Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-015-0897-x

ORIGINAL ARTICLE

Assessment of genetic homogeneity and analysis of phytomedicinal

potential in micropropagated plants of Nardostachys jatamansi,
a critically endangered, medicinal plant of alpine Himalayas
Biswajit Bose1 • Suman Kumaria1 • Hiranjit Choudhury1 •

Pramod Tandon1

Received: 29 June 2015 / Accepted: 20 October 2015

Ó Springer Science+Business Media Dordrecht 2015

Abstract Nardostachys jatamansi (D. Don) DC., a small, while callus mediated organogenesis (CMO)-derived plants
perennial, rhizomatous herb of immense medicinal showed slight variations as compared to mother plant. The
importance since ancient times, is restricted to specialized genome size of N. jatamansi was found to be
habitats of alpine Himalayas ranging from 3000 to 5200 m 2C = 1.40 ± 0.01 pg and therefore 684.6 Mbp (1C).
asl. The species has been recently listed as critically Although organogenic calli showed mixoploidy but no
endangered under IUCN Red list of threatened species due major phenotypic and genetic rearrangements were detec-
to over exploitation of its rhizomes for medicinal uses, ted by flow cytometry in callus-derived plants. Signifi-
habitat degradation, trade and other biotic and anthro- cantly higher antioxidant activity was observed in callus-
pogenic interferences. An efficient protocol using both derived plants as compared to mother and DSO-derived
indirect and direct shoot organogenesis has been optimized plants. Plant parts, regeneration pathways and various
for N. jatamansi. Best callusing was achieved from the cut solvent systems greatly affected the yields of total pheno-
ends of leaf and petiole explants within 15 days of culture lics, flavonoids, alkaloids, tannins contents present in the
in MS medium supplemented with 1.5 mg/l a-naphthalene in vitro raised plantlets.
acetic acid and 1.0 mg/l meta-Topolin. Culturing the
explants at low temperature (13 ± 1 °C) resulted in better Keywords Nardostachys jatamansi  Meta-Topolin 
callus growth, shoot regeneration, hyperhydricity control TDZ  Direct organogenesis  Callus  Flow cytometry 
and improvement in photosynthetic pigment content in SCoT  ISJ  Antioxidant  Total phenolics  Total
regenerated shoots. Also, direct organogenesis from shoot flavonoids
tip and petiole explants was achieved in MS medium
containing 1.0 mg/l meta-Topolin. Optimum rooting was Abbreviations
achieved in the same medium supplemented with 1.0 mg/l SCoT Start Codon Targeted polymorphism
indole acetic acid wherein averages of 4.52 roots/shoot ISJ Intron Splice Junction
were induced. Genetic stability of in vitro-derived plantlets FCM Flow cytometry
was assessed and compared to mother plant using molec- LB01 Lysis buffer 01
ular markers and flow cytometry. Intron Splice Junction MS medium Murasshige and Skoog medium (1962)
(ISJ) and Start Codon Targeted polymorphism (SCoT) NAA a-naphthaleneacetic acid
marker based profiling revealed uniform banding profile in 2-4D 2,4-dichlorophenoxyacetic acid
case of direct shoot organogenesis (DSO)-derived plants mT Meta-Topolin
KN Kinetin
2-iP N6–[2-isopentenyl] adenine
& Suman Kumaria BAP 6-benzylaminopurine
sumankhatrikumaria@hotmail.com TDZ Thidiazuron
1 IAA Indole-3 acetic acid
Plant Biotechnology Laboratory, Department of Botany,
North-Eastern Hill University, Shillong 793022, Meghalaya, IBA Indole-3-butyric acid
India PGRs Plant growth regulators

