Acta Physiologiae Plantarum (2018) 40:137

https://doi.org/10.1007/s11738-018-2703-y

ORIGINAL ARTICLE

Transverse thin cell layer (t-TCL)-mediated improvised

micropropagation protocol for endangered medicinal orchid
Dendrobium aphyllum Roxb: an integrated phytomolecular approach
Paromik Bhattacharyya1,3 · Prasenjit Paul1 · Suman Kumaria1 · Pramod Tandon1,2

Received: 4 April 2017 / Revised: 3 June 2018 / Accepted: 4 June 2018

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2018

Abstract
Clonal propagation is an important biotechnological tool for the conservation of rare, endangered and threatened as well as
commercially important plant species of which orchids are an important representative. In the present research, an attempt
was made to develop a fast, clonally stable as well as phytochemically enriched regeneration protocol for propagating Den-
drobium aphyllum, an important medicinal orchid using transverse thin cell layer (t-TCL) explants. Murashige and Skoog
(MS) medium supplemented with 15 µM meta-topolin along with 10 µM TDZ and 10 µM ­AgNO3 was found to be most
conducive for shoot proliferation. After 8 weeks of culture, an average of 39.41 shoots/explant proliferated; till date this is the
best shooting frequency for D. aphyllum and one of the best amongst the orchids. The highest rooting frequency of 82.34%
was accomplished in half-strength MS medium supplemented with 15 µM IBA. The plantlets were successfully acclimatized
in greenhouse exhibiting normal developmental patterns and flowering occurred after 6 months of acclimatization with 3–4
flowers/stalk irrespective of the flowering season. The genetic homogeneity of the micropropagated plants was ascertained
using IRAP and ISSR markers. A comparative assessment of the antioxidant activity (mother and micropropagated plant)
was performed using DPPH and FRAP assays which was recorded to be higher amongst the in vitro-regenerated plants. A
rapid, genetically stable regeneration protocol coupled with comprehensively higher phytochemical yield is reported in the
present study on D. aphyllum. The results of this study confirm the use of in vitro propagation protocol for sustainable com-
mercial utilization as well as conservation of medicinal orchid bio-resources within a limited time period.

Keywords  Gene-targeted markers · Medicinal orchids · Traditional Chinese medicine (TCM) · Micropropagation · Plant
growth regulators

Introduction

Communicated by M. Capuana.
Orchids are one of the most diversified groups amongst the
flowering plant families and are valued highly for their long-
Electronic supplementary material  The online version of this lasting flowers exhibiting an extensive diversity in color,
article (https​://doi.org/10.1007/s1173​8-018-2703-y) contains shape, size and fragrance. Amongst the various orchid gen-
supplementary material, which is available to authorized users.
era, Dendrobium is one of the most important genera. It is
* Paromik Bhattacharyya one of the largest in the orchidaceous family comprising
[email protected] 900–2000 species and is the most popular cut flower orchid
1
(Paul et al. 2017; Bhattacharyya et al. 2015a; Baker and
Plant Biotechnology Laboratory, Department of Botany, Baker 1996). However, due to higher deforestation activity
Centre for Advanced Studies, North-Eastern Hill University,
Shillong, Meghalaya 793022, India and other anthropogenic pressures, various orchid genera
2 including Dendrobium aphyllum Roxb. are facing risk of
Biotech Park, Kursi Road, Lucknow, Uttar Pradesh 226021,
India habitat destruction (Nayak et al. 1997; Hossain et al. 2012).
3 Apart from having immense horticultural importance, dend-
Research Centre for Plant Growth and Development, School
of Life Sciences, University of KwaZulu-Natal, Scottsville, robes also have a significant use in various traditional phar-
Private Bag X01, Pietermaritzburg 3209, South Africa macopeia (Bulpitt 2005). Primarily, the chemical entities

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that are found in dendrobes are phenolic compounds, poly- conventional regeneration systems and plant transformation
saccharides, alkaloids having multiple biological activities studies and have proved to be a better suited propagule in
including immunomodulatory, antioxidant and anti-tumor terms of regeneration frequency including orchids (Bui Van
effects (Ng et al. 2012; Yang et al. 2015). Chinese pharma- et al. 1999; Rout et al. 2006; Rangsayatorn 2009; Bose et al.
copeia (2010 version) has two monographs of medicinally 2017b).
important dendrobes, of which, one is known as Dendrobii Furthermore, to achieve a fast, rapid and stable regenera-
caulis (Shihu in Chinese) constituting D. nobile, D. fimbria- tion protocol, the importance of choosing appropriate plant
tum, D. aphyllum and other related dendrobes and the other growth regulator (PGR) complements is essential. Tradition-
one known as Dendrobii officinalis caulis (Tiepi Shihu in ally, the orchids have been propagated using purine-based
Chinese) derived from D. officinale (Yang et al. 2015). Tra- cytokinins such as 6-benzyl amino purine (BAP) and kine-
ditionally, the paste of D. aphyllum is used in the treatment tin (Kn). Recently, topolins have emerged as an effective
of organ deformities (Hossain 2009; Hossain et al. 2012; alternative to conventional cytokinins. Amongst the vari-
Yang et al. 2015). This medicinal attribute of D. aphyllum ous forms of topolins, meta-topolin (mT) has proved to be
is primarily due to the presence of phytoconstituents such an extremely reliable PGR and has been applied in various
as bibenzyls, phenanthrenes and flavones (Ng et al. 2012; plant species including orchids (Vasudevan and Van Staden
Yang et al. 2015). Apart from its medicinal use, the flow- 2011; Aremu et al. 2012; Bhattacharyya et al. 2016). Cou-
ers are also very attractive, having demand in horticulture pled with that, in vitro shoot proliferation rate can be fur-
industry (Hossain et al. 2012). Effective usage of plant tissue ther magnified by synergistic use of silver nitrate (­ AgNO3)
culture strategies facilitates an up-regulation of the second- which acts as an ethylene suppressor and enhances the
ary metabolite-synthesizing activity of the propagated plants rate of shoot proliferation (Cruz de Carvalho et al. 2000;
including medicinal orchids (Bhattacharyya et al. 2014, Chithra et al. 2004; Sridevi and Giridhar 2014; Sgamma
2016). Only a few reports exist on the phytochemical profil- et al. 2015). However, the sustainability of in vitro propaga-
ing of the micropropagated medicinal orchids (Giri et al. tion techniques principally depends on the production of
2012; Bhattacharyya et al. 2014, 2015a, 2016; Bose et al. true-to-type plants so that the advantage in the use of elite
2017a, b). Being a highly medicinal orchid, having multi- genotypes over natural seedlings is maintained (Goel et al.
target drug effects, D. aphyllum is reported to have a high 2009). Several factors such as species type, donor genotypes,
degree of antioxidant activity (Yang et al. 2015). Recent explants source and media composition contribute to soma-
researches have revealed the role played by various antioxi- clonal variability (Larkin and Scowcroft 1981; Devi et al.
dants in reducing the oxidative damage caused by various 2014; Bhattacharyya et al. 2014) which might be heritable
disorders such as cancer, AIDS and cataracts. This neces- (Jain 2001). Thus, to ensure clonal fidelity, assessment of
sitates the development of strategies which would lead to genetic stability of the propagated plants is necessary. In
magnify the antioxidant activity within the micropropagated recent years, molecular markers such as randomly ampli-
clones through in vitro regeneration pathways. fied polymorphic DNA (RAPD), inter-simple sequence
Keeping in view the immense pharmaco-horticultural repeats (ISSR), simple sequence repeats (SSR) have been
importance of D. aphyllum, tissue culture is the best suited successfully applied to evaluate the genetic homogeneity of
approach to supply quality planting materials from both the regenerated plants. Targeting a particular region of the
mass propagation and conservation perspectives (Dohling genome, the conventional molecular marker such as RAPD
et al. 2012). Although a few reports exist on in vitro propa- has a certain limitation which has been greatly improvised
gation of D. aphyllum (Nayak et al. 1997; Talukdar 2001; by the usage of gene-targeted markers of which inter-ret-
Hossain et al. 2012), there is a need for a fast, clonally stable rotransposon-targeted amplified region (IRAP) deserves
and phytochemically superior regeneration protocol facilitat- special mention (Poczai et al. 2013; Bhattacharyya and Van
ing the concept of sustainable conservation (Benson 2002; Staden. 2018). IRAP has been proved to be an effective low-
Nalawade and Tsay 2004). Plant tissue culture has played the cost molecular marker for detecting genetic fidelity with
most important role in achieving the goal of sustainable con- high precision amongst micropropagated plants including
servation. Generally, the plants are either propagated through orchids (Bhattacharyya et al. 2016).
organogenesis or somatic embryogenesis, but in recent past, Although a few propagation protocols have been devel-
thin cell layers (TCL) or thin cross sections (TCSs) of plant oped for the propagation of D. aphyllum, there is no report
tissues such as shoots, stem nodes, protocorm-like bodies pertaining to TCL-based micropropagation of D. aphyllum
(PLBs) have been successfully used for clonal propagation along with phytomolecular assessment of the in vitro-raised
of dendrobes (Bui Van et al. 1999; Zhao et al. 2007). The plantlets. Therefore, the present study was undertaken taking
TCL comprise tissue segments of small size (0.1–5 mm) into consideration the following objectives: (1) to develop a
excised transversally or longitudinally from different plant high-frequency micropropagation protocol using transverse
tissues. It has been used as an effective alternative to the thin cell layer (t-TCL) culture of nodal segments of this

