxic by-products, which induce programmed loss of cel-
lular controls (Phillips et al., 1994; Kaeppler et al., 1998; Martins et al.,
2004). The occurrence of somaclonal variations during in vitro propaga-
tion is a serious impediment in the practical utilization of plant tissue
culture techniques (Rahman and Rajora, 2001). Both phenotypic as
well as genetic variations may occur during clonal propagation giving
rise to genetically unstable clones (Kaeppler et al., 2000). Thus, detect-
ing the causes, mechanisms, and also the particular stage of propagation
pathway at which the somaclonal variations occur will further help in
minimizing and eliminating the issues of clonal variability during
micropropagation. Both chromosomal rearrangements and single gene
mutations have been reported to contribute to the effect of somaclonal
variations (Phillips et al., 1994). Besides DNA hypomethylation, genome
adaptation to differential microelement environments and the presence
of hot spots are the other probable causes of clonal variability (Bogani
et al., 1996; Linacero et al., 2000; Jaligot et al., 2004; Keyte et al., 2006;
Lukens and Zhan, 2007).
The clonal variations induced in the tissue-culture-raised plants are
reected in their genome proling patterns using different marker
systems, which propel DNA-based marker systems for the assessment
of clonal delity, a technique of choice. Furthermore, it is more afford-
able in comparison to other available protocols, e.g. ow cytometry
(FCM), and easy to carry out. In the present study, we have developed
a comprehensive approach using gene-targeted SCoT markers which
enabled us to detect the point of origin of somaclonal variations in a
model orchid tissue culture system. In order to ascertain a perspective
Fig. 2. SCoT proles of A. africana obtained with SCoT primer S5 for mT-derived plants and (Lane L: 500 bp ladder; A) BA-derived plants (L: 500 bp ladder; B); Lane 1: mother plant; Lanes
210: micropropagated plants of the rst regeneration; Lanes 1119: micropropagated plants of the second regeneration; Lanes 2028: micropropagated plants of the third regeneration.
Avg bands/primer Total no of PB PP Distance range (Jaccard's coefcient)
3.5 3 5.35 0.951.00
3.8 4 6.45 0.941.00
4.5 5 6.94 0.951.00
3.5 4 7.14 0.951.00
4.37 5 7.14 0.941.00
approach by taking into account maximum possible factors, we had
attempted to trace the clonal variability in this model tissue culture
system at various stages of sub-culturing as well as after successful
acclimatization. In the rst regeneration, 56 bands were generated by
the mT-derived plants, whereas 62 amplied bands were produced by
the plants derived from the BA pathway (Table 3). The number of
polymorphic bands was found to be 3 and 4, respectively (Table 3;
Fig. 2A, B). The Jaccards distance matrix ranged from 0.95 to 1.00 and
0.94 to 1.00, respectively (Table 3). The UPGMA clustering organized
the sampled representatives of the rst regeneration derived from
both the pathways into two broad clusters (Fig. 3A, B). Similar to the
rst generation, molecular analysis of the micropropagated plants in
the second generation obtained after second subculture parsing were
also assessed using SCoT markers. Using the same primers, 72 and
56 bands were obtained in case of mT and BA-derived plants of
which 5 and 4 bands were polymorphic (Table 3; Fig. 2A, B). Using
the binary matrix generated in the process, Jaccard's similarity ma-
trix was calculated, which ranged from 0.95 to 1.00 and 0.95 to
1.00, respectively (Table 3). Similar to the rst regeneration of
orchids, the sampled representatives from both the pathways
were broadly grouped into two major clusters (Fig. 3C, D). Finally,
the 4-month-old greenhouse acclimatized plants were analyzed.
The hardened plants produced a total of 70 amplicons with the 16
assayed SCoT primers of which 5 bands were found polymorphic
with a percentage polymorphism value of 7.14 and similarity coef-
cient ranging from 0.94 to 1.00 (Table.3). As with the other two re-
generations of micropropagated plants, the representatives were
broadly organized into two broad clusters (Fig. 4).
 P. Bhattacharyya et al. / South African Journal of Botany 108 (2017) 294302 299

Fig. 3. UPGMA dendogram generated for band data from SCoT marker illustrating coefcient similarities among regenerated plants and the mother plant [rst regeneration: A, B (mT and
BA- derived); second regeneration: C, D (mT and BA-derived).

