Assessment of genetic stability amongst micropropagated Ansellia africana, a vulnerable medicinal orchid species of Africa using SCoT markers

South African Journal of Botany 108 (2017) 294–302

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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

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Article history: Received 18 July 2016 Received in revised form 30 September 2016 Accepted 2 November 2016 Available online xxxx Edited by JS Boatwright Keywords: Ansellia africana Genetic stability Gene targeted molecular markers Medicinal orchid micropropagation SCoT-PCR Somaclonal variation

a b s t r a c t Micropropagation is an important tool for the conservation of threatened and commercially important plant species of which orchids deserve special attention. Ansellia africana is one such medicinally important orchid species having much commercial significance. However, for large-scale micropropagation to become not only successful but also acceptable by end-users, somaclonal variations occurring in the plantlets need to be eliminated. In the present study, the clonal integrity of micropropagated A. africana plants derived from two different pathways was assessed using a gene-targeted marker system, i.e. Start Codon Targeted polymorphism (SCoT). Our studies recorded a significantly higher gene flow value (Nm = 1.596) amongst the generations with an increment in clonal variability. The developed protocol helps to understand the main start point of clonal variability in a model tissue culture system and the role played by the various plant growth regulators (PGRs). The protocol also documents a fast and cost-effective regeneration pathway for commercially important medicinal orchids with reproducible molecular detection system to monitor and detect clonal variability for obtaining clonally stable true-to-type plantlets for sustainable commercial use. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Amongst horticultural and floral crops, the orchids are inarguably one of the most charismatic, having captivated the attention of conservationists, growers, and enthusiasts worldwide. They are one of the most pampered plants and occupy a top position amongst all the flowering plants valued for cut flower and potted plants, fetching a very high price in the international market. The world consumption of orchids was reported to be valued at more than $500 million in 2000 (Hew and Yong, 2004; Wang, 2004). Apart from their ornamental value, orchids are also known for their medicinal importance, especially in the traditional systems of medicine. It is believed that the Chinese were the first to cultivate and describe the orchids, and they were almost certainly the first 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 figures 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 antidotes to bad dreams (Hutchings, 1996). The smoke from burning roots is also used for the same purpose (Hutchings, 1996;

⁎ Corresponding author. E-mail address: [email protected] (J. Van Staden).

http://dx.doi.org/10.1016/j.sajb.2016.11.007 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

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 significant horticultural usage in the cut flower industry. Thus having such a wide array of usage, its natural populations are under tremendous 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 categorized 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, efficiency 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.,

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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 efficient tools in detection of clonal variability, primarily as they are not influenced by any environmental factors and also because of their high reproducibility. Traditionally, various conventional molecular markers like random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR) are used extensively in the assessment of clonal fidelity (Bhattacharyya et al., 2014, 2015; Devi et al., 2015). However, as the conventional molecular markers target a specific region of the genome, 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 specifically, it is a type of targeted molecular marker technique with the ATG context as one part of a functional gene; markers generated 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 fidelity in various medicinal plants including orchids (Bhattacharyya et al., 2016a, 2016b). The present study was therefore aimed at developing a genetically stable, sustainable regeneration protocol for A. africana and to assess how explant source and plant growth regulators (PGRs) influence the clonal fidelity 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 Sigma–Aldrich Co., Steinheim, Germany. The topolins, meta-Topolin (mT), were prepared as described previously (Doležal et al., 2006, 2007). All chemicals used in the assays were of analytical grade.