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medicinally important orchid species, (2) to assess genetic Culture medium and growth conditions
conformity amongst the regenerates and donor plants using
ISSR and IRAP molecular markers, and (3) to evaluate To assess the effect of mT on multiple-shoot prolifera-
the production of photosynthetic pigments and secondary tion, the t-TCL explants were cultured in MS medium
metabolites in wild and tissue culture-raised plantlets. (Murashige and Skoog 1962) supplemented with 3%
sucrose as carbon source and varying concentrations of mT
(High Media, Mumbai, India). Furthermore, to determine
Materials and methods the synchronized effect of mT with thidiazuron (TDZ) and
α-naphthaleneacetic acid (NAA), the t-TCL explants were
Plant material and explant preparation cultured in MS medium supplemented with optimum con-
centration of mT (15 µM) along with varying concentrations
Ten-year-old plant of D. aphyllum (Fig.  1a) maintained of TDZ or NAA (5–20 µM). The pH of the culture medium
in the greenhouse of the Plant Biotechnology Laboratory, was adjusted to 5.8 using 0.1N NaOH or HCl before auto-
Department of Botany, North-Eastern Hill University claving. The culture vessels containing the medium were
(NEHU), Shillong, India, was used as a donor plant from autoclaved at 121 °C for 20 min at 104 kPa pressure. The
which nodal segments were excised. The explants were thor- experiments were repeated three times and each treatment
oughly washed under flowing tap water for a 30 min time consisted of 10 replicates with each culture period lasting
period and were surface sterilized in accordance with the for 8 weeks’ time. The cultures were maintained under ambi-
protocol outlined by Dohling et al. (2012). Thereafter, the ent culture room conditions [temperature 25 ± 2 °C; relative
excised nodal segments were further transversally sliced into humidity 80%; light intensity 35–50 µmol/m2/s]. The light
thin sections of 0.1–0.4 mm size and used as explants for illuminance was provided by cool white fluorescent tubes
micropropgation. (Philips, Kolkata, India).

Fig. 1  In vitro propagation of D. aphyllum from t-TCL explants. a (bar = 1 cm). f Development and proliferation of regenerated multiple
Mature plant growing in green house. b Emergence of adventitious shoots in MS + 10 µM + TDZ + 15 µM mT after 2 weeks (bar = 1 cm).
buds at the peripheral region of the t-TCL after 2  weeks of culture g Multiplication of shoots in MS + 15  µM mT and 10  µM TDZ
(bar = 10  mm). c Closeup view of the initiation of leaf from t-TCL after 8  weeks of culture (bar  = 1  cm). h Rooting in half-strength
section (bar = 1  cm). d Proliferation of adventitious buds at the MS + 15 µM IBA after 8 weeks of culture. i Complete plantlets with
basal part of the explant (bar = 1 cm). e Regeneration of the adven- roots (bar = 1 cm)
titious buds into a tissue clump after 2  weeks in the same media

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Effect of ­AgNO3 on shoot proliferation manufacturer’s protocol. The purity of the isolated DNA was
checked and quantified using UV spectrophotometer. A total
To assess the effect of A­ gNO3 on the shoot proliferation of 35 ISSR and 10 IRAP primers were screened and out of
and multiplication of D. aphyllum, the proliferated shoots these 9 ISSR and 5 IRAP primers which produced the best
obtained as a result of treatments with various PGRs were amplification and reproducible patterns were selected for
cultured in MS medium supplemented with varying concen- the final amplification. ISSR-PCR was carried out in accord-
trations of ­AgNO3 (5–25 µM). Number of proliferated shoots ance with the methodology described by Bhattacharyya et al.
proliferated were recorded both at the time of culture as well (2015b) whereas IRAP-PCR was carried out following the
as after 4th and 8th weeks of culture initiation. Experimental protocol described by Kalendar and Schulman (2006) with
parameters such as number of replicates and culture condi- minor modifications. The amplified PCR products were
tions were similar as mentioned above. separated using 1.8% denaturating agarose gel electropho-
resis with 1X Tris–acetate–EDTA (TAE) tank buffer. The
Effect of auxins on rooting gel profiles were subsequently documented using Biostep
DH-20, Gel Documentation System, Germany.
Proliferating shoots of D. aphyllum with two or three leaves
were cultured in half-strength MS medium complemented Data analysis
with 5–20 µM indole butyric acid (IBA) or NAA and main-
tained under a 12-h photoperiod with an illuminance of The generated band profiles were scored into a binary matrix
50 µmol/m2/s. The data on total number of roots generated on the basis of their presence (1) and absence (0). Only clear
and length were recorded after 4-week time period. and unambiguous bands were taken into consideration whilst
the smeared bands were discarded. To assess the robust-
Hardening and acclimatization ness and comparative efficacy of the marker systems used to
assess genetic stability of the regenerants, various paramet-
The full-grown plants with proliferated roots were carefully ric measures such as resolving power (Rp) and polymorphic
removed from the culture bottles. The plants were washed information content (PIC) for the IRAP and ISSR markers
thoroughly to remove any traces of solidifying agar frag- were used (Smith et al. 1997; Prevost and Wilkinson 1999).
ments and were then transferred to thermocol pots contain- The binary molecular data obtained from IRAP and ISSR
ing potting mixture [vermiculite and sawdust in 1:1 ratio] markers were pooled and dendrogram was constructed using
along with top layer of moss for moisture retention. This NTSYS software package version 2.20K (Rohlf 1998). To
step is outlined as primary hardening. The primary hardened determine the robustness of the cladding pattern of the gen-
plants were kept at a temperature of 25 ± 2 °C under a 12-h erated dendrogram, the marker data were further subjected
photoperiod (50 µmol/m2/s) for 2 weeks’ time. The plants to 1000 replicate bootstrap analysis using Free-Tree pro-
were sprayed with 1/10th MS medium without sucrose and gramme (Pavlicek et al. 1999).
were then transferred to the growth chamber [(Conviron,
Canada); temperature 20 °C; light 50 µmol/m2/s] for second- Determination of photosynthetic pigments
ary hardening. Finally, after successful secondary hardening, within the regenerated plants
the plants were transferred to greenhouse.
The level of the photosynthetic pigments present within the
Statistical analysis mother as well as the micropropagated plants was deter-
mined and quantified. Chlorophyll a, b and carotenoids
The recorded experimental data were all subjected to anal- were estimated in accordance with the protocol described
ysis of variance (ANOVA) using SAS software package by Lichtenthaler (1987) and modified further by Aremu et al.
(SAS-JMP Software Package, USA). The means were com- (2012). The photosynthetic pigment contents were expressed
pared using Duncan’s multiple range test (Duncan 1955). All in µg/gm FW and calculated in accordance with the formulae
the experiments were repeated three times. outlined below:
( )
Chlorophyll a Ca = 11.24A662 − 2.04A645 .
Assessment of genetic fidelity using IRAP and ISSR
markers ( )
Chlorophyll b Cb = 20.13A645 − 4.19A662 .