123

DSO Direct shoot organogenesis
CMO Callus mediated organogenesis
PCR Polymerase chain reaction
Mbp Mega base pairs
Introduction
Nardostachys jatamansi (D.Don) DC. (Valerianaceae
family) commonly known as ‘Jatamansi’ and ‘Indian
spikenard’ is a small, erect, perennial, hairy, rhizomatous
herb. Rhizomes of this plant are stout, thick, aromatic, dark
grey in colour and crowned with the reddish brown fibrous
remains of dead leaves and petioles. The herb is grown in
the alpine Himalayas spanning Himachal Pradesh, Uttar-
akhand, Sikkim, Arunachal Pradesh in India, Nepal, and
Bhutan at altitudes of 3000–5200 m asl and it is more
frequent in steep rocky soils rich in organic carbon and
nitrogen (Chauhan and Nautiyal 2005). Phytochemical
investigations have revealed that both rhizomes and roots
of N. jatamansi contained a large amount of essential oils
rich in sesquiterpenes and coumarins, which are used for
the medicinal purposes (Singh 2011; Rekha et al. 2013).
Other classes of compounds as steroids, alkaloids, lignans
and neolignans have been also reported (Shabhag et al.
1965; Hirose et al. 1978; Bagchi et al. 1991). The principal
sesquiterpene is jatamansone or valeranone (Govindachari
et al. 1961), although other bioactive compounds are found
such as jatamansin, jatamansic acid, jatamansinol, jata-
mansinone, nardosinone, nardal, nardol, nardin, spirojata-
mol, oroselol, seselin, jatamol A and B, patchuli alcohol,
dihydropyranocoumarin and actinidine have also been
reported (Singh 2011). Since Vedic period (1000–800
B.C), N. jatamansi has a very long history of use in ethno
medicine, ayurveda and in the alternative medicine indus-
try (Sharma et al. 2000; Chen and Mukerji 2013). It has
also been mentioned in Holy Bible (John 12:5) and in
poems of ‘‘Song of Solomon’’ (1:12 and 4:13) (Duke 2007;
Musselman 2012). This plant is appreciated for its stimu-
lant, sedative, hepatoprotective, cardioprotective, neuro-
protective, antidiabetic, antioxidant, antifungal,
antibacterial and anticancer properties (Song et al. 2010;
Singh 2011; Khan et al. 2012; Pandey et al. 2013;
Chaudhary et al. 2015; Purnima and Kothiyal 2015). It is
used as an herbal medicine singly or as an ingredient of
poly-herbal formulations by many companies in domestic
as well as in international market. Recently, N. jatamansi
has been listed in the IUCN Red list of threatened species
as Critically Endangered (CR) due to over exploitation for
medicinal uses, habitat degradation, climate change, trade,
and other biotic and anthropogenic interferences (Ved et al.
2015). This species is also considered as Critically
123
Plant Cell Tiss Organ Cult
Endangered (CR) in the Red Data Book of Indian Plants
(Nayar and Sastry 1990) and also listed in the Convention
on International Trade in Endangered Species of Wild
Fauna and Flora (CITES) Appendix II since 1997. A Con-
servation Assessment and Management Plan (CAMP)—
WWF India has identified this specie as a Critically
Endangered in North West Himalayas, Sikkim and Aru-
nachal Pradesh (Molur and Walker 1998). In addition, N.
jatamansi as one of the major (top 7 Asian) CITES enlisted
plants, which requires a proper harvesting and conservation
(Mulliken and Crofton 2008).
The maintenance of genetic integrity of in vitro-raised
plants with respect to the mother plant is the most crucial
consideration for large scale clonal propagation of geneti-
cally elite lines and genetic transformations (Bairu et al.
2011). The primary factors influencing induction of soma-
clonal variations in vitro include culture methods and envi-
ronment, explants source, ploidy level, culture age and
concentrations of plant regulators supplemented in the
medium (Rani and Raina 2000; Venkatachalam et al. 2007;
Singh et al. 2013). Apart from phenotypical observations,
several strategies are available for detecting somaclonal
variations including chromosome numbers, isozyme profiles
and PCR-based molecular markers. The use of molecular
markers has evolved as rapid, sensitive, cost effective and
reliable method for assessing clonal fidelity as these markers
are not influenced by environmental factors. Start Codon
Targeted polymorphism (SCoT) is a novel, gene targeted
marker system based on short conserved regions surrounding
ATG-translation sites of plant genes (Collard and Mackill
2009) and has proved as a reliable marker in detecting
genetic variations in in vitro-derived plants (Bhattacharyya
et al. 2014; Rathore et al. 2014; Agarwal et al. 2015). On the
other hand, the semi-random Intron Splice Junction (ISJ)
markers, originally developed by Weining and Langridge
(1991), are based on highly conserved core of exon–intron
boundary and indispensable for post-transcriptional DNA
processing in plants. The main advantage of ISJ markers is
that introns are present in most plant genomes and one can
therefore, target extremely diverse range of plant genomes
by avoiding heterochromatic regions to get comprehensive
account of polymorphisms. The utility of these two maker
systems, which amplify two different regions of the genome,
can lead to better efficiency of molecular markers in
detecting genetic polymorphism in in vitro-derived plants. In
recent years, flow cytometry (FCM) has become the method
of choice over molecular markers for detecting existing
somaclonal variations at genome level (Alan et al. 2007;
Obae and West 2010; Faisal et al. 2014). Ease of sample
preparations and rapid analyses of a large number of samples
within a short time makes it better suited for estimation of
genome size, ploidy level and nuclear replication state
(Doležel et al. 2007).
Plant Cell Tiss Organ Cult
N. jatamansi propagates by means of underground rhi-
zomes or by the dispersal of winged fruits over long dis-
tance by winds. It has a long juvenile phase of 3–4 years
followed by a short reproductive phase, and seed germi-
nation in nature is very low (10–20 %) (Nautiyal et al.
2003). Despite having a large number of medicinal prop-
erties, religious importance and critically endangered sta-
tus, N. jatamansi has not received much attention with
respect to conservation and sustainable utilization. Proto-
cols for regeneration of N. jatamansi via callus-derived
roots and somatic embryogenesis have been reported
(Mathur 1992, 1993); however, the protocols remain inef-
ficient in eliciting multiple shoot proliferation directly from
the callus inspite of several manipulations. These previ-
ously published protocols also lack in hardening and
acclimatization of tissue culture regenerated plants and
clonal fidelity analysis, which is an important component
of any plant tissue culture protocol.
In view of the medicinal and conservation importance of
N. jatamansi, the present study was designated (1) to
optimize multiple shoot induction via callus mediated and
direct shoot organogenesis pathways, (2) to determine the
genetic stability of in vitro-regenerated plants by flow
cytometry and molecular profiling (ISJ and SCoT), and (3)
to evaluate the photosynthetic pigments and different bio-
chemical parameters (total phenolics, flavonoids, alkaloids,
tannins content) and antioxidant activities in both the wild
and tissue-cultured raised plants over different regeneration
pathways.
Materials and methods
Plant material and explant preparation
Plants of N. jatamansi were collected from Nathang Valley
(3789 m asl, Sikkim) and Tawang (4085 m asl, Arunachal
Pradesh) in the month of June during their vegetative
growth phase. Plant materials were authenticated by
Botanical Survey of India, Kolkata and accessions (NJSK1
and NJAP1) were deposited at Central National Herbarium
(CNH, Kolkata). The plants were maintained in the glass
house of Plant Biotechnology Laboratory, Department of
Botany, North-Eastern Hill University, Shillong. Petiole,
young leaf and shoot tip explants were selected from
healthy mother plants (25–35 cm) and were thoroughly
washed under running tap water for 40–45 min. These
were then surface sterilized using initial treatment with
0.5 % (w/v) cetrimide (Himedia, India) for 5 min followed
by through washing with distilled water. The cleaned
explants were finally treated with 10 % (v/v) sodium
hypochlorite for 6 min and finally rinsed 3–4 times with
sterile distilled water.
Optimization of callus mediated organogenesis
(CMO)
Callus induction
The leaf (1–2 cm2) and petiole (1–2 cm) were cultured in
MS medium (Murashige and Skoog 1962) supplemented
with different auxins (NAA, 2-4D, Picloram) either alone
or in combination with various cytokinins (BAP, 2-iP and
mT) in a range of concentrations (0.5–2.0 mg/l). The
medium was fortified with 3 % (w/v) sucrose, 0.8 % (w/v)
agar, and pH was adjusted to 5.8 using 1 N NaOH or HCl
before autoclaving at 121 °C and 103 kPa for 15 min. Data
on the frequency of callus induction were recorded after
4 weeks of culture. Callus growth was calculated by
measuring fresh weight of callus raised in each treatment.
Calli were sub-cultured every 3 weeks in the same medium
for further proliferation.
Effect of temperature on callus induction, growth and shoot
regeneration
In order to assess the effect of temperature on callus induc-
tion, growth and shoot regeneration, two sets of cultures per
treatment were made. One set of cultures was maintained in
growth room at room temperature (22 ± 1 °C) under 14 h
light and 10 h dark cycles with 50 lmol m-2 s-1 light
intensity using cool, white fluorescent tubes (Philips, India)
and, another set was incubated in a growth chamber (Con-
viron-Adaptis, USA) at low temperature (5–15 °C) under
14 h photoperiod with 50 lmol m-2 s-1 light intensity
using cool, white fluorescent tubes (Philips, India) so as to
mimic the natural conditions. Data were recorded after
4 weeks of incubation.
Shoot regeneration from callus
Around 200 mg of calli developed in optimum callus
induction medium were transferred to regeneration MS
medium supplemented with different plant growth regula-
tors (BAP, KN, TDZ, 2-iP, mT and NAA) alone or in
combinations (0.5–2.0 mg/l). For all the experiments,
plantlets cultured for 4 weeks in all treatments were tested
for hyperhydricity according to Tabart et al. (2015):
% hyperhydricity ¼ ðnumber of hyperhydric leaves=
total number of leavesÞ  100
Optimization of direct shoot organogenesis (DSO)
Direct shoot organogenesis was optimized using shoot tips
(1–2 cm) and petioles (1–2 cm) as sources of explants. The
123