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N6-benzyladenine (BA), meta-topolin (mT) in a range of 5–20 μM and indole acetic acid (IAA) at 5 μM concentration for shoot induction. Indole butyric acid (IBA) and indole acetic acid (IAA) in a range of 5– 20 μM were used to promote root induction along with phenolic elicitors such as phloroglucinol (PG) ranging in concentrations ranging from 10 to 40 μM. 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 μmol m−2 s−1 irradiance provided by cool-white fluorescent tubes (OSRAM, Germany). Data on number of explants and number of developing shoots per explant were recorded. The regenerated plants with well-developed roots were removed from culture tubes, washed to remove the solidifying agar, and transferred to plastic pots containing different potting mixtures constituted of vermiculite, sand, and decaying litter in a 1:1:1 ratio. Plants were acclimatized for 2 weeks at 25 ± 2 °C under a 12 h photoperiod (40 μmol 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 variance using SAS and means were compared using Duncan's multiple range test.

2.4. Assessment of clonal fidelity of the micropropagated plants 2.4.1. DNA extraction and PCR amplification 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 reproducible primers were selected for the final amplification (Table S1). The PCR amplifications 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 Amplification 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.

2.2. 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 KwaZuluNatal (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 μmol m− 2 s− 1 of light intensity provided by cool-white fluorescent 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

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, profiles 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 coefficient. To estimate genetic variation level within the micropropagated plants, genetic diversity parameters 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 flow (Nm) was estimated using the expression Nm = 0.25 × (1 − Gst)/Gst. Analysis of molecular variance (AMOVA) was performed using Arlequin version 3.01 (Excoffier 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 significance of this analog was evaluated by 1000 random permutations of sequences among populations (Miller, 1998). Pairwise similarity matrices were generated by Jaccard's coefficient of similarity, using the SIMQUAL format of NTSYS-pc (Rohlf, 1998).

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Fig. 1. Micropropagation of A. africana. (A) Mother plant. (B) Initiation of shoot buds in MS + 15 μM BA (Bar = 1 cm). (C) Initiation of shoot buds in MS + 10 μM mT (Bar = 1 cm) (Bar = 1 cm). (D) Development of multiple shoots after 15 days of initiation in MS medium + 15 μM BA (Bar = 1 cm). (E) Development of multiple shoots after 15 days of initiation MS medium + 10 μM mT (Bar = 1 cm). (F) Proliferation of multiple shoots after 30 days when maintained in 20 μM BA + 5 μM NAA (Bar = 1 cm). (G) Proliferation of multiple shoots after 30 days when maintained in 10 μM mT + 5 μM NAA (Bar = 1 cm). (H) Root induction from shoots at 15 μM IBA (Bar = 1 cm). (I) Root induction from shoots at 30 μM PG (Bar = 1 cm). (J) Rooted shoots in MS medium + 15 μM IBA + 30 μM 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 Formulation of an effective micropropagation strategy of any rare, endangered threatened (RET) category plant species, including orchids, is of utmost importance for their sustainable utilization and conservation. Development of successful clonal propagation protocols is largely influenced by usage of proper PGR combinations. In achieving desired rates of shoot proliferation, cytokinins play a significant role. Although cytokinins have vital roles in various plant metabolic pathways, their regulatory role on various cellular physiological and development processes in plants are well reported (Werner et al., 2001). Furthermore, regeneration and proliferation of multiple shoots is closely correlated with the type and concentrations of cytokinin used (Amoo et al., 2014). In orchids, formation and development of protocorm-like bodies (PLB)s from explants such as shoot-tips, axillary buds, stem segments is one of the accepted methods of in vitro propagation in orchids (Dohling et al., 2012). In the present study, we tested the effects of a conventional cytokinin (BA) as well as an aromatic cytokinin (mT) on the in vitro regeneration pathways of A. africana. The explants responded differentially to BA and mT; however, both PGRs showed response within 15 days of culture. When the explants were cultured in MS medium supplemented with BA, formation of direct shoots and PLBs were also observed. However, the frequency of PLB formation and number of shoots was less in comparison to that of mT (Table 1; Fig. 1B). However,

Table 1 Effects of various concentrations of mT and BA alone or in combination with NAA in MS medium on formation and shoot multiplication of A. africana. Plant growth regulators (μM) mT 0.0 5 10 15 20 – – – 5 10 15 20 – – – –