Total genomic DNA was extracted from the young leaves Total chlorophyll = 7.05A662 + 18.09A645 .
of both mother as well as micropropagated plants D. aphyl-
lum (15 randomly collected plants) using GENEAXY
( )
Total carotenoids = 1000A470 − 1.90Ca − 63.14Cb ∕214.
DNA extraction kit (GENEAXY, USA) following the

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Extract preparation and secondary metabolite horticultural importance. The t-TCL explants were cul-
assessment tured in MS medium supplemented with various concen-
trations of mT which showed induction of adventitious
The plant parts (leaf and stem) were harvested from both buds within 2 weeks of culture (Table 1). Initially, the
mother as well the micropropagated plants and were cleaned adventitious buds appeared in the peripheral region of the
thoroughly under flowing tap water. These were blotted t-TCL (Fig. 1b, c), but later on emerged from the basal
using paper towels and dried at ambient room temperature part of the explant (Fig. 1d) and proliferated into a tissue
and were ground into fine powder using REMI mixture clump (Fig. 1e). Similar reports have been documented in
grinder (REMI, India). One hundred milligram of this pow- other related plant species including orchids. The num-
dered material was weighed and dissolved in 100 ml of dif- ber of proliferating shoots/explant was comprehensively
ferent solvents (methanol, chloroform and acetone). All the higher in comparison to the other reported t-TCL proto-
extractions were carried out using an orbital shaker (REMI, cols in orchids which substantiates unequivocally the role
India) adjusted at 170 rpm for a 24-h time period. Finally, played by mT in influencing the rate of tissue regenera-
the obtained extracts were centrifuged at 10,000 rpm for tion pathway-based micropropagation reports in orchids
10 min and the obtained supernatants were filtered through (Bui Van et  al. 1999; Nayak et  al. 2002; Zhang et  al.
Whatman filter paper no.1. The obtained plant extracts were 2005; Zhao et al. 2007). Amongst the various tested mT
assayed for three broad phytochemical parameters, i.e., total concentrations, the highest response with 11.22 shoots/
phenolic content (TPC), total flavonoid content (TFC) and explant was recorded in MS medium supplemented with
total alkaloid content (TAC) in accordance with the protocol 15 µM (Table 1). The present shoot initiation rate is sig-
described by Bhattacharyya et al. (2016). TPC, TFC and nificantly higher in comparison to that recorded previously
TAC were quantified on the basis of gallic acid equivalent for micropropagation of D. aphyllum (Nayak et al. 1997;
(GAE), quercetin equivalent (QE) and atropine equivalent Talukdar 2001; Dutta et al. 2011; Hossain et al. 2012).
(AE), respectively. The standard curves for the analysis were In recent times, mT has evolved as a potential alternative
prepared using absolute methanol (v/v) and the obtained to conventional cytokinins such as BAP and Kn (Werb-
results were expressed in milligram standard equivalent/g of rouck et al. 1996; Aremu et al. 2012). Due to lesser levels
dry weight. The absorbance was recorded using Lambda-35 of residual toxicity and higher multiple-shoot-inducing
double spectrophotometer (Perkin-Elmer, USA). property, it has become a PGR of choice for plant tissue
culture experiments. Similar findings were reported in D.
Estimation of antioxidant activity nobile and Ansellia africana, two related medicinal orchid
species (Vasudevan and Van Staden 2011; Bhattacharyya
The antioxidant activities of the mother and the micropro- et al. 2016). The shoot regeneration potentiality was fur-
pagated D. aphyllum plants were compared using1,1-diphe- ther magnified when the proliferated shoots were cultured
nyl-2-picrylhydrazyl (DPPH) assay according to the method further into medium supplemented with varying concentra-
described by Jagtap et al. (2011) and also by ferric reducing tions of TDZ and NAA along with 15 µM of mT (Table 2;
antioxidant power (FRAP) assay following the protocol out- Fig. 1F). Amongst all the treatments, MS medium contain-
lined by Benzie and Stain (1996). A correlation coefficient ing 10 µM of TDZ and 15 µM of mT resulted in the highest
between the TPC, TFC and TAC with the estimated anti-
oxidant activities determined by DPPH and FRAP assays
was calculated. The experimental results were expressed as Table 1  Effects of mT on shoot proliferation and multiplication from
± SE of three consecutive experiments. Furthermore, the t-TCL nodal explants of D. aphyllum 
generated data were subjected to ANOVA and the statisti- Concentrations % response for No. of shoots per Average
cal tests of significance between the obtained mean values of mT (µM) shoot proliferation t-TCL explant shoot length
(cm)
using Duncan’s multiple range test (Duncan 1955).
0.0 – – –
5.0 43.21 ± 0.25e 2.22 ± 0.43e 1.22 ± 0.32c
Results and discussion 10.0 47.32 ± 0.35d 4.21 ± 0.23d 1.55 ± 0.22c
15.0 79.43 ± 0.12a 11.22 ± 0.39a 2.53 ± 0.23a
Multiple‑shoot induction and proliferation 20.0 67.54 ± 0.22b 7.32 ± 0.44b 1.97 ± 0.43b
25.0 53.43 ± 0.32c 6.88 ± 0.26c 1.89 ± 0.42b
The efficacy of fast and direct shoot regeneration with-
Values of mean within a column followed by the same letter are not
out an intermediate callus phase using t-TCL explants
significantly different by Duncan’s multiple range test (P ≥ 0.05). Val-
helped to produce a large number of plantlets of D. ues corresponding to means (± SE) of three independent experiments.
aphyllum, widely revered for its immense medicinal and Ten replicates were used for each experiment

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Table 2  Interactive effect of optimized concentration (15 µM) of mT proliferation was observed when t-TCL explants were fur-
with TDZ or NAA on shoot multiplication from t-TCL nodal explants ther transferred to a medium supplemented with 15 μM mT
of D. aphyllum 
for a time period of 2 weeks (Table 2; Fig. 1f).
Concentration of % of shoot prolif- No. of shoots Average
PGRs (µM) eration per t-TCL shoot length
explant (cm)
TDZ NAA mT Effect of ­AgNO3 on multiple‑shoot proliferation
5 – 15 82.21 ± 0.28d 17.11 ± 0.11d 1.22 ± 0.22b
When the cultures were maintained in the same medium
10 – 15 93.42 ± 0.15a 23.22 ± 0.15a 2.49 ± 0.17a
for prolonged time, there was no significant change in
15 – 15 89.11 ± 0.12b 21.11 ± 0.43b 2.29 ± 0.25a
shoot regeneration frequency. To further accelerate the
20 – 15 86.34 ± 0.23c 20.23 ± 0.32b 1.92 ± 0.45b
frequency of regeneration along with an increased shoot
– 5 15 74.35 ± 0.43e 14.11 ± 0.12f 1.11 ± 0.33b
length, the effect of A
­ gNO3 on the multiple-shoot prolifer-
– 10 15 83.53 ± 0.21b 15.32 ± 0.18e 1.23 ± 0. 24b
ation of D. aphyllum was tested. Incorporation of A ­ gNO3
– 15 15 87.67 ± 0.65c 21.42 ± 0.28b 2.15 ± 0.15a
in the medium resulted in a significant increment in the
– 20 15 81.29 ± 0.72d 19.11 ± 0.32c 1.89 ± 0.27b
number of multiple shoots. Optimum response (35.21 and
Values of mean within a column followed by the same letter are not 39.41 shoots after 4 and 8 weeks of culture) was recorded
significantly different by Duncan’s multiple range test (P ≥ 0.05). Val- in MS medium appended with 15 µM mT and 10 µM TDZ
ues corresponding to means (± SE) of three independent experiments.
along with 10 µM A ­ gNO3 (Table 3; Fig. 1g). This shoot-
Ten replicates were used for each experiment
ing frequency observed in the present report is found to
be significantly higher than previously published reports
rate of shoot proliferation, i.e., 23.22 shoots/explant with on D. aphyllum (Nayak et al. 1997; Talukdar 2001; Dutta
an average shoot length of 2.49 cm. The superiority of the et al. 2011; Hossain et al. 2012). The reason behind get-
TDZ in orchid micropropagation is well documented. It ting significantly higher multiplication rate using ­AgNO3
has played an important role in the in vitro propagation of may be primarily because of the fact that ­AgNO3 acts as
various orchid species including dendrobes (Cheruvathur an ethylene inhibitor, a gas which is produced during plant
et al. 2010; Bhattacharyya et al. 2014). The present experi- regeneration and inhibits shoot proliferation during clonal
mental finding further justifies the potency of TDZ to be propagation (Seong et al. 2005). Addition of A ­ g 2+ ions
used as a PGR of choice in combination with topolins to in the culture medium acts as an antagonist to ethylene
enhance the pace of in vitro propagation. The conducive biosynthesis and significantly enhances shoot proliferation
effect of TDZ in tissue proliferation is reported to be pri- and multiplication (Kumar et al. 2009). The present study
marily because of its role in synthesis, accumulation and also documents the synergistic effect of mT and TDZ with
concentration specific balancing in the ratio of exogenous ­AgNO3 serving as cell proliferation trigger magnifying the
PGRs which allow specific modes of regeneration (Mok multiple-shoot number significantly. The conducive effect
and Mok 1985; Tao et al. 2011). A brief exposure to TDZ ­ g2+on the shoot bud multiplication and elongation
of the A
ranging from 6–20 days, depending on concentration has has been reported earlier by various other coworkers and
been reported to be beneficial for shoot proliferation (Liu our research finding is in agreement with their findings
et al. 2003; Tao et al. 2011) which is in congruence to (Hyde and Phillips 1996; Brar et al. 1999; Giridhar et al.
our present finding where a significant increment in shoot 2003; Sridevi and Giridhar 2014; Sgamma et al. 2015).