shoots were regenerated in MS medium supplemented with
3 % (w/v) sucrose, 0.8 % (w/v) agar, and different plant
growth regulators (BAP, KN, TDZ, 2-iP and mT) at con-
centrations ranging from 0.5 to 2.0 mg/l. The pH was
adjusted to 5.8 using 1 N NaOH or HCl before autoclaving
at 121 °C and 103 kPa for 15 min. Plantlets raised via
DSO-derived pathways were also checked for hyperhy-
dricity issues.
Rooting and acclimatization
Regenerated shoots measuring about 2 cm in height (both
CMO and DSO-derived) in optimum regeneration medium
were excised and transferred to MS medium supplemented
with IAA or IBA (0.5–2.0 mg/l). The frequency of rooting,
average number of roots per shoot and root length were
recorded after 4 weeks of culture. The regenerated plantlets
with well developed roots were removed and washed with
water to remove traces of agar. The young plantlets were
transplanted into thermocol pots containing soil, sand and
leaf litter mixture (2:1:1) with a cover of a layer of moss so
as to retain humidity. Plantlets were acclimatized for
3 weeks in a growth chamber (Conviron-Adaptis, USA) for
14 h photoperiod (light 50 lmol m-2 s-1; humidity
70–80 %; temperature 8–14 °C). The young plantlets were
watered regularly with half-strength MS liquid medium
without sucrose and then transferred to glasshouse (light
70 lmol m-2 s-1; humidity 70 %; temperature 13 °C).
The survival rate (%) of the hardened plantlets was esti-
mated after 60 days of acclimatization.
Genetic stability analysis of regenerated plantlets
Genomic DNA extraction and PCR amplification
Genomic DNA was isolated from young leaves of mother
and twenty in vitro propagated (10 DSO ? 10 CMO-
derived) plants of N. jatamansi following the method
described by Porebski et al. (1997). The purity of the iso-
lated DNA was checked using a nanodrop (Nanodrop
2000-C, Thermo Scientific, USA). Regenerated plantlets
were tested for clonal fidelity using primers 10 each of
SCoT and ISJ markers (Metabion, Gamburg, Germany) for
their unambiguous and reproducible banding patterns. All
the PCR reactions contained 30 ng of template DNA, 1X
PCR buffer, 0.2 mM dNTPs, 1 U Taq polymerase (Ban-
galore Genei, India) and 15 pmol primer (SCOT and ISJ).
The reaction programs for SCoT and ISJ were set at 94 °C
for 4 min followed by 40 cycles of 94 °C for 1 min,
48–57 °C for 1 min and 2 min at 72 °C with a final
extension 72 °C for 5 min in a Veritti Thermal Cycler
(Applied Biosystems, USA). All the amplified PCR
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Plant Cell Tiss Organ Cult
products obtained from SCOT and ISJ markers were
resolved by electrophoresis on 1.8 % agarose gel at
80–85 V for 2.5 h in a 1X TAE buffer, stained with
ethidium bromide. DNA fragments were visualized under
UV light and photographed using the Gel Documentation
System (Biostep DH-20, Germany).
Flow cytometry analysis
Intact nuclei suspension was prepared following the pro-
tocol developed by Doležel et al. (1989) with some minor
modifications from youngest fully developed leaves of
donor as well as in vitro-derived plants (both CMO and
DSO), friable and compact nodular calli. Seeds of refer-
ence standard [Pisum sativum Citrad (2C = 9.09 pg)] was
kindly provided by Jaroslav Dolezel (Institute of Experi-
mental Botany, Academy of Science, Czech-Republic).
The selected plant tissue was co-chopped with reference
standard with a sharp razor blade in a plastic petridish with
2 ml of lysis buffer (LB01) containing 15 mM Tris (Hi-
media, India), 2 mM Na2EDTA (Himedia, India), 0.5 mM
spermine tetrahydrochloride (Sigma-Aldrich, India), 8 mM
KCl (Himedia, India), 20 mM NaCl (Himedia, India),
0.1 % (v/v) Triton-X100 (Himedia, India). After chopping,
the nuclei suspension was filtered through a 40 lm nylon
mesh filter (BD Falcon, USA). Then, the nuclei were
stained with 50 lg/ml propidium iodide (PI) (Sigma-
Aldrich, India) and 50 lg/ml of RNase A (Himedia, India)
to prevent staining of double stranded RNA. Antioxidant
like 1 % PVP-40 (Himedia, India) was added to buffer to
minimise negative effect of cytosolic compounds to PI
fluorescence. Estimation of nuclear DNA content was
performed within 15 min period with BD FACS Callibur
flow cytometer (Becton–Dickinson, NJ, USA) equipped
with a 15 mW 488 nm air-cooled Argon ion laser and
results were acquired using BD Cell Quest Pro software
(version 6.0, BD Bioscience, USA). For each sample at
least 10,000 nuclei were analyzed. The analysis was per-
formed with five replicates on three different days. Fre-
quency versus Fl histogram was generated to compare the
mean position of sample relative to the internal standard.
Evaluation of secondary metabolites
Plant material and extract preparation
Fresh plant parts (leaf and rootstock) of donor plant as well
as in vitro-derived plants (both CMO and DSO) were
washed under running tap water and blotted with tissue
towel. The tissue was shed dried at room temperature.
Briefly, 100 mg of dried tissue was homogenized in 100 ml
of respective solvent (methanol, acetone, chloroform,
acetonitrile and water) and extractions were carried on an
Plant Cell Tiss Organ Cult
orbital shaker (REMI, India) with constant stirring at
180 rpm for 24 h. The mixtures were centrifuged at
10,000 rpm for 15 min and the supernatant was filtered
through Whatman No. 1 filter paper. Measurements for
biochemical parameters were taken in Lamda-35 double
beam spectrophotometer (Perkin-Elmer, USA).
Determination of total polyphenols, total flavonoids, total
alkaloids and total tannins
The extracts were analyzed for estimation of total phenolic
content (TPC), total flavonoid content (TFC), total alkaloid
content (TAC) and total tannin content (TTC) according to
the methods described by Singleton and Rossi (1965),
Chang et al. (2002), Sreevidya and Mehrotra (2003) and
Singleton et al. (1999) respectively. The estimation of TPC,
TFC, TAC and TTC were expressed as mg standard gallic
acid equivalent (GAE), quercetin equivalent (QE), atropine
equivalent (AE) and tannic acid equivalent (TAE) per gm
of dry tissue respectively.
Determination of antioxidant activity
DPPH free radical scavenging assay
The DPPH (2, 2-diphenyl-1-picryl hydrazyl) free radical
scavenging activity of the extracts (mother and in vitro-
derived) was determined as described by Brand-Williams
et al. (1995). Briefly, 150 ll DPPH solution (1 mM in
methanol) was mixed with 100 ll of plant extract in
varying concentrations (5–250 lg/ml) in a final reaction
volume of 3 ml. The mixture was shaken vigorously and
incubated in the dark at room temperature for 30 min. A
control was prepared as above without any extract and
absorbance was measured spectrophotometrically at
517 nm. The radical scavenging activity (% inhibition) was
plotted against different concentrations of plant extract and
IC50 (the concentration providing 50 % of radical scav-
enging activity) value was calculated from the graph by
linear regression analysis. The IC50 value of plant extract
in different solvents was compared against a standard
antioxidant compound, ascorbic acid (5–100 lg/ml).
DPPH radical scavenging activity was calculated using the
following formula (Brand-Williams et al. 1995):
% Radical scavenging activity
¼ ðControl OD  Sample ODÞ=Control OD
Metal chelating assay
The ferrous ion chelating assay was performed by the
method described by Dinis et al. (1994). Briefly, 500 ll of
plant extract was mixed with 100 ll 2 mM FeCl2. The
reaction was initiated by adding 40 ll of 5 mM ferrozine
into the mixture and kept at room temperature for 10 min.
The absorbance was measured at 562 nm. The ratio of
inhibition of ferrozine-Fe2? complex formation was cal-
culated according to Dinis et al. (1994):