BA 0.0 – – – 5 10 15 20 – – – – 5 10 15 20

NAA 0.0 – – – – – – – 5 5 5 5 5 5 5 5

Response frequency (%)

Shoots/explant

PLB formation (%)

– 56 74 71 69 32 47 54 51 73 81 80 77 39 46 55 57

– 3.1 6.2 6.1 5.3 1.3 2.2 3.2 2.1 7.2 9.3 8.3 7.6 2.2 3.3 5.4 4.3

– 68 74 72 67 31 42 51 48 76 83 79 77 34 45 57 51

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4d 0.2b 0.3b 0.2c 0.4f 0.3e 0.4d 0.1d 0.5b 0.3a 0.3a 0.2b 0.3f 0.2e 0.4d 0.2d

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 g 0.2d 0.4d 0.5e 0.6i 0.4 h 0.3 g 0.4 h 0.2c 0.4a 0.3b 0.2c 0.2 h 0.3 g 0.2e 0.5f

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2c 0.5b 0.4b 0.3c 0.4f 0.2e 0.3d 0.2e 0.5b 0.6a 0.5b 0.4b 0.3f 0.2e 0.6d 0.5d

Mean values within a column followed by the same letter are not significantly different by Duncan's multiple range test (P ≤ 0.05). Values correspond to means (±SE) of three independent experiments. Five replicates were used for each experiment.

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using mT, formation of both PLBs as well as direct shoot formation was observed at a significantly higher rate (Table 1; Fig. 1C). Furthermore, treatment with mT alone resulted in a significant increment in the conversion of PLBs into multiple shoot. The explants cultured in plain basal MS medium did not show any response. Coupled with that, mT also exhibited a higher response frequency of 74% generating both shoot buds as well as primary PLBs in comparison to BA where the highest observed regeneration frequency was 54%. After 3 weeks, multiple shoot buds were initiated along with the formation of secondary PLBs in which the efficacy of mT was established over BA. The rate of formation of multiple shoots was significantly lower in case of BA-derived plants with no signs of development of secondary PLBs (Table 1; Fig. 1D, E). When the explants were grown in cytokinin and auxin (5 μM NAA) supplemented medium, a higher rate of response frequency of shoot buds and PLBs was observed in all PGR combinations. Being potent cytokinins, the presence of either BA or mT has been reported to trigger the meristematic activation in the tissue cells, which ultimately assists in the formation of multiple shoots (Košir et al., 2004). In the present study, synergistic effects of cytokinin and auxin combinations induced an effective multiple shoot induction rate (Table 1; Fig. 1F, G). However, a comparative assessment of functional efficacy between BA and mT puts forward the latter as a more superior PGR than the former. The induction rate varied with type and concentration of growth regulators. The combination, concentrations, and the ratio between the growth regulators are critically important for the formation of shoots and PLBs in orchids (Dohling et al., 2007; Bhattacharyya et al., 2016b). The efficacy of the mT over the conventional purine-based cytokinins such as BA and KN has been reported by various coworkers for various medicinal plant species including orchids (Werbrouck et al., 1995, 1996; Bogaert et al., 2004; Bairu et al., 2007; Aremu et al., 2012; Bose et al., 2016; Bhattacharyya et al., 2016b). Being less toxic in nature, mT accounts for a much higher multiple shoot proliferation rate, and simultaneously, it is becoming a much accepted PGR in comparison to conventional purinebased cytokinins like BA or Kinetin (Amoo et al., 2011). The primary reason behind the superiority of mT over BA and other conventional cytokinins is the fact that the localized accumulation of the topolins in plant tissues is prevented by its fast translocation rate (Kamínek et al., 1997). Coupled with this, the metabolic end products of mT are easily degradable and the hydroxyl group in the side chain of mT makes possible the formation of O-glucoside metabolites, a metabolite which is considered to be a cytokinin storage form, stable under certain conditions but rapidly getting converted to active cytokinin bases when required (Parker et al., 1978; Werbrouck et al., 1996; Bairu et al., 2009). The reversible sequestration of the O-glucosides, in turn, allows the continuous availability of cytokinins at a physiologically active level over a prolonged period of time resulting in a high shoot multiplication rate in in vitro cultures (Strnad, 1997). The present experimental finding also advocates the efficiency of mT over BA and is in close proximity with the findings of Bhattacharyya et al. (2016a) in Dendrobium nobile, a medicinal orchid species. As previously mentioned, the rate of shoot proliferation magnified significantly in synergy with 5 μM NAA, which acted as an auxin trigger. In the present study, the highest number of multiple shoots were induced when the proliferating shoot buds were cultured in MS media supplemented with 10 μM of mT and 5 μM NAA (Table. 1). Along with alleviated shoot multiplication rates, formation of secondary PLBs was also observed facilitating a higher clonal propagation rate. The present finding is also supported by the findings of Bairu et al. (2008) and Amoo et al. (2011), where a significant increment in callus yield as well as adventitious shoot proliferation was recorded using mT. The multiple shoots generated from both the pathways, i.e. mT-derived as well as BA-derived were sub-cultured after 1 month interval. After two