Table 3  Effects of different MS + 15 µM mT + 10 µM TDZ

concentrations of ­AgNO3 on
shoot multiplication from AgNO3 (µM) Shoots/explant at the time Shoots/explant at after Shoots/explant at
explants sub-cultured in of culture 4 weeks of culture after 8 weeks of
MS + 15 µM mT + 10 µM TDZ culture

0.0 23.22 ± 0.15d 27.39 ± 0.43d 30.26 ± 0.35d

5.0 28.32 ± 0.23c 32.22 ± 0.11c 36.23 ± 0.46b
10.0 35.23 ± 0.45a 35.21 ± 0.25a 39.41 ± 0.53a
15.0 31.65 ± 0.21b 33.15 ± 0.17b 36.22 ± 0.32b
20.0 28.23 ± 0.32c 31.33 ± 0.43c 33.28 ± 0.22c
25.0 19.22 ± 0.22e 24.23 ± 0.48e 31.49 ± 043d

Values of mean within a column followed by the same letter are not significantly different by Duncan’s
multiple range test (P ≥ 0.05). Values corresponding to means (± SE) of three independent experiments.
Ten replicates were used for each experiment

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Acta Physiologiae Plantarum (2018) 40:137 Page 7 of 14  137

Rooting and acclimatization of plantlets to moderate temperatures, it becomes necessary to create

such conditions artificially, referred to as primary hardening
The proliferated full-grown shoots were transferred to the (Lavanya et al. 2009; Bhattacharyya et al. 2016). A signifi-
rooting medium supplemented with either IBA or NAA cant etiolation in the micropropagated shoots of D. aphyllum
ranging in concentrations between 5 and 20 µM. However, was observed during the primary hardening process which
the percentage of rooting in the full-strength MS medium might be because of the fact that transfer of the plantlets
was found to be not desirable (data not shown); therefore, from the in vitro conditions to direct sunlight leads to pho-
half-strength MS medium was used. Maximum rooting fre- toinhibition and chlorophyll bleaching (Van Huylenbroeck.
quency of 82.34% with 15.22 roots/shoot was recorded in 1995). Coupled with that, abcissic acid (ABA) metabolism
half-strength MS medium containing 15 µM IBA (Table 4; also plays a very critical role in the plant growth and devel-
Fig. 1g). Comparatively, IBA is found to be a better auxin opment mainly through optimizing plant water balance and
in inducing roots over NAA from the recorded parameters regulating the plant response mechanism to various adap-
such as number of roots/shoot and root length taken into tive stress factors including temperature (Hetherington 2001;
consideration (Table 4; Fig. 1h, e). The efficacy of IBA over Finkelstein and Gibson 2002). In the present study, along
other auxins in promoting rooting in orchids is well docu- with the other factors, the use of vermiculite with saw dust
mented and is in congruence with the findings of other cow- in the ratio of 1:1 highly enhanced the hardening efficiency
orkers (Nayak et al. 1997; Cheruvathur et al. 2010). Being of the micropropagated plantlets. Furthermore, vermiculite
an epiphyte, development of well-branched root system is played a significant role in the successful acclimatization
of utmost importance. In the present protocol, the best root- of the in vitro-raised plantlets. The probable reason behind
ing frequency recorded was 82.34% with an average root such an efficacy might be due to the higher moisture reten-
length of 5.12 cm and root number of 15.22 roots/shoot tion capacity of the vermiculite and constant absorption of
which is the best rooting frequency for D. aphyllum reported oxygen which can be better availed by the hardened plant-
till date (Table 4). The fully grown plants with developed lets including dendrobes during the acclimatization process
root systems were transferred to a potting mixture contain- (Paul et al. 2017; Runkles et al. 1958; Zhao et al. 2007).
ing vermiculite and saw dust mixture in 1:1 ratio and were Addition of top layer of moss helped to increase humidity
maintained in growth chamber under ambient conditions for because of its high water retention capacity. The present
primary hardening. The micropropagated plants on being finding is in close congruence with the findings of Bhat-
transferred to ex vitro conditions are subjected to various tacharyya et al. (2015a) in D. thyrsiflorum. After successful
abiotic and biotic stresses including temperature variations, primary hardening, the plants were transferred to greenhouse
which greatly affect the viability of the micropropagated conditions resulting in about 95% survival rate (Fig. 2a).
plants (Deb and Imchen 2010). Being epiphytic in nature,
acclimatization in dendrobes needs special attention. In Flowering of the micropropagated plants
nature, as these plants grow in shady environment under low
The greenhouse-hardened plants of D. aphyllum developed
keikis within 4 months of secondary hardening and started
Table 4  Effects of different concentrations of IBA and NAA on root- flowering after 6 months of secondary hardening (Fig. 2b,
ing of D. aphyllum shoots after 8 weeks of culture c). Initiation and induction of flowers in plants are primarily
Auxin (µM) Frequency of Roots/shoot Root length (cm)
controlled by various factors such as photoperiod, tempera-
shoot with roots ture, irradiance, and water availability (Bernier et al. 1993;
IBA NAA Bernier and Périlleux 2005). Coupled with the above physi-
0.0 0.0 – – – ological conditions, cytokinins also played an important role
5.0 – 72.32 ± 0.23c 4.23 ± 0.12f 2.23 ± 0.16d in induction and maturation of floral buds and might have
10.0 – 79.23 ± 0.45b 7.39 ± 0.28e 3.45 ± 0.19c influenced flowering. The endogenous hormonal level pro-
15.0 – 82.34 ± 0.32a 15.22 ± 0.32a 5.12 ± 0.25a vides an floral stimulus which is then transported through
20.0 – 78.45 ± 0.41b 13.23 ± 0.17b 4.43 ± 0.32b the vascular tissue to the apical meristem and subsequently
– 5.0 69.56 ± 0.34d 3.23 ± 0.29 g 2.12 ± 0.43d initiates flower initiation (Bernier and Périlleux 2005). In
– 10.0 71.84 ± 0.23c 8.67 ± 0.35d 2.43 ± 0.33d flowering plant species including orchids, a positive syn-
– 15.0 63.29 ± 0.43e 9.43 ± 0.52c 2.22 ± 0.29d chrony has been reported with the cytokinin concentration
– 20.0 61.49 ± 0.37f 8.23 ± 0.43d 2.14 ± 0.52d and apical meristem during floral transition phase (Blan-
chard and Runkle 2008). Along with cytokinins, A ­ gNO3
Values of mean within a column followed by the same letter are not also plays a significant role in controlling flowering. Being
significantly different by Duncan’s multiple range test (P ≥ 0.05). Val-
ues corresponding to means (± SE) of three independent experiments. an antagonist of ethylene biosynthesis, Beyer (1976) had
Ten replicates were used for each experiment reported that higher ethylene concentrations which act as

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 137   Page 8 of 14 Acta Physiologiae Plantarum (2018) 40:137