% inhibition ¼ Acontrol  Asample =Acontrol  100
Quantification of photosynthetic pigments
in regenerated plantlets
Chlorophyll a and b as well as total carotenoids were
quantified as outlined by Lichtenthaler (1987). Expanded
shoot leaves (300 mg) were collected from the first apical
node of shoots at the end of third subculture in shoot
multiplication medium supplemented with different cyto-
kinins (BAP, TDZ, 2-iP and mT). Pigment content was
expressed in mg/gm FW using the formulae outlined by
Lichtenthler (1987):
Chlorophyll a ðCaÞ ¼ 11:24A662  2:04A645
Chlorophyll b ðCbÞ ¼ 20:13A645  4:19A662
Total chlorophyll ¼ 7:05A662 þ 18:09A645
Total carotenoids ¼ ð1000A4701:90Ca63:140CbÞ=214
Data analysis
All experiments were carried out with six replicates and
were repeated three times. Data were analyzed using one-
way analysis of variance (ANOVA) at 0.05 significance
level in JMPÒ version 7.0.1 (SAS Institute, Cary, NC,
USA). The significant differences among the means were
assayed by Tukey’s Honestly Significant Difference (HSD)
test on significant findings.
Results and discussion
Callus mediated organogenesis (CMO)
The disinfection technique used was efficient for in vitro
establishment with 92 % of the explants retaining mor-
phogenic potential. Within 4 weeks of culture, callus
induction commenced from the cut regions of leaves and
petioles. Explants cultured in MS medium without any
growth regulators showed no callus proliferation. The
developing calli were found to be dark green, friable under
the light conditions (14 h photoperiod), while light green
compact nodular calli were observed in cultures kept in
darkness. Callusing was achieved in MS medium contain-
ing 1.5 mg/l NAA along with 1 mg/l mT with 48 %
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induction frequency and callus growth (0.221 ± 0.07 gm
FW) at room temperature (22 ± 1 °C temperature and 14 h
photoperiod) (Fig. 1).
Temperature had a profound effect on callus growth,
subsequent proliferation and shoots regeneration of this
alpine plant. A wide range of low temperature (5–15 °C) was
tested for better callus growth and proliferation and subse-
quent shoot regeneration. It was found that culturing the
explants in medium, supplemented with different auxins and
cytokinins resulted in better callus induction frequency (%),
further proliferation (callus fresh weight) and shoot regen-
eration preferably at low temperature (13 ± 1 °C) under
14 h photoperiod. MS medium containing 1.5 mg/l NAA
along with 1.0 mg/l mT was beneficial for optimum callus-
ing response (88 %) and growth (0.446 ± 0.06 gm FW) at
low temperature of 13 ± 1 °C (Figs. 1, 2c–e). Callus cul-
tured at 22 ± 1 °C turned brown within 15 days of incu-
bation which might be due to temperature stress. It was also
observed that amount of calli incubated at 13 ± 1 °C were
relatively higher (approx. twice in amount) and remained
healthy for more than 45 days without subculture. Healthy
and fresh calli were subcultured in the optimum callus
induction medium for further proliferation. The use of NAA
as one of the most effective auxins for callus induction has
been extensively reported for e.g. Silene vulgaris (Jack et al.
2005), Punica granatum (Kanwar et al. 2010) and Doren
ammoniacum (Irvani et al. 2010). Similarly, application of
mT in enhancing callus growth has also been earlier reported
by Bairu et al. (2007).
The regenerative ability of proliferative calli was stud-
ied by the application of various cytokinins (BAP, KN,
Fig. 1 Callus induction of N.
jatamansi as influenced by
different plant growth regulators
and temperature
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Plant Cell Tiss Organ Cult
2-iP, TDZ and mT) alone or in combination with NAA at
0.5 mg/l. The highest regeneration frequencies (92 %),
maximum number of shoots (25.2 ± 0.4) with 6.1 ± 0.2
leaves per shoot were achieved in medium supplemented
with combination of 1.0 mg/l of mT and 0.5 mg/l of NAA
(Table 1). Shoot primordia only appeared from both friable
as well as compact nodular calli after 2 weeks of culture in
the regeneration medium when calli were cultured at
13 ± 1 °C temperature for 14 h photoperiod (Fig. 2f–h).
Calli cultured at 22 ± 1 °C either did not respond or
resulted in rhizogenesis. At lower temperature, higher
shoot multiplication rate was previously reported in Vitis
vinifera (Moriguchi et al. 1988) and various Saussurea spp.
(Arora and Bhojwani 1989; Chen and Li 2005; Guo et al.
2007). Although in TDZ containing medium better
response of the explants was observed at lower concen-
trations, the use of its higher concentrations resulted in
increased hyperhydricity (60 % at 1.5 mg/l and 75 % at
2 mg/l) (Fig. 2i). In the present findings, mT was nearly
twice more effective than conventional cytokinins (BAP
and KN) in shoot regeneration. No hyperhydric shoots
were observed at higher concentrations of the growth
regulators in the medium. These results are in accordance
with the previous reports on various other plant species
(Baroja-Fernández et al. 2002; Bairu et al. 2007, 2009).
Direct shoot organogenesis (DSO)
Regeneration of adventitious shoots was observed from
shoot tip segments of N. jatamansi within 4 weeks of
culture without any callus phase at 13 ± 1 °C temperature
Plant Cell Tiss Organ Cult
Fig. 2 Callus mediated organogenesis in N. jatamansi. a–b Plants
with inflorescence growing in the alpine Himalayas in Arunachal
Pradesh, c Caullogenesis from petiole explants in MS ? 1.5 mg/l
NAA ? 1.0 mg/l mT, Bar = 5 mm, d Induction of dark green friable
organogenic callus from leaf explants in MS ? 1.5 mg/l
NAA ? 1.0 mg/l mT, Bar = 5 mm, e Induction of nodular callus
from leaf explant kept in dark in MS ? 1.5 mg/l NAA ? 1.0 mg/l
mT, Bar = 5 mm, f Multiple shoot induction from compact nodular
(Fig. 3a–c). Among all cytokinins tested, mT (1.0 mg/l) in
the medium proved to be beneficial in inducing the highest
number of multiple shoots per explant (14.50 ± 0.8) with
maximum shoot length (8.0 ± 0.4) after 6 weeks of culture
(Table 2). The superiority of mT over other cytokinins
have been attributed to its faster translocation in plant
tissues (Kaminek et al. 1987), and formation of easily
degradable o-glucoside metabolites which are considered
to be stable cytokinin storage forms that can be converted
to active cytokinin bases when required (Werbrouck et al.
1996; Bairu et al. 2009). Similar to the present findings, the
use of mT and its derivatives over commonly used
callus in MS ? 1.0 mg/l mT ? 0.5 mg/l NAA, Bar = 1 cm, g–
h Induction of multiple shoot and further proliferation from friable
callus in MS ? 1.0 mg/l mT ? 0.5 mg/l NAA, Bar = 1 cm, i Hyper-
hydricity of in vitro plant cultured in MS ? 1.0 mg/l TDZ
Bar = 1 cm, j 6 week old plant in culture, Bar = 1 cm, k Rooting
of N. jatamansi in MS ? 1.0 mg/l IAA, Bar = 1 cm, l Acclimatized
plants in growth chamber after 60 days
cytokinins (BAP and KN) in improving shoot proliferation
has been demonstrated in other plant species such as potato
cultivars (Baroja-Fernández et al. 2002), Aloe polyphylla
(Bairu et al. 2007), Musa sp. (Roels et al. 2005; Aremu
et al. 2012a) Aloe arborescence (Amoo et al. 2012),
Huernia hystrix (Amoo and Van Staden 2013) and
Coleonema album (Fajinmi et al. 2014).
Adventitious shoots were also found to emerge from
petiole explants directly within 2 weeks of culture. Petiole
enlarged at one end and bud-like structures (Fig. 3d–e)
subsequently evolved into shoots (Fig. 3f–g). Highest
numbers of multiple shoots (12.43 ± 0.4) with maximum
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 Plant Cell Tiss Organ Cult