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cycles of sub-culturing, the proliferated shoots were transferred to rooting media for root induction. 3.2. Rooting and acclimatization The multiple shoots generated from both the pathways, i.e. mT- and BA-mediated were transferred to a medium supplemented with IBA, IAA, and PG at varying concentrations (Table 2). As the rooting percentage was less in full-strength MS medium (data not shown), the medium strength was reduced to half strength. Both IAA and IBA and phenolic elicitor PG were found to be conducive to root induction (Table 2). In this medium, 3–5 roots per shoot were obtained after 6 weeks of culture using root inducers separately (Table 2; Fig. 1H). Being an epiphyte, development of adequate branching is of utmost importance. Thus, in order to increase the rooting frequency, varying concentrations of IBA were used, along with PG to enhance the rate of root induction and proliferation. The efficacy of PG on in vitro rooting has been reported in various plant species including orchids (Bhattacharyya et al., 2015, 2016a; Kumar et al., 2016). The conducive effect of PG in the in vitro rooting of the orchids might be due to the auxin-phenol synergism resulting in the down-regulation of the peroxidase activity in the rooting medium, thereby protecting the endogenous auxin from peroxidase-catalyzed oxidation (De Klerk et al., 1999). Furthermore, being a precursor in the lignin biosynthesis pathway, PG also prevents hyperhydricity within the micropropagated plants by providing precursors which are generally at extremely low levels or not synthesized at all in hyperhydric tissues, and thus by increasing the activity of enzymes involved in lignin biosynthesis (Phan and Hegedus, 1986; Ross and Grasso, 2010). In the present study, the synergized effect of IBA and PG promoted a significantly higher rate of rooting with highest recorded rooting frequency of 76% with an average of 8 roots/shoot within 1 month (Table 2; Fig. 1I, J, K). The plants were maintained in the optimum rooting medium for another 2 months and the full-grown plants (Fig. 1L) were transferred to plastic cups in the greenhouse, resulting in 87% survival. There were no visual or morphological abnormalities observed within the micropropagated plants (Fig. 1M). 3.3. Assessment of clonal fidelity among the micropropagated plants of A. africana In spite of having various advantages, a major criticism of the tissueculture-raised plants is the development of somaclonal variations. Table 2 Effects of auxins and phenolic elicitor on root formation and proliferation of A. africana. Root inducers (Auxins/phenolic elicitor) IBA 0.0 5 10 15 20 – – – – – – – – 5 10 15 20

IAA 0.0 – – – – 5 10 15 20 – – – – – – – –

PG 0.0 – – – – – – – – 10 20 30 40 10 20 30 40

Frequency of shoots with roots

Shoot/root

Root length (cm)