Fig. 2  a Greenhouse-acclimatized plantlets grown with a potting secondary hardening. c Flowering of the in vitro-raised plants within
mixture constituting of vermiculite and saw dust mixture in 1:1 ratio 6 months of hardening under greenhouse condition
with a top layer of moss. b Development of keikis after 4 months of

obstacle for floral initiation could be modulated with an opti- the final amplification, 5 most reproducible IRAP primers
mized use of A ­ gNO3. In our present experimental findings, produced 26 amplifiable bands whereas 9 screened ISSR
we have found that micropropagation of the D. aphyllum primers generated 50 bands. Both IRAP and ISSR primers
plants using a synchronized system comprising mT, TDZ produced two polymorphic bands; however, the number of
along with ethylene suppressor A ­ gNO3 not only enhances primers used differed in case of two markers (Table 5, S1;
the rate of micropropagation but also helps in the induc- Fig. 3a, b). The obtained marker data were pooled together
tion of an early flowering. Although, Hossain et al. (2012) and genetic variability was determined revealing a total of
achieved flowering within the in vitro-propagated plants of 5.26% clonal variability amongst the regenerants whereas
D. aphyllum, but the number of the flowers were restricted individually IRAP and ISSR markers detected 7.69 and 4%
to 1–2. In this protocol, 3–4 flowers on an average were variability amongst the regenerants (Table 5). The Jaccard’s
obtained within 6 months of hardening (Fig. 2c). Further- similarity index ranged from 0.93 to 1.00 (Table 5; Fig. 3c,
more, it justifies the use of mT in the induction of flowering d). Comparative study between IRAP and ISSR primer
in horticulturally important orchid species like D. aphyllum efficiency revealed that IRAP detected higher variability
and is the first report for the induction of in vitro flowering and proved to be a better marker system in the detection of
within epiphytic orchids to the best of our knowledge. clonal variability in comparison to ISSR. Being a conven-
tional molecular marker, ISSR targets a specific section of
Genetic fidelity of the micropropagated plantlets the genome which might not exhibit variability. This limita-
tion has been largely resolved by the usage of retrotrans-
The occurrence of genetic variability within the micropro- poson-targeted molecular markers, i.e., IRAP (Kilinç et al.
pagated plants is a major obstacle towards the sustainable 2014). Furthermore, Larkin and Scowcroft (1981) identified
commercial utilization of the medicinal plants (Negi and the transposable element movement within the genome as
Saxena 2010). Various factors including explant type and an important factor contributing to somaclonal variations
PGR stress can contribute to the occurrence of cryptic varia- within micropropagated plants. Being a retrotransposon-
tions (Goto et al. 1998). While validating the genetic fidelity targeted marker, IRAP proved to be a marker of choice for
of the micropropagated plants with that of the mother plant, detecting somaclonal variability. As IRAP could amplify
two marker systems, i.e., IRAP and ISSR have been used. In genomic distances between two long terminal repeats

Table 5  Comparison of IRAP and ISSR markers, individually as well as collectively

SL Method No. of primer Total bands Avg.bands/ Total no. of poly- % of polymor- Distance range (Jac- PIC
used amplified primer morphic bands phism card’s coefficient)

1 IRAP 5 26 5.20 2 7.69 0.94–1.00 0.74

2 ISSR 9 50 5.55 2 4.00 0.93–1.00 0.82
3 IRAP + ISSR 14 76 5.42 4 5.26 0.93–1.00 0.81

PIC polymorphic information content

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Acta Physiologiae Plantarum (2018) 40:137 Page 9 of 14  137

Fig. 3  Banding profiles of the acclimatized D. aphyllum plants using drogram illustrating genetic similarity amongst the mother plant and
a IRAP-LTR-2 primers, b ISSR M [L = 100 bp ladder; lane 1 mother regenerated plants of D. aphyllum; d cluster analysis of the regener-
plant (M); lanes 2–16 micropropagated plants of D. aphyllum]; c den- ated plants after 1000-repetition bootstrap analysis

(LTR), it proved to be more efficient over conventional ISSR Secondary metabolite and photosynthetic pigment
marker in the precise detection of clonal variability within profiling
the micropropagated plants of D. aphyllum. A compara-
tive study of Rp and PIC values of IRAP and ISSR markers One of the primary reasons of acclimatization failure within
(Tables 5, S1) proved IRAP to be more effective in detection the micropropagated plants is in vitro stress induced during
of clonal variability as supported by the findings of Smith plant tissue culture pathways along with a poorly developed
et al. (1997), Prevost and Wilkinson (1999) and Kalendar photosynthetic apparatus (Pospisilova et al. 2007). Being
and Schulman (2006). This further advocates the use of an epiphyte in nature, a robust photosynthetic module for
multi-marker approach to assess genetic stability within orchids is of utmost importance. Thus, to validate the effec-
micropropagated plants as a single-marker system cannot tiveness of the present micropropagation protocol in the
detect the entire range of somaclonal variation (Mallón et al. development of photosynthetic capacity of the micropro-
2010). The present findings reveal, that within the micro- pagated plants, we had quantified the photosynthetic pig-
propagated plantlets of D. aphyllum, negligible degree of ment contents within the mother and the micropropagated
genetic variability exists counter-justifying the suitability of plants of D. aphyllum. The study revealed higher levels of
t-TCL as explant source along with the stability of the propa- chlorophyll a, b, total chlorophyll as well as carotenoid lev-
gated plants assessed after prolonged micropropagation and els within the in vitro-raised plants of D. aphyllum in com-
acclimatization passage stress. Similar results were reported parison to the mother plant. The leaf extract of the micro-
by Singh et al. (2012) in Eclipta alba, and Chavan et al. propagated plants showed a comprehensive higher yield of
(2014) in Ceropegia santapaui, two related medicinal plant carotenoids, chlorophyll a, b and total chlorophyll whereas
species. The above protocol also puts forward the benefi- the stem extracts of the mother plant resulted in the lowest
cial role of mT in arresting clonal variability and promoting pigment yields (Table 6). The probable reason behind such
higher growth rate which is in congruence with the findings finding may be justified by the broad-spectrum activity of
of other coworkers asserting improvised histogenic stabil- cytokinins by promoting the formation of photosynthetic
ity as well as anti-senescent effects (Werbrouck et al. 1995; pigments as well as the chloroplast proteins (Aremu et al.
Bogaert et al. 2004; Bhattacharyya et al. 2016). 2012). Coupled with the conducive effect of the cytokinins,

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­AgNO3 also played a triggering role in upregulating the

Total chlorophyll/
phyll (µg/g FW) carotenoid ratio
photosynthetic pigment synthesis (Giridhar et al. 2003).
The synchronized effect of cytokinin–AgNO3 significantly

5.84 ± 0.5
4.84 ± 0.3
resulted in an exponential increase in the photosynthetic
pigments within the micropropagated plantlets and thereby
resulted in an increased rate of survivability. This finding is

429.21 ± 0.1b
534.45 ± 0.2a
Total chloro-

Values represent ± SE of three replicates. Values of mean within a column followed by the same letter are not significantly different by Duncan’s multiple range test (P ≥ 0.05)
in close analogy with that of Amoo et al. (2014).
Along with the positive compliance found in the yield
of photosynthetic pigments, a comparative assessment of
various secondary metabolite contents between the mother
Chlorophyll a/b

and micropropagated plants of D. aphyllum recorded a sig-

0.99 ± 0.1
0.95 ± 0.2
nificant alteration in the TPC, TFC and TAC levels. Metha-
ratio

nolic extracts of the micropropagated plants exhibited higher

levels of TPC (49.47 mg GAE/g DW), TFC (8.41 mg QE/g
Chlorophyll b

415.19 ± 0.2b
529.32 ± 0.3a

DW) and TAC (57.17 mg/AE/g DW) whereas the lowest

(µg/g FW)

values were obtained in the chloroform fractions of the

mother plant (Table 7). The highest values of TPC and TFC
were recorded within the methanolic stem and leaf extracts
Chlorophyll a

412.14 ± 0.5b
505.42 ± 0.3a

respectively of the micropropropagated D. aphyllum plants

(µg/g FW)

whereas TAC was noted to be highest within the metha-

Micropropagated plant

nolic leaf extracts of the in vitro raised plants. Various lit-

erature studies suggested that D. aphyllum is an important
biological reserve for precious therapeutic molecules having
110.21 ± 0.5a
73.43 ± 0.2b
Total carot-
enoid (µg/g

a broad-spectrum of usage in traditional Chinese pharma-

copeia (Hossain 2011; Yang et al. 2015). The secondary
FW)

metabolites are important biomolecules having an impor-

tant role in regulating vital cell functions and regulation of
Total chloro-
phyll (µg/g FW) phyll/carot-
enoid ratio