Fig. 4. UPGMA dendogram generated for band data from SCoT marker illustrating
coefcient similarities among mother plant and regenerated plants of the third
regeneration.
3.4. Population genetic structure of the micropropagated plants
Coupled with this, population genetic structure among the various
generations of the micropropagated A. africana was also ascertained.
The present ndings are also justied by the study of the genetic
structure given by SCoT markers revealing that the number of observed
alleles (Na) steadily increased from 1.13/1.23 (mT/BA) in the rst gener-
ation to 1.28 in the third generation with an overall frequency of 1.39
(Table 4). Similarly, the number of alleles (Ne) that effectively contrib-
uted to the genetic diversity also showed steady increase from 1.09/
1.15 (mT/BA) in the rst generation up to 1.23 in the third generation
with an overall rate of 1.15. The present parameters of population struc-
ture also indicate that BA-derived plantlets were experiencing more
clonal variability in comparison to mT-derived plants. Other important
parameters like Shannon (I) and Nei (H) diversity indices, which were
used to evaluate the abundance and uniformity of alleles present at
one locus, also showed a steady increase. Present ndings revealed
that these indices signicantly increased from rst to third generation
and this level of variability was found to be proportionate to the gener-
ations analyzed in the study (Table 4). In the present study, using
AMOVA, it was revealed that there exists a higher distribution of genetic
variation within the generations as compared to among the generations
of micropropagated A. africana which is also supported by GST value. A
signicantly higher gene ow was recorded between the generations
(Nm = 1.596), which might have resulted in a marked change in allele
frequencies, which further justies the argument of steady increments

Table 4
Genetic diversity and differentiation parameters for three generations of micropropagated A. africana using SCoT marker.

Generations Na SD Ne SD H SD I SD Pp Gst Nm FST

1st generation mT pathway 1.13 0.33 1.09 0.27 0.05 0.14 0.07 0.20 5.40
BA pathway 1.23 0.21 1.15 0.11 0.12 0.14 0.11 0.15 6.53
2nd generation mT pathway 1.13 0.33 1.10 0.28 0.05 0.19 0.15 0.22 6.73
BA pathway 1.15 0.15 1.17 0.21 0.1 0.16 0.23 0.15 7.23
3rd generation Acclimatized plants 1.28 0.45 1.23 0.37 0.12 0.20 0.18 0.29 7.23
Overall 1.39 0.49 1.15 0.24 0.10 0.14 0.16 0.22 6.62 0.238 1.596 0.535