shoot length (7.34 ± 0.6) were observed in MS medium
containing 1 mg/l mT (Table 2). Direct shoot proliferation
using petiolar segments has been previously reported in
Piper colubrinum (Kelkar and Krishnamurthy 1998),
Dioscorea spp. (Anike et al. 2012), and P. longum (Rani
and Dantu 2012). But, to the best of our knowledge, till
date no report on direct organogenesis from shoot tip and
petiolar segments of N. jatamansi exists.
Rooting and acclimatization
In vitro raised shoots (2–3 cm) were excised individually
and cultured in MS medium containing either IAA or IBA
alone for root induction. The regenerated shoots were
found to produce root within 2 weeks of incubation. The
Fig. 3 Direct shoot organogenesis in N. jatamansi. a Initial enlarge- c
ment of shoot tip explants, Bar = 5 mm, b–c Multiple shoots
induction and further shoot proliferation from shoot tip explant in
MS ? 1.0 mg/l mT, Bar = 5 mm, d Emergence of green protuber-
ance at the end of petiole, Bar = 5 mm, e Bud-like structures from
green protuberance of petiole explants in MS ? 1.0 mg/l mT,
Bar = 5 mm, f–g Induction of multiple shoots from bud-like
structure, Bar = 1 cm, i 6 week old plant in culture, Bar = 1 cm
highest number of roots (4.51 ± 0.01) and maximum
length of roots (3.70 ± 0.11 cm) with 82.40 % rooting
frequency was observed in explants cultured in MS med-
ium fortified with 1.0 mg/l of IAA (Table 3). Similar
findings were observed where IAA has been successfully
employed for rooting in N. jatamansi (Mathur 1992). IAA
has also been reported to enhance the root formation in

Table 1 Influence of different plant growth regulators on shoot regeneration from callus of N. jatamansi
Plant growth regulators(mg/l) Regeneration frequency (%) No. of shoots/callus Length of shoots (cm) No. of leaves/shoot
BAP KN 2-iP TDZ mT NAA

0 0 0 0 0 0 0 0 0 0
x s
0.5 – – – – – 42 3.5 ± 0.1 3.9 ± 0.4 2.4 ± 0.2n
1.0 – – – – – 54 5.0 ± 0.1v 4.7 ± 0.2q 3.0 ± 0.1l
1.5 – – – – – 47 3.9 ± 0.3w 4.18 ± 0.4r 2.5 ± 0.1n
y t
2.0 – – – – – 45 3.0 ± 0.4 3.7 ± 0.1 2.2 ± 0.3o
u r
– 0.5 – – – – 38 5.3 ± 0.2 4.2 ± 0.2 2.8 ± 0.5m
– 1.0 – – – – 45 8.9 ± 0.6s 5.6 ± 0.4n 3.7 ± 0.4i
t p
– 1.5 – – – – 40 6.4 ± 0.1 4.9 ± 0.2 3.5 ± 0.2j
u q
– 2.0 – – – – 36 5.4 ± 0.3 4.6 ± 0.2 3.2 ± 0.4k
o j
– – 0.5 – – – 62 13.5 ± 0.3 6.56 ± 0.3 4.1 ± 0.3f
k h
– – 1.0 – – – 66 16.3 ± 0.5 7.03 ± 0.4 4.4 ± 0.2e
– – 1.5 – – – 58.5 12.4 ± 0.6q 6.0 ± 0.6l 3.7 ± 0.1i
r o
– – 2.0 – – – 52 10.1 ± 0.4 5.43 ± 0.3 3.3 ± 0.3k
m k
– – – 0.5 – – 64 15.1 ± 0.3 6.2 ± 0.7 3.9 ± 0.2gh
k h
– – – 1.0 – – 68 16.3 ± 0.6 6.9 ± 0.5 4.3 ± 0.3e
l j
– – – 1.5 – – 60 15.6 ± 0.5 6.47 ± 0.2 4.0 ± 0.3fg
n m
– – – 2.0 – – 57 14.2 ± 0.4 5.85 ± 0.1 3.7 ± 0.3i
– – – – 0.5 – 74 19.1 ± 0.2i 7.66 ± 0.6f 4.9 ± 0.4e
– – – – 1.0 – 82 21.4 ± 0.7e 8.60 ± 0.4c 5.3 ± 0.2c
k i
– – – – 1.5 – 64.8 16.2 ± 0.4 6.73 ± 0.2 4.1 ± 0.6f
p k
– – – – 2.0 – 59.5 13.2 ± 0.6 6.23 ± 0.4 3.8 ± 0.5hi
g e
– – – 0.5 – 0.5 78 20.1 ± 0.7 8.06 ± 0.3 5.0 ± 0.2d
– – – 1.0 – 0.5 84 21.8 ± 0.5d 8.72 ± 0.3b 5.5 ± 0.4b
f d
– – – 1.5 – 0.5 82 21.0 ± 0.5 8.47 ± 0.4 5.4 ± 0.5bc
j h
– – – 2.0 – 0.5 68 18.1 ± 0.6 7.03 ± 0.2 4.4 ± 0.4e
c d
– – – – 0.5 0.5 82 22.2 ± 0.3 8.46 ± 0.4 5.4 ± 0.2bc
a a
– – – – 1.0 0.5 92 25.2 ± 0.4 9.6 ± 0.6 6.1 ± 0.2a
– – – – 1.5 0.5 84 22.9 ± 0.5b 8.73 ± 0.2b 5.5 ± 0.6b
h g
– – – – 2.0 0.5 71 19.4 ± 0.6 7.33 ± 0.3 5.0 ± 0.3d
Values represent mean ± SE of 6 replicates/treatment and all the experiments were repeated three times. Means followed by the same letter in
the column are not significantly different as indicated by Tukey–Kramer HSD (P B 0.05)