7.00 ± 0.5
5.55 ± 0.3

important physiological functions. A wide number of such

biomolecules also represent potent therapeutic attributes in
combatting cardiovascular disorders and cancer (Palacio
et al. 2008; Moyo et al. 2010; Yang et al. 2015). Amongst
365.23 ± 0.2b
469.42 ± 0.4a
Total chloro-

all secondary metabolites, the presence of alkaloids is the

most pronounced within orchids (Bose et al. 2017a, b; Hos-
Table 6  Photosynthetic pigment content in different parts of D. aphyllum 

sain 2011). Coupled with that, the phytochemical profiling

Chlorophyll a/b

of the micropropagated plants obtained by the present cyto-

kinin–AgNO3-mediated regeneration system documents
1.02 ± 0.4
1.01 ± 0.3

a higher yield of secondary metabolites in comparison to

ratio

other conventional approaches. The probable reason behind

such result might be the positive synchrony between the
Chlorophyll b

319.14 ± 0.3b
413.12 ± 0.4a

mT and A ­ gNO3 resulting in an enhanced level of secondary

(µg/g FW)

metabollite. Cytokinins such as mT and TDZ are known for

their positive effects in enhancing the yield of secondary
metabolites within micropropagated orchids which was fur-
Chlorophyll a

326.41 ± 0.6b
419.53 ± 0.2a

ther pronounced by the addition of A ­ gNO3. Furthermore, the

(µg/g FW)

present experimental findings are also closely supported by

our previous findings in D. nobile, a related orchid species
used in traditional Chinese medicine (TCM) (Bhattacharyya
Mother plant

52.15 ± 0.3b

et al. 2016).
84.49 ± 0.4a
Total carot-
enoid (µg/g

FW fresh weight
FW)

Antioxidant activity
Plant part

The primary reason of a plant to be categorized as medici-

Stem
Leaf

nal is being estimated by its antioxidant potentials. The

13
Acta Physiologiae Plantarum (2018) 40:137 Page 11 of 14  137

Table 7  Profiling of total phenolic, flavonoids and alkaloids content in different parts of D. aphyllum with respect to different solvent systems
Plant parts Solvent Mother plant Micropropagated plant
TPC TFC TAC​ TPC TFC TAC​

Stem Methanol 33.52 ± 0.17a 2.13 ± 0.21b 32.53 ± 0.12b 49.47 ± 0.18a 5.24 ± 0.16b 44.11 ± 0.23b

Acetone 4.32 ± 0.11d 0.74 ± 0.16d 14.86 ± 0.15d 9.62 ± 0.23c 0.91 ± 0.18e 18.79 ± 0.18f
Chloroform 2.97 ± 0.19e 0.25 ± 0.18d 12.15 ± 0.16d 3.54 ± 0.25f 0.76 ± 0.24 19.16 ± 0.19e
Leaves Methanol 18.79 ± 0.11b 7.13 ± 0.22a 41.67 ± 0.25a 33.54 ± 0.26b 8.41 ± 0.22a 57.17 ± 0.19a
Acetone 8.14 ± 0.16c 2.13 ± 0.21b 29.13 ± 0.21c 8.43 ± 0.24d 2.86 ± 0.19d 29.54 ± 0.17d
Chloroform 4.12 ± 0.21d 1.11 ± 0.27c 18.23 ± 0.18d 5.27 ± 0.23e 4.29 ± 0.21c 30.56 ± 0.11c

Values represent ± SE of three replicates. Values of mean within a column followed by the same letter are not significantly different by Duncan’s
multiple range test (P ≥ 0.05). Phenolics: mg GAE/g DW, flavonoids: mg QE/g DW and alkaloids: mg AE/g DW
GAE gallic acid equivalent, QE quercetin equivalent, ATP atropine equivalent, DW dry weight, TPC total phenolic content, TAC​ total alkaloid
content

antioxidant potential of a medicinal herb develops from within the chloroform extracts (Fig. 4a, b). The findings
the presence of various secondary metabolites of which reveal a significant increase in antioxidant activity within
polyphenols form an important class of biomolecules the micropropagated plants in comparison to the mother
(Brewer 2011). Having exhibited powerful antioxidant plant which is in congruence with the published reports
activity in different in vitro cellular models, they have in medicinal plant species including orchids (Chavan
shown potentiality in downregulation of various reactive et al. 2014; Bhattacharyya et al. 2015a, 2016). The pre-
oxygen species such as hypochlorous acid, superoxide sent report is a preliminary baseline finding which depicts
anion, peroxylnitrile, peroxyl and hydroxyl radical (Hal- the positive analogy of mT–TDZ–AgNO3 complement in
liwell 2008). Besides that, recorded antioxidant poten- increasing the antioxidant potential within the micropro-
tial serves as an important benchmark character for the pagated plant which justifies the findings of Amoo et al.
assessment of medicinal activity of the in vitro-propagated (2012). Both DPPH and FRAP methods detected higher
plants (Amoo et al. 2012). In the present study, the com- degree of antioxidant activity within the leaf extracts of
parative assessment of antioxidant activity of various the micropropagated D. aphyllum plants which might be
plant parts of D. aphyllum (mother and micropropagated) due to the presence of various photosynthetic pigments.
was estimated using DPPH and FRAP antioxidant assays Furthermore, when the various secondary metabolite
using plant extracts. The highest antioxidant activity was parameters as well as the recorded antioxidant activities
exhibited by the methanolic leaf extract of the micropro- were correlated, TPC and TAC showed positive correlation
pagated plants whereas the lowest activity was recorded with antioxidant activity whereas TFC did not influence
the antioxidant activity significantly (Table S2).

Fig. 4  Antioxidant activity of D. aphyllum within mother and micropropagated plants using methanol, acetone and chloroform as solvents. a
DPPH and b FRAP

13
 137   Page 12 of 14 Acta Physiologiae Plantarum (2018) 40:137

Conclusions Bernier G, Havelange A, Houssa C, Petitjean A, Lejeune P (1993)