– 53 61 72 65 32 45 41 36 42 47 56 43 53 62 76 69

– 2.1 3.2 5.3 5.1 2.1 3.1 2.1 1.3 2.1 3.5 3.2 3.5 4.2 6.1 8.1 7.1

– 1.2 1.4 1.5 1.3 1.1 1.1 1.2 1.3 2.1 2.4 3.2 2.0 4.2 4.6 5.7 5.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3c 0.2b 0.5a 0.4b 0.3e 0.5d 0.3d 0.2e 0.5d 0.3d 0.4c 0.3d 0.2c 0.4b 0.3a 0.4b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 g 0.3f 0.4d 0.3d 0.5 g 0.3f 0.4 g 0.3 h 0.5 g 0.3f 0.5f 0.3f 0.4e 0.2c 0.6a 0.5b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.5e 0.3e 0.4e 0.5e 0.3e 0.2e 0.3e 0.5e 0.4d 0.5d 0.6c 0.5d 0.4b 0.3b 0.5a 0.6a

Mean values within a column followed by the same letter are not significantly different by Duncan's multiple range test (P ≤ 0.05). The values represent the means (±SE) of three independent experiments.

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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 amplified

Avg bands/primer

Total no of PB

PP

Distance range (Jaccard's coefficient)

1st

mT pathway BAP pathway mT pathway BAP pathway Acclimatized

16

56 62 72 56 70

3.5 3.8 4.5 3.5 4.37

3 4 5 4 5

5.35 6.45 6.94 7.14 7.14

0.95–1.00 0.94–1.00 0.95–1.00 0.95–1.00 0.94–1.00

2nd 3rd

*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 cellular controls (Phillips et al., 1994; Kaeppler et al., 1998; Martins et al., 2004). The occurrence of somaclonal variations during in vitro propagation 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, detecting 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 reflected in their genome profiling patterns using different marker systems, which propel DNA-based marker systems for the assessment of clonal fidelity, a technique of choice. Furthermore, it is more affordable in comparison to other available protocols, e.g. flow 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

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 first regeneration, 56 bands were generated by the mT-derived plants, whereas 62 amplified 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 first regeneration derived from both the pathways into two broad clusters (Fig. 3A, B). Similar to the first 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 matrix was calculated, which ranged from 0.95 to 1.00 and 0.95 to 1.00, respectively (Table 3). Similar to the first 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 coefficient ranging from 0.94 to 1.00 (Table.3). As with the other two regenerations of micropropagated plants, the representatives were broadly organized into two broad clusters (Fig. 4).

Fig. 2. SCoT profiles 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 2–10: micropropagated plants of the first regeneration; Lanes 11–19: micropropagated plants of the second regeneration; Lanes 20–28: micropropagated plants of the third regeneration.

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Fig. 3. UPGMA dendogram generated for band data from SCoT marker illustrating coefficient similarities among regenerated plants and the mother plant [first 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 coefficient 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 findings are also justified 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 first generation to 1.28 in the third generation with an overall frequency of 1.39 (Table 4). Similarly, the number of alleles (Ne) that effectively contributed to the genetic diversity also showed steady increase from 1.09/ 1.15 (mT/BA) in the first generation up to 1.23 in the third generation with an overall rate of 1.15. The present parameters of population structure 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 findings revealed that these indices significantly increased from first to third generation and this level of variability was found to be proportionate to the generations 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 significantly higher gene flow was recorded between the generations (Nm = 1.596), which might have resulted in a marked change in allele frequencies, which further justifies the argument of steady increments