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Plant Cell Tiss Organ Cult

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 Plant Cell Tiss Organ Cult

Table 2 Effect of different

Plant growth regulators(mg/l) No. of shoots/explant Length of shoots (cm)
cytokinins on shoot
regeneration from shoot tip and BAP KN 2-iP TDZ mT ST PT ST PT
petiole explants of N. jatamansi
0 0 0 0 0 1.0 ± 0.1q 0±0 0.98 ± 0.01n 0±0
o q
0.5 – – – – 3.2 ± 0.1 3.1 ± 0.2 3.02 ± 0.1l 2.50 ± 0.1o
1.0 – – – – 5.4 ± 0.2l 5.1 ± 0.4m 4.08 ± 0.1j 3.70 ± 0.2k
n p l
1.5 – – – – 3.6 ± 0.3 3.4 ± 0.1 3.04 ± 0.2 2.96 ± 0.2n
2.0 – – – – 2.9 ± 0.2p 2.1 ± 0.1r 2.86 ± 0.3m 1.96 ± 0.3p
m n k
– 0.5 – – – 4.6 ± 0.3 4.33 ± 0.2 3.8 ± 0.2 3.40 ± 0.6l
j k i
– 1.0 – – – 7.2 ± 0.4 6.8 ± 0.3 4.98 ± 0.2 4.16 ± 0.4i
k l j
– 1.5 – – – 6.8 ± 0.5 5.33 ± 0.6 4.12 ± 0.2 3.90 ± 0.3j
m o k
– 2.0 – – – 4.5 ± 0.2 4.0 ± 0.5 3.7 ± 0.3 3.10 ± 0.3m
– – 0.5 – – 9.45 ± 0.6g 7.23 ± 0.2j 5.20 ± 0.3g 4.86 ± 0.4h
d e c
– – 1.0 – – 11.40 ± 0.5 9.80 ± 0.6 6.80 ± 0.4 5.94 ± 0.2e
– – 1.5 – – 10.70 ± 0.9e 7.80 ± 0.4i 5.88 ± 0.5f 5.10 ± 0.4g
– – 2.0 – – 8.50 ± 0.5i 6.8 ± 0.7k 5.0 ± 0.4hi 4.22 ± 0.2i
f g e
– – – 0.5 – 9.80 ± 0.3 8.7 ± 0.7 6.20 ± 0.4 5.86 ± 0.3e
b c b
– – – 1.0 – 12.80 ± 0.7 10.60 ± 0.6 7.10 ± 0.3 6.82 ± 0.4b
– – – 1.5 – 10.60 ± 0.5e 9.30 ± 0.5f 6.76 ± 0.6c 6.10 ± 0.6d
h i gh
– – – 2.0 – 8.7 ± 0.6 7.73 ± 0.3 5.14 ± 0.5 5.70 ± 0.4f
c d b
– – – – 0.5 12.60 ± 0.4 10.33 ± 0.3 7.06 ± 0.4 6.43 ± 0.3c
a a a
– – – – 1.0 14.50 ± 0.8 12.43 ± 0.4 8.0 ± 0.4 7.34 ± 0.6a
c b b
– – – – 1.5 12.53 ± 0.6 11.0 ± 0.6 7.16 ± 0.3 6.73 ± 0.2b
– – – – 2.0 9.58 ± 0.7g 8.01 ± 0.8h 6.54 ± 0.2d 5.72 ± 0.3f
ST shoot tip, PT petiole explants
Values represent mean ± SE of 6 replicates/treatment and all the experiments were repeated three times.
Means followed by the same letter in the column are not significantly different as indicated by Tukey–
Kramer HSD (P B 0.05)

Table 3 Effect of different

Auxins Concentration (mg/l) Rooting frequency (%) Number of roots Length of roots (cm)
auxins on in vitro rooting from
micro-shoots of N. jatamansi in Control – – –
MS medium
IAA 0.5 68.0 2.85 ± 0.01d 1.76 ± 0.11d
1.0 82.40 4.51 ± 0.01a 3.70 ± 0.11a
b
1.5 74.40 4.10 ± 0.02 3.28 ± 0.13b
c
2.0 54.0 3.33 ± 0.42 2.44 ± 0.11c
d
IBA 0.5 66.50 2.75 ± 0.01 1.78 ± 0.19d
ab
1.0 79.70 4.23 ± 0.02 3.02 ± 0.08b
1.5 70.10 3.94 ± 0.01b 2.44 ± 0.11c
cd
2.0 53.37 3.06 ± 0.05 2.22 ± 0.04c
Values represent mean ± SE of 6 replicates/treatment and all the experiments were repeated three times.
Means followed by the same letter in the column are not significantly different as indicated by Tukey–
Kramer HSD (P B 0.05)

several other plant species (Cheruvathur et al. 2012; Kehie
et al. 2012; Barpete et al. 2014). Rooted plantlets with 4–5
fully expanded leaves were successfully transferred into
thermocol cups containing sterile soil, sand and leaf litter
(2:1:1). The acclimatized plantlets were established in the
glasshouse at controlled temperature (13 ± 1 °C) with
80 % survival rate.
Genetic stability analysis of regenerated plants
ISJ and SCoT analyses
The quantification of polymorphic loci is an essential
parameter used in the clonal stability analysis of a regener-
ated population. In the present study, 20 randomly selected

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Plant Cell Tiss Organ Cult
Fig. 4 Banding profile in N.
jatamansi using ISJ and SCoT
markers a ISJ-6 primer in DSO
derived plants, b ISJ-9 primer in
CMO derived plants, c SCoT S9
primer in DSO derived plants,
d SCoT S8 primer in CMO
derived plants; Lane 1 mother
plant, lanes 2-11 in vitro-
derived plants
in vitro regenerated plantlets (10 DSO ? 10 CMO-derived)
as well as donor plant were used to assess the clonal fidelity.
Out of the 30 ISJ primers screened, 10 primers gave clear,
reproducible, unambiguous and scorable bands in both CMO
and DSO-derived plants, and were considered for further
amplification. These 10 ISJ primers generated 63 and 57
amplicons in DSO and CMO-derived plants, out of which 3
(DSO) and 4 (CMO) were polymorphic. (Figure 4a, b;
Table 4). The number of bands varied from 4 to 8 with an
average 6.3 (DSO) and 5.7 (CMO) bands per primer
respectively. Using SCoT, a total of 36 primers were
screened, 10 primers resulted in clear, unambiguous, con-
stantly reproducible and scorable bands. A total of 51 (DSO)
and 55 (CMO) bands were scored; out of which 2 (DSO) and
4 (CMO) bands were polymorphic, while rest were
monomorphic (Fig. 4c, d; Table 4). The number of bands
varied from 3 to 7 with an average 5.1 (DSO) and 5.5 (CMO)
bands per primer respectively. The pooled ISJ and SCoT
matrix data produced a total 114 (DSO) and 112 (CMO)
bands, out of which 109 (DSO) and 104 (CMO) fragments
were found to be monomorphic respectively. The degree of
variability detected within the in vitro derived plants has
been found to be 4.38 % (DSO) and 7.14 % (CMO)
respectively which is very less. The present findings
revealed that the DSO derived plants were genetically more
stable than CMO derived plants. This might be due to the
exogenous application of PGRs during callus incubation,
shoot regeneration and prolonged culture passage. The
variability information generated by the SCoT is primarily
derived from the functional gene or its immediate flunking
region, making it a more targeted marker system. On the
other hand, ISJ markers target ubiquitous conserved intron–
exon boundary in the plant genome and thus by combining
two marker systems, it is possible to assess the genetic
variability by random scanning of the whole genome.
Robustness of SCoT marker system in detecting genetic
variability in in vitro system has been previously reported in
Cleome gynandra (Rathore et al. 2014), Dendrobium nobile
(Bhattacharyya et al. 2014), and Alhagi maurorum (Agarwal
et al. 2015). The results of our study also supports the fact
that direct multiplication by shoot tips or petioles produce
more stable clones in comparison to other regeneration
pathways. Our findings are further supported by the obser-
vations of other workers (Ghimire et al. 2012; Ramakrishnan
et al. 2014; Skała et al. 2015). However, till date no infor-
mation is available on the use of ISJ marker system for the
assessment of genetic fidelity in the in vitro-derived plants.
In this present study, it was found that ISJ marker system is
also as efficient as SCoT in detecting variation in case of N.
jatamansi.
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 Plant Cell Tiss Organ Cult

Table 4 Data on ISJ and SCoT primers used in the detection of genetic stability in micropropagated plants of N. jatamansi
Sl Primer Primer Sequence (50 -30 ) DSO CMO
No. Name
Total No. of No. of % of Total No. of No. of % of
no. of mono- poly- poly- no. of mono- poly- poly-
bands morphic morphic morphism bands morphic morphic morphism
bands bands bands bands

Intron splice junction (ISJ)