Physiological signals that induce flowering. Plant Cell 5:1147
Beyer EM (1976) A potent inhibitor of ethylene action in plants. Plant
The present research focuses on the development of a t- Physiol 58:268–271
TCL micropropagation protocol for sustainable exploita- Bhattacharyya P, van Staden J (2018) Molecular insights into genetic
tion of D. aphyllum, an important medicinal orchid taxon. diversity and population dynamics of five medicinal Eulophia spe-
cies: a threatened orchid taxa of Africa. Physiol Mol Biol Plants.
A significant shoot proliferation rate has been achieved https​://doi.org/10.1007/s1229​8-018-0523-6
using synergistic effects of mT, TDZ and A ­ gNO3, which Bhattacharyya P, Kumaria S, Diengdoh R, Tandon P (2014) Genetic
is the highest till date in D. aphyllum. The report also stability and phytochemical analysis of the in vitro regenerated
comprehensively documents the effect of mT and ­AgNO3 plants of Dendrobium nobile Lindl., an endangered medicinal
orchid. Meta Gene 2:489–504
on dendrobium micropropagation using D. aphyllum as a Bhattacharyya P, Kumaria S, Job N, Tandon P (2015a) Phyto-molecular
model plant. A comparative asessment of phytomolecu- profiling and assessment of antioxidant activity within microprop-
lar parameters revealed eliteness of the micropropagated agated plants of Dendrobium thyrsiflorum: a threatened, medicinal
clones in comparison to the donor plants. In precise, the orchid. Plant Cell Tissue Organ Cult 122:535–550
Bhattacharyya P, Kumaria S, Tandon P (2015b) Applicability of ISSR
research ascertains high genetic stability and phytochemi- and DAMD markers for phyto-molecular characterization and
cal enrichment of the clones which makes this protocol association with some important biochemical traits of Dendro-
ideally suitable for mass propagation. The approach is fast bium nobile, an endangered medicinal orchid. Phytochemistry
and simple and can be adopted for the production of large 117:306–316
Bhattacharyya P, Kumaria S, Tandon P (2016) High frequency regen-
volumes of orchid materials for commercial exploitation eration protocol for Dendrobium nobile: a model tissue culture
and conservation, thereby upholding the principle of sus- approach for propagation of medicinally important orchid species.
tainable commercial exploitation. S Afr J Bot 104:232–243
Blanchard MG, Runkle ES (2008) Benzyladenine promotes flowering
in Doritaenopsis and Phalaenopsis orchids. J Plant Growth Regul
Author contribution statement  PB, PP, PT and SK designed 27:141–150
the experiments. PB and PP executed the experiments, Bogaert I, Van Cauter S, Werbrouck SPO, Dolezal K (2004) New
recorded and analyzed the data. PB and SK wrote and edited aromatic cytokinins can make the difference. In: V International
the MS. Symposium on in vitro culture and horticultural breeding 725.
pp 265–270
Bose B, Choudhury H, Tandon P, Kumaria S (2017a) Studies on
secondary metabolite profiling, anti-inflammatory potential,
Acknowledgements  These authors sincerely thank the funding agen- in vitro photoprotective and skin-aging related enzyme inhibi-
cies, Department of Biotechnology (DBT), Government of India and tory activities of Malaxis acuminata, a threatened orchid of nutra-
Center for Advanced Studies (CAS) grant from University Grants Com- ceutical importance. J Photochem Photobiol B Biol. https​://doi.
mission (UGC), New Delhi. org/10.1016/j.jphot​obiol​.2017.07.010
Bose B, Kumaria S, Choudhury H, Tandon P (2017b) Insights into
nuclear DNA content, hydrogen peroxide and antioxidative
enzyme activities during transverse thin cell layer organogen-
esis and ex vitro acclimatization of Malaxis wallichii, a threat-
References ened medicinal orchid. Physiol Mol Biol Plants. https​://doi.
org/10.1007/s1229​8-017-0474-3
Amoo SO, Staden J, Van (2012) Influence of plant growth regulators Brar MS, Moore MJ, Al-khayri JM, Morelock TE, Anderson EJ (1999)
on shoot proliferation and secondary metabolite production in Ethylene inhibitors promote in  vitro regeneration of cowpea
micropropagated Huernia hystrix. Plant Cell Tissue Organ Cult (Vigna unguiculata L.). In Vitro Cell Dev Biol Plant 35:222–225
112:249–256 Brewer MS (2011) Natural antioxidants: sources, compounds, mecha-
Amoo SO, Aremu AO, Moyo M, Szu¨cˇova´ L, Dolezˇal K, Van Staden nisms of action, and potential applications. Compr Rev Food Sci
J (2014) Physiological effects of a novel aromatic cytokinin ana- Food Saf 10:221–247
logue in micropropagated Aloe arborescens and Harpagophytum Bui Van L, Phuong NTH, Hong LTA, Tran Thanh Van K (1999) High
procumbens. Plant Cell Tissue Organ Cult 116:17–26 frequency shoot regeneration from Rhynchostylis gigantea (orchi-
Aremu AO, Bairu MW, Szüčová L, Doležal K, Finnie JF, Van Staden daceae) using thin cell layers. Plant Growth Regul 28:179–185
J (2012) Assessment of the role of meta-topolins on in vitro pro- Bulpitt CJ (2005) The uses and misuses of orchids in medicine. QJM
duced phenolics and acclimatization competence of micropropa- 98:625–631
gated “Williams” banana. Acta Physiol Plant 34:2265–2273 Chavan JJ, Gaikwad NB, Umdale SD, Kshirsagar PR, Bhat KV, Yadav
Baker M, Baker M (1996) Dendrobium species culture. Timber Press, SR (2014) Efficiency of direct and indirect shoot organogenesis,
Portland. https​://www.cabdi​rect.org/cabdi​rect/abstr​act/19970​ molecular profiling, secondary metabolite production and anti-
30672​3 oxidant activity of micropropagated Ceropegia santapaui. Plant
Benson E (2002) Plant conservation biotechnology. CRC Press, Boca Growth Regul 72:1–15
Raton Cheruvathur MK, Abraham J, Mani B, Thomas TD (2010) Adventi-
Benzie IFF, Strain JJ (1996) The ferric reducing ability of plasma tious shoot induction from cultured internodal explants of Malaxis
(FRAP) as a measure of “antioxidant power”: the FRAP assay. acuminata D. Don, a valuable terrestrial medicinal orchid. Plant
Anal Biochem 239:70–76 Cell Tissue Organ Cult 101:163–170
Bernier G, Périlleux C (2005) A physiological overview of the genetics Chithra M, Martin KP, Sunandakumari C, Madhusoodanan PV (2004)
of flowering time control. Plant Biotechnol J 3:3–16 Silver nitrate induced rooting and flowering in  vitro on rare