H = Nei's gene diversity; I = Shannon's information index; Na = observed number of alleles; Ne = effective number of alleles; Pp = percentage of polymorphic loci; SD = standard
deviation; Gst = diversity among populations; Nm = gene ow 0.25(1Gst)/Gst; Hpop = variability within population, FST = xation index or F statistics.
300 P. Bhattacharyya et al. / South African Journal of Botany 108 (2017) 294302
Table 5
Analysis of molecular variance (AMOVA) for three regenerations of micropropagated
A. africana.
Source of Sum of Variance Percentage of
variation d.f squares components variation
Among generations 2 15.867 0.64811 25.51
Within generations 25 47.311 1.89244 74.49
Total 27 63.179 2.54055
d.f = degrees of freedom.
in genetic variations among generations of micropropagated plants
(Table 5). The UPGMA data matrix was analyzed further to study the ge-
netic relatedness of the generations of micropropagated A. africana
plants which revealed that the plants from all three regenerations can
be grouped into two groups (Fig. 3AD; Fig. 4). The above ndings are
well supported by the AMOVA results, which reveal that a greater de-
gree of variation exists within the generations as compared to that of
among the generations. The present outcome is closely supported by
the ndings of other workers in a variety of plant taxa where axillary
branching did not ensure the desired genetic stability (Kanwar and
Bindiya, 2003; Modgil et al., 2005; Feyissa et al., 2007; Devi et al., 2014).
3.5. Comparative assessment of the PGRs in inducing clonal variability
The present research puts forward that increment of clonal variabil-
ity over generations, even when using axillary buds considered to be a
stable explant, may be due to different reasons. Oxidative stress
induced during the propagation pathway can be the most signicant
factor contributing to the development of clonal variability. In the
plant tissue culture experiment, PGRs play a pivotal role in plant propa-
gation and is also considered to be one of the primary causes of
somaclonal variations in micropropagated plants (Stover, 1986; Koir
et al., 2004). The mode of regeneration, tissue culture environment,
and culture conditions may account for the occurrence of clonal vari-
ability (Rani and Raina, 2000; Bairu et al., 2006). In order to achieve op-
timum growth of plants, various synthetic PGRs are used which might
have been induced in vitro stress by biochemical or other nutritional
conditions (Devarumath et al., 2002; Koir et al., 2004). In the present
study, in order to ascertain the role of the PGRs in development of clonal
variability, we used both BA and mT in the micropropagation experi-
ments. The experimental ndings revealed, in both rst and second
regenerations, that the clonal variability is increasing in BA-derived
plants which might be due to the residual toxicity of BA and its low
translocation rate. Whereas in the case of mT-derived plants, the rate
of clonal variability, although undergoing similar degree of in vitro
stress, was signicantly low. The probable reason behind such a
phenomenon might be the high levels of oxidative stress contributing
to DNA damage, including microsatellite instability within the
micropropagated plantlets (Jackson et al., 1998). Apart from this,
Werbrouck et al. (1996) and Bogaert et al. (2004) reported improved
histogenic stability and anti-senescent effects through the use of mT,
which has also accounted for the superiority of the mT over BA. Our
present nding is closely corroborated by the ndings of our previous
studies and other coworkers (Aremu et al., 2012; Bhattacharyya et al.,
2016b). The present analysis revealed that point of origin of clonal
variability in this model tissue culture system is during the in vitro
shoot induction and multiplication. Sub-culturing increased the
variability rate (Table 3). However, when the plants were subjected to
acclimatization, there was no signicant increase in clonal variability.
The probable reason behind such an outcome might be the use of PG.
Recent research showed the protective role played by PG in arresting
DNA damage both in in vitro and in vivo models (Kim et al., 2012; Piao
et al., 2014, 2015). In this research, the use of PG might have arrested
the occurrence of variability and thereby checked the rate of clonal var-
iability. The utility of the present report can be further justied by the
fact that the molecular information generated by the conventional
markers like RAPD or ISSR is usually obtained from non-coding DNA re-
gions, as a result of which the obtained information can only be held
useful when it is linked with some trait. To its contrary, SCoT is a type
of targeted molecular marker, which is derived either from the gene it-
self or from its anking sequences (Collard and Mackill, 2009). There-
fore, the information obtained from the SCoT marker could be
correlated with the functional genes and their corresponding traits
(Collard and Mackill, 2009). Considering the importance of assessment
of clonal delity using a very reliable and advanced marker system like
SCoT (Collard and Mackill, 2009; Bhattacharyya et al., 2016a, 2016b),
the present protocol appears to be of signicant importance in provid-
ing an appropriate sustainable, clonally stable regeneration protocol
for this and other related plant species.
4. Conclusions
The formulation of plant tissue culture strategies for sustianable
utilization RET plant resources is of utmost importance in the present
context. In the present study, we have developed a fast, reproducible,
and genetically stable regeneration protocol which can be successfully
utilized in the large-scale production of this medicinaly important spe-
cies of orchid and related plant species. In this protocol, we were able to
induce a higher root proliferation rate which deserves special mention
as in Traditional African Pharmacopiaea (TAP), root infusions of
A. africana are used in the treatment of neural disorders. Furthermore,
to the best of our knowledge, this is the rst report of production of ge-
netically stable germplasm of any African medicinal orchid. Coupled
with that, the present approach also provides a basic molecular insight
into the role of the synthetic PGRs in inducing clonal variability. Our re-
search ndings clearly justify the novelty of mT in the micropropagation
pathway along with higher genetic stability in comparison to conven-
tional cytokinins like BA. The present molecular assay using SCoT
markers helped us to develop a genetic stability evaluation system
which is fast, economic, reproducible, and reliable. The approach nul-
lies the criticisms raised against the use of dominant markers such as
RAPD and ISSR and successfully detects variability with high precission.
In a nutshell, the present protocol provides a mass propagation as well
as clonal delity detection system using gene-targeted molecular mark-
er SCoT which can be effectively used in the sustainble utilization of
plant genetic resources by detecting and elliminating the adversities
of somaclonal variations.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.sajb.2016.11.007.
Acknowledgments
PB and VK thank the University of KwaZulu-Natal (UKZN), South
Africa and the National Research Foundation (NRF), Pretoria for nan-
cial support in the form of Postdoctoral Fellowships.
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