1 ISJ-4 GTCGGCGGACAGGTAAGT 6 6 – – 6 6 – –
2 ISJ-5 CAGGGTCCCACCTGCA 7 7 – – 5 4 1 20.0
3 ISJ-6 ACTTACCTGAGCCAGCGA 8 8 – – 6 5 1 16.66
4 ISJ-7 TGCAGGTCAGGACCCT 7 6 1 14.28 5 5 – –
5 ISJ-9 AGGTGACCGACCTGCA 6 6 – – 8 7 1 12.5
6 ISJ-12 GGACTGGAGCAGGTAAGT 7 6 1 14.28 4 4 – –
7 IT-31 GAAGCCGCAGGTAAG 5 5 – – 4 4 – –
8 IT-32 GACTCGCCAGGTAAG 4 4 – – 8 7 1 12.5
9 ET-36 ACTTACCTGGGGCTC 8 7 1 12.5 5 5 – –
10 ET-38 ACCTACCTGGCCGAT 5 5 – – 6 6 – –
Start codon targeted polymorphism (SCoT)
11 S4 CAACAATGGCTACCACCT 4 4 – – 6 6 – –
12 S5 CAACAATGGCTACCACGA 6 6 – – 5 5 – –
13 S6 CAACAATGGCTACCACGC 5 4 1 20.0 6 5 1 16.66
14 S7 CAACAATGGCTACCACGG 4 4 – – 4 4 – –
15 S8 CAACAATGGCTACCACGT 6 5 1 16.66 7 5 2 28.57
16 S9 CAACAATGGCTACCAGCA 3 3 – – 3 3 – –
17 S13 ACGACATGGCGACCATCG 6 6 – – 6 5 1 16.66
18 S18 ACCATGGCTACCACCGCC 6 6 – – 6 6 – –
19 S23 CACCATGGCTACCACCAG 5 5 – – 7 7 – –
20 S27 ACCATGGCTACCACCGTG 6 6 – – 5 5 – –
Total 114 109 5 4.38 112 104 8 7.14
DSO direct shoot organogenesis, CMO callus mediated organogenesis

FCM analysis Although organogenic calli showed mixoploidy but no

N. jatamansi plants produce high quantities of secondary
metabolites which can interfere with the FCM analysis
(Price et al. 2000). Therefore, the addition of 1 % PVP-40
to LB01 buffer solution improved the quality of DNA
histograms by minimising the negative effect of cytosolic
compounds produced by N. jatamansi shoots. Clearly
defined histograms for accurate determination of genomic
size were obtained following FCM analysis of intact leaf
nuclei. Using Pisum sativum Citrad nuclei (9.09 pg/2C) as
an internal standard, the nuclear DNA content of N. jata-
mansi (2n) was calculated to be 1.40 ± 0.01 pg/2C. The
1C genome size of N. jatamansi was found to be 684.6
Mbp (1 pg = 978 9 109 Mbp, Doležel et al., 2007). FCM
analysis with internal standardization provide mean nuclear
DNA indices (DI) averaging 0.146. There was no signifi-
cant difference (P B 0.05) in nuclear DNA content
between wild and in vitro raised plantlets (Fig. 5; Table 5).
major phenotypic and genetic instability were detected by
FCM in CMO-derived plants even after long-term sub-
culture. Similar genomic stability was reported by callus-
derived plants in Oil palm (Rival et al. 1997), Rubus
chamaemorus (Thiem and Śliwińska 2003), Trieyrtis hirta
(Nakano et al. 2006), Digitaria exilis (Ntui et al. 2010) and
Hydrastis canadensis (Obae and West 2010). Cytogeneti-
cally normal (diploid) cells usually maintain totipotency
and regenerate well. Polyploid and aneuploid cells may not
regenerate at the same efficiency and hence the population
of regenerated plants is usually biased towards normal
karyotype.
Evaluation of secondary metabolites
Being one of the phytochemically rich medicinal plants,
the knowledge of the biochemical constitution of various
parts of N. jatamansi is essential. Plant phenolics,

123
Plant Cell Tiss Organ Cult
Fig. 5 Flow cytometric
histogram of N. jatamansi.
a Donor plants simultaneously
chopped with internal standard,
b Mixoploid callus, c Direct
shoot organogenesis, d Callus
mediated organogenesis; a: N.
jatamansi G0/G1 peak; b: N.
jatamansi G2 peak; c: P.
sativum G0/G1 peak and d: P.
sativum G2 peak
flavonoids, alkaloids and tannins represent a diverse array
of plant secondary metabolites which predominantly act as
powerful antioxidants or scavengers of free radicals (Rice-
Evans et al. 1997; Pietta 2000). These phytochemicals have
a myriad of beneficial functions in improving human health
by modulating human metabolism and used for treatment
against many inflammatory disorders, cardiovascular dis-
eases, diabetes and cancer which occur due to deregulation
of free radical generation in the cells (Singh et al. 2002;
Alothman et al. 2009; Xanthopoulou et al. 2010; Bahado-
ran et al. 2013). Table 6 shows the total phenolics, flavo-
noids, alkaloids and tannin contents of different parts viz,
leaf and rootstock of donor plant, CMO and DSO-derived
plants. The results indicated that donor and DSO-derived
plants have lower levels of phytoconstituents as compared
to CMO-derived plants. Methanolic CMO-derived root-
stock extract exhibited highest TPC (58.34 ± 0.9 mg TAE/
gm DW) where as aqueous leaf extract of donor plants
showed lowest phenolic content (8.54 ± 0.6 mg TAE/gm
DW). The flavonoid content was recorded highest in the
methanolic rootstock extract of CMO-derived plants
(35.56 ± 0.8 mg QE/gm DW) while lowest flavonoid
content was recorded in aqueous leaf extract of donor
plants (8.78 ± 0.2 mg QE/gm DW). The reports on TPC
and TFC in donor plants are in accordance with the pre-
viously published reports on N. jatamansi (Sharma and
Singh 2012).
Apart from phenolics and flavonoids, alkaloids and
tannins are also very important plant phytochemicals.
Earlier alkaloid actinidine was reported in N. jatamansi
though detailed quantitative approach for estimation of
alkaloid and tannin were lacking till date (Hirose et al.
1978). Total alkaloid and tannin contents in CMO-derived
plants increased convincingly compared to donor and
DSO-derived plants under in vitro PGR stressed conditions.
Methanolic rootstock extract of CMO-derived plants
showed highest TAC (41.12 ± 0.4 mg AE/gm DW) and
TTC (31.44 ± 0.6 mg TE/gm DW) while aqueous leaf
extract resulted in lowest deposition. The present study
revealed that yield of various secondary metabolites varies
with the polarities of the different solvent systems used as
well as with the plant parts. Similar observations were
recorded in Dendrobium nobile (Bhattacharyya et al.
2014), Salacia chinensis (Chavan et al. 2014), and Thymus
123