13
Acta Physiologiae Plantarum (2018) 40:137 Page 13 of 14  137

rheophytic woody medicinal plant, Rotula aquatica Lour. Indian Kilinç FM, Süzerer V, Çiftçi Y, Onay A, Yıldırım H, Uncuog˘lu AA,
J Biotechnol 3:418–421 Tilkat E, Metin OK, Ibrahim K, Akdemir OF (2015) Clonal micro-
Cruz de Carvalho MH, Van Le B, Zuily-Fodil Y, Thi AP, Thanh Van propagation of Pistacia lentiscus L. and assessment of genetic
KT (2000) Efficient whole plant regeneration of common bean stability using IRAP markers. Plant Growth Regul 75:75–88
(Phaseolus vulgaris L.) using thin-cell-layer culture and silver Kumar V, Parvatam G, Ravishankar GA (2009) AgNO3: a potential
nitrate. Plant Sci 159:223–232 regulator of ethylene activity and plant growth modulator. Elec-
Deb CR, Imchen T, 2010. An Efficient In vitro hardening technique of tron J Biotechnol 12:1–15
tissue culture raised plants. Biotechnology 9, 79–83 Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source
Devi SP, Kumaria S, Rao SR, Tandon P (2014) Single primer ampli- of variability from cell cultures for plant improvement. Theor
fication reaction (SPAR) methods reveal subsequent increase in Appl Genet 60:197–214
genetic variations in micropropagated plants of Nepenthes kha- Lavanya M, Venkateshwarlu B, Devi BP (2009) Acclimatization of
siana Hook. f. maintained for three consecutive regenerations. neem microshoots adaptable to semi-sterile conditions. Indian J
Gene 538:23–29 Biotechnol 8:218–222
Dohling S, Kumaria S, Tandon P (2012) Multiple shoot induction Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of
from axillary bud cultures of the medicinal orchid, Dendrobium photosynthetic biomembranes. Methods Enzym 148:350–382
longicornu. AoB Plants 2012:1–7. https​://doi.org/10.1093/aobpl​ Liu CZ, Murch SJ, El-Demerdash M, Saxena PK (2003) Regeneration
a/pls03​2 of the Egyptian medicinal plant Artemisia judaica L. Plant Cell
Duncan DB (1955) Multiple range and multiple F tests. Biometrics Rep 21:525–530
11:1–42 Mallón R, Rodríguez-Oubiña J, González ML (2010) In vitro propaga-
Dutta S, Chowdhury A, Bhattacharjee B, Natha PK, Dutta BK (2011) tion of the endangered plant Centaurea ultreiae: assessment of
In vitro multiplication and protocorm development of Dendro- genetic stability by cytological studies, flow cytometry and RAPD
bium aphyllum (Roxb.) CEC Fisher. Assam Univ J Sci Technol analysis. Plant Cell Tissue Organ Cult 101:31–39
7:57–62 Mok MC, Mok DWS (1985) The metabolism of [­ 14C]-tMdiaziiroii
Finkelstein RR, Gibson SI (2002) ABA and sugar interactions regu- in callus tissues of Phaseolus lunatus. Physiol Plant 65:427–432
lating development: cross-talk or voices in a crowd? Curr Opin Moyo M, Ndhlala AR, Finnie JF, Van Staden J (2010) Phenolic com-
Plant Biol 5:26–32 position, antioxidant and acetylcholinesterase inhibitory activities
Giri L, Jugran A, Rawat S, Dhyani P, Andola H, Bhat ID, Rawal RS, of Sclerocarya birrea and Harpephyllum caffrum (Anacardiaceae)
Dhar U (2012) In vitro propagation, genetic and phytochemical extracts. Food Chem 123:69–76
assessment of Habenaria edgeworthii: an important Astavarga Murashige T, Skoog F (1962) A revised medium for rapid growth and
plant. Acta Physiol Plant 34:869–875 bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Giridhar P, Indu EP, Vijaya Ramu D, Ravishankar GA (2003) Effect Nalawade SM, Tsay H-S (2004) In vitro propagation of some impor-
of silver nitrate on in vitro shoot growth of Coffee. Trop Sci tant Chinese medicinal plants and their sustainable usage. In Vitro
43:144–146 CellDev Biol—Plant 40:143–154
Goel MK, Kukreja AK, Bisht NS (2009) In vitro manipulations in St. Nayak NR, Rath SP, Patnaik S (1997) In vitro propagation of three
John’s wort (Hypericum perforatum L.) for incessant and scale epiphytic orchids, Cymbidium aloifolium (L.) Sw., Dendrobium
up micropropagation using adventitious roots in liquid medium aphyllum (Roxb.) Fisch. and Dendrobium moschatum (Buch-
and assessment of clonal fidelity using RAPD analysis. Plant Cell Ham) Sw. through thidiazuron-induced high frequency shoot
Tissue Organ Cult 96:1–9 proliferation. Sci Hortic (Amsterdam) 71:243–250
Goto S, Thakur RC, Ishii K (1998) Determination of genetic stability in Nayak NR, Sahoo S, Patnaik S, Rath SP (2002) Establishment of thin
long-term micropropagated shoots of Pinus thunbergii Parl. using cross section (TCS) culture method for rapid micropropagation
RAPD markers. Plant Cell Rep 18:193–197 of Cymbidium aloifolium (L.) Sw. and Dendrobium nobile Lindl
Halliwell B (2008) Are polyphenols antioxidants or pro-oxidants? (Orchidaceae) Sci Hortic (Amsterdam) 94:107–116
What do we learn from cell culture and in vivo studies? Arch Negi D, Saxena S (2010) Ascertaining clonal fidelity of tissue cul-
Biochem Biophys 476:107–112 ture raised plants of Bambusa balcooa Roxb. using inter simple
Hetherington AM (2001) Guard cell signaling. Cell 107:711–714 sequence repeat markers. New For 40:1–8
Hossain MM (2009) Traditional therapeutic uses of some indig- Ng TB, Liu J, Wong JH, Ye X, Sze SCW, Tong W, Zhang KY (2012)
enous orchids of Bangladesh. Med Arom Plant Sci Biotechnol Review of research on Dendrobium, a prized folk medicine. Appl
3:100–106 Microbiol Biotechnol 93:1795–1803
Hossain MM (2011) Therapeutic orchids: traditional uses and recent Palacio L, Baeza MC, Cantero JJ, Cusidó R, Goleniowski ME (2008)
advances—an overview. Fitoterapia 82:102–140 In vitro propagation of “jarilla”(Larrea divaricata CAV.) and sec-
Hossain MM, Sharma M, Pathak P (2012) In vitro propagation of Den- ondary metabolite production. Biol Pharm Bull 31:2321
drobium aphyllum (Orchidaceae)—seed germination to flowering. Paul P, Joshi M, Gurjar D et al (2017) In vitro organogenesis and
J Plant Biochem Biotechnol 22:157–167 estimation of β-sitosterol in Dendrobium fimbriatum Hook.: an
Hyde CL, Phillips GC (1996) Silver nitrate promotes shoot develop- orchid of biopharmaceutical importance. South African J Bot
ment and plant regeneration of chile pepper (Capsicum annuum 113:248–252
L.) via organogenesis. In Vitro CellDev Biol Plant 32:72–80 Pavlicek A, Hrda S, Flegr J (1999) Free-Tree–freeware program for
Jagtap UB, Waghmare SR, Lokhande VH, Suprasanna P, Bapat VA construction of phylogenetic trees on the basis of distance data
(2011) Preparation and evaluation of antioxidant capacity of and bootstrap/jackknife analysis of the tree robustness. Applica-
Jackfruit (Artocarpus heterophyllus Lam.) wine and its protec- tion in the RAPD analysis of genus Frenkelia. Folia Biol (Praha)
tive role against radiation induced DNA damage. Ind Crops Prod 97–99
34:1595–1601 Poczai P, Varga I, Laos M, Cseh A, Bell N, Valkonen JPT, Hyvönen J
Jain SM (2001) Tissue culture-derived variation in crop improvement. (2013) Advances in plant gene-targeted and functional markers:
Euphytica 118:153–166 a review. Plant Methods. https​://doi.org/10.1186/1746-4811-9-6
Kalendar R, Schulman AH (2006) IRAP and REMAP for retrotranspo- Pospisilova J, Synková H, Haisel D, Semoradova S (2007) Acclima-
son-based genotyping and fingerprinting. Nat Protoc 1:2478–2484 tion of plantlets to ex vitro conditions: effects of air humidity,

13
 137   Page 14 of 14 Acta Physiologiae Plantarum (2018) 40:137

irradiance, ­CO2 concentration and abscisic acid (a Review). Acta Talukdar A (2001) Multiple shoot induction in Dendrobium aphyllum
Hortic 748:29 Roxb. J Orchid Soc India 15:35–38
Prevost A, Wilkinson MJ (1999) A new system of comparing PCR Tao J, Yu L, Kong F, Zhao D (2011) Effect of plant growth regulators
primers applied to ISSR fingerprinting of potato cultivars. Theor on in vitro propagation of Cymbidium faberi Rolfe. Afr J Biotech-
Appl Genet 98:107–112 nol 10:15639–15646
Rangsayatorn N (2009) Micropropagation of Dendrobium draconis Van Huylenbroeck JM, Huygens H, Debergh PC (1995) Photoinhibition
Rchb. f. from thin cross-section culture. Sci Hortic (Amsterdam) during acclimatization of micropropagated Spathiphyllum “Petite”
122:662–665 plantlets. In Vitro CellDev Biol Plant 31:160–164
Rohlf FJ (1998) NTSYS-pc version 2.0 Vasudevan R, Van Staden J (2011) Cytokinin and explant types influ-
Rout GR, Mohapatra A, Jain SM (2006) Tissue culture of ornamental ence in vitro plant regeneration of Leopard Orchid (Ansellia afri-
pot plant: A critical review on present scenario and future pros- cana Lindl.). Plant Cell Tissue Organ Cult 107:123–129
pects. Biotechnol Adv 24:531–560 Werbrouck SPO, van der Jeugt B, Dewitte W, Prinsen E, Van Onck-
Runkles JR, Scott AD, Nakayama FS (1958) Oxygen sorption by moist elen HA, Debergh PC (1995) The metabolism of benzyladenine
soils and vermiculite. Soil Sci Soc Am J 22:15–18 in Spathiphyllum floribundum “Schott Petite”in relation to accli-
Seong ES, Song KJ, Jegal S, Yu CY, Chung IM (2005) Silver nitrate matisation problems. Plant Cell Rep 14:662–665
and aminoethoxyvinylglycine affect Agrobacterium-mediated Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC (1996)
apple transformation. Plant Growth Regul 45:75–82 Meta-topolin, an alternative to benzyladenine in tissue culture?
Sgamma T, Thomas B, Muleo R (2015) Ethylene inhibitor silver nitrate Physiol Plant 98:291–297
enhances regeneration and genetic transformation of Prunus Yang D, Liu L-Y, Cheng Z-Q, Xu FQ, Fan WW, Zi CT, Dong FW,
avium (L.) cv Stella. Plant Cell Tissue Organ Cult 120:79–88 Zhou J, Ding ZT, Hu JM (2015) Five new phenolic compounds
Singh SK, Rai MK, Sahoo L (2012) An improved and efficient micro- from Dendrobium aphyllum. Fitoterapia 100:11–18
propagation of Eclipta alba through transverse thin cell layer cul- Zhang Y, But PP, Wang Z-T, Shaw P (2005) Current approaches for the
ture and assessment of clonal fidelity using RAPD analysis. Ind authentication of medicinal Dendrobium species and its products.
Crops Prod 37:328–333 Plant Genet Resour Charact Util 3:144–148
Smith JSC, Chin ECL, Shu H, Smith OS, Wall SJ, Senior ML, Mitchell Zhao P, Wang W, Feng F-S, Wu F, Yang ZQ, Wang WJ (2007) High-
SE, Kresovich S, Ziegle J (1997) An evaluation of the utility of frequency shoot regeneration through transverse thin cell layer
SSR loci as molecular markers in maize (Zea mays L.): com- culture in Dendrobium candidum Wall Ex Lindl. Plant Cell Tissue
parisons with data from RFLPs and pedigree. Theor Appl Genet Organ Cult 90:113–131
95:163–173
Sridevi V, Giridhar P (2014) In vitro shoot growth, direct organogen-
esis and somatic embryogenesis promoted by silver nitrate in Cof-
fea dewevrei. J Plant Biochem Biotechnol 23:112–118

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