Table 4 Genetic diversity and differentiation parameters for three generations of micropropagated A. africana using SCoT marker. Generations 1st generation 2nd generation 3rd generation Overall

mT pathway BA pathway mT pathway BA pathway Acclimatized plants

Na ± SD

Ne ± SD

H ± SD

I ± SD

1.13 1.23 1.13 1.15 1.28 1.39

1.09 1.15 1.10 1.17 1.23 1.15

0.05 ± 0.14 0.12 ± 0.14 0.05 ± 0.19 0.1 ± 0.16 0.12 ± 0.20 0.10 ± 0.14

0.07 0.11 0.15 0.23 0.18 0.16

± ± ± ± ± ±

0.33 0.21 0.33 0.15 0.45 0.49

± ± ± ± ± ±

0.27 0.11 0.28 0.21 0.37 0.24

± ± ± ± ± ±

0.20 0.15 0.22 0.15 0.29 0.22

Pp

Gst

Nm

FST

5.40 6.53 6.73 7.23 7.23 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 flow 0.25(1–Gst)/Gst; Hpop = variability within population, FST = fixation index or F statistics.

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Table 5 Analysis of molecular variance (AMOVA) for three regenerations of micropropagated A. africana. Source of variation

d.f⁎

Sum of squares

Variance components

Percentage of variation

Among generations Within generations Total

2 25 27

15.867 47.311 63.179

0.64811 1.89244 2.54055

25.51 74.49

⁎ 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 genetic 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. 3A–D; Fig. 4). The above findings are well supported by the AMOVA results, which reveal that a greater degree of variation exists within the generations as compared to that of among the generations. The present outcome is closely supported by the findings 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 variability 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 significant factor contributing to the development of clonal variability. In the plant tissue culture experiment, PGRs play a pivotal role in plant propagation and is also considered to be one of the primary causes of somaclonal variations in micropropagated plants (Stover, 1986; Košir et al., 2004). The mode of regeneration, tissue culture environment, and culture conditions may account for the occurrence of clonal variability (Rani and Raina, 2000; Bairu et al., 2006). In order to achieve optimum 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; Košir 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 experiments. The experimental findings revealed, in both first 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 significantly 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 finding is closely corroborated by the findings 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 significant 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 variability. The utility of the present report can be further justified by the fact that the molecular information generated by the conventional

markers like RAPD or ISSR is usually obtained from non-coding DNA regions, 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 itself or from its flanking sequences (Collard and Mackill, 2009). Therefore, 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 fidelity 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 significant importance in providing 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 species 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 first report of production of genetically 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 research findings clearly justify the novelty of mT in the micropropagation pathway along with higher genetic stability in comparison to conventional 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 nullifies 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 fidelity detection system using gene-targeted molecular marker 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 financial support in the form of Postdoctoral Fellowships. References Amoo, S.O., Finnie, J.F., Van Staden, J., 2011. The role of meta-topolins in alleviating micropropagation problems. Plant Growth Regulation 63, 197–206. Amoo, S.O., Aremu, A.O., Moyo, M., Szüčová, L., Doležal, K., Van Staden, J., 2014. Physiological effects of a novel aromatic cytokinin analogue in micropropagated Aloe arborescens and Harpagophytum procumbens. Plant Cell, Tissue and Organ Culture 116, 17–26. Aremu, A.O., Bairu, M.W., Doležal, K., Finnie, J.F., Van Staden, J., 2012. Topolins: a panacea to plant tissue culture challenges? Plant Cell, Tissue and Organ Culture 108, 1–16. Bairu, M.W., Fennell, C.W., Van Staden, J., 2006. The effect of plant growth regulators on somaclonal variation in Cavendish banana (Musa spp. AAA). Scientia Horticulturae 108, 347–351. Bairu, M.W., Stirk, W.A., Doležal, K., Van Staden, J., 2007. Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell, Tissue and Organ Culture 90, 15–23. Bairu, M.W., Stirk, W.A., Doležal, K., Van Staden, J., 2008. The role of topolins in micropropagation and somaclonal variation of banana cultivars “Williams” and “Grand Naine” (Musa spp. AAA). Plant Cell, Tissue and Organ Culture 95, 373–379.

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