Table 5 Flow cytometric determination of nuclear DNA content in callus cultures, in vitro regeneranants and mother plant
Regeneration pathway/tissue organ Pisum sativum Citrad (internal reference standard)-2C = 9.09 pg)
2C DNA content (pg)
Donor plant 1.401 ± 0.01a
Direct shoot organogenesis (DSO) 1.398 ± 0.22a
Callus mediated organogenesis (CMO) 1.410 ± 0.23a
Friable callus 1.310 ± 0.12b
Nodular compact callus 1.280 ± 0.16c
pg Picogram, Mbp megabasepairs, 1 pg = 978 Mbp. (Doležel et al. 2007)
numidicus (Ali et al. 2014). The significant variation in the
phytoconstituents in the different parts might be attributed
to hormonal contents, organ type, ontogeny as well as
endogenous physiological changes taking place in the
plants. The present findings showed the effectiveness of
mT in improving secondary metabolites in vitro in case of
N. jatamansi. The application of mT and its derivatives has
been reported previously to regulate developmental pro-
cesses and thus modify the concentration of secondary
metabolites in vitro (Amoo and Van Staden 2013; Bas-
karan et al. 2014).
Antioxidant activity of in vitro derived plants
DPPH free radical scavenging activity
The DPPH free radical scavenging activity of different
parts of donor and in vitro raised plants (both CMO and
DSO) was tested through DPPH method and the IC50
values are presented in Fig. 6a. In this method, antioxidants
react with deep violet coloured 2, 2-diphenyl-b-picrylhy-
drazyl (stable free radical) and convert it to 2, 2-diphenyl-
b-picrylhydrazine with discoloration. The degree of dis-
coloration implies the free radical scavenging potential of
the plant extract. Plant parts, regeneration pathways and
solvent system had significant effect on the antioxidant
potential of this medicinal plant. The results revealed that
the root extract had the higher DPPH scavenging potential
than the leaf extracts. Among various solvents and plant
parts tested, methanolic rootstock extract of CMO-derived
plants showed the significant antioxidant activity i.e.,
34.2 lg/ml (IC50) in comparison to standard ascorbic acid
[36.49 lg/ml (IC50)]. The leaf extracts showed moderate
DPPH radical scavenging activity. The lowest DPPH
activity was observed in aqueous leaf extract of donor
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Plant Cell Tiss Organ Cult
1C genome size (Mbp) DNA index
684.6 0.15
679.71 0.15
689.49 0.16
640.59 0.14
625.92 0.13
plants with a very high IC50 value of 186.83 lg/ml.
Similarly highest antioxidant activity in methanolic
extracts of in vitro raised plants of Aloe arborescence
(Amoo et al. 2012), Ceropegia santapaui (Chavan et al.
2014) and Dendrobium thyrsiflorum (Bhattacharyya et al.
2015) have been reported. DPPH free radical has been
widely used as model system to investigate the radical
scavenging activity in the various natural compounds. The
strong antioxidant potential could be attributed to the
presence of higher amount of polyphenols, flavonoids,
tannins and alkaloids. It is widely accepted that these
secondary metabolites show powerful antioxidant activity
due to their redox properties and ability to quenching sin-
glet oxygen, possibly acting as primary antioxidants (Rice-
Evans et al. 1996; Halliwell 2008). These results on
antioxidant potential are also in agreement with the pre-
viously published report on N. jatamansi (Lyle et al. 2009;
Sharma and Singh 2012; Mishra et al. 2014).
Metal chelating activity
Iron is essential requirement for oxygen transport, cellular
respiration and activity of many enzymes. Ferrous ion is
active pro-oxidants in the food systems (Yamauchi et al.
1988). Strong chelating activity is attributed to formation
of ferrous-ferrozine complex and therefore, rate of
decolouration helps to estimate metal chelating activity of
samples. Methanolic rootstock extract of CMO-derived
plants showed very strong inhibitory effect
(93.97 ± 3.4 %) while aqueous leaf extract of donor plants
less inhibitory activity (50 ± 1.4 %) (Fig. 6b). In the pre-
sent study, metal chelating activity was significantly
affected by the various solvent systems used and plant parts
similar to DPPH radical scavenging activity. The increas-
ing antioxidant potential in DSO and CMO-derived plants
could be due to exogenous supply of different plant growth
Plant Cell Tiss Organ Cult

Table 6 Total phenolics, flavonoids, alkaloids and tannin content in different parts of N. jatamansi with respect to different solvent systems (phenolics: mg GAE/gm DW; flavonoids: mg QE/

17.58 ± 0.3
11.77 ± 0.5
11.0 ± 0.5
7.5 ± 0.4
7.3 ± 0.2
31.44 ± 0.6
22.12 ± 0.5
19.77 ± 0.2
13.44 ± 0.2
9.8 ± 0.1

DSO direct shoot organogenesis, CMO callus mediated organogenesis, GAE gallic acid equivalent, QE quercetin equivalent, AE atropine equivalent, TAE Tannic acid equivalent, DW dry weight
regulators and regeneration pathways followed. Similar
observations have been reported in Ceropegia sp (Chavan

TTC
et al. 2013), Helicterus isora and Ceiba pentendra (Lo-
ganayaki et al. 2013).

28.76 ± 0.5
23.09 ± 0.3
21.08 ± 0.5
18.33 ± 0.6
15.63 ± 0.2
41.12 ± 0.4
31.12 ± 0.7
28.76 ± 0.6
23.05 ± 0.2
16.78 ± 0.3
Quantification of photosynthetic pigments
TAC

in regenerated plantlets
33.13 ± 0.4
27.76 ± 0.3
25.56 ± 0.4
17.0 ± 0.6
13.77 ± 0.3
35.56 ± 0.8
29.83 ± 0.6
25.54 ± 0.4
18.11 ± 0.3
14.56 ± 0.3
Acclimatization failures is one of the major issues of the
in vitro-derived plants due to poor development of
TFC

photosynthetic efficiency coupled with induced stress

from artificial environment (Valero-Aracama et al. 2006;
37.89 ± 0.9
31.37 ± 0.6
28.78 ± 0.8
19.92 ± 0.7
17.62 ± 0.2
58.34 ± 0.9
46.78 ± 0.7
39.43 ± 0.4
27.53 ± 0.5
21.34 ± 0.4
Pospisilova et al. 2007). The present experimental
CMO

findings document a positive correlation of cytokinins on

TPC

the yield of photosynthetic pigments at the end of third

subculture in shoot multiplication medium (Table 7). At
12.22 ± 0.3
8.12 ± 0.2
7.99 ± 0.2
4.12 ± 0.1
4.07 ± 0.1
25.22 ± 0.3
17.76 ± 0.4
14.98 ± 0.2
9.5 ± 0.1
6.8 ± 0.2

an optimum concentration of mT (1 mg/l), significantly

higher levels of chlorophyll a, chlorophyll b, total
TTC

chlorophyll and total carotenoids were observed over the

other conventional cytokinins (BAP, TDZ and 2-iP)
22.0 ± 0.6
17.17 ± 0.3
15.56 ± 0.4
12.17 ± 0.2
9.45 ± 0.5
38.23 ± 0.3
26.77 ± 0.2
24.44 ± 0.4
20.12 ± 0.5
13.77 ± 0.4

treated plantlets. The positive role of mT and mT

TAC

derivatives in delaying leaf senescence by accumulating

and maintaining photosynthetic pigments have been
reported earlier in Banana (Aremu et al. 2012a; b),
29.0 ± 0.7
23.44 ± 0.3
22.04 ± 0.6
14.16 ± 0.4
10.0 ± 0.5
32.21 ± 0.7
25.87 ± 0.2
22.0 ± 0.2
15.88 ± 0.1
11.11 ± 0.1

Pelargonium (Mutui et al. 2012) and Prunus (Gentile

TFC

et al. 2014).
Values represent mean ± SE of 5 replicates and all the experiments were repeated three times
33.0 ± 0.5
25.80 ± 0.4
23.98 ± 0.7
15.66 ± 0.8
12.36 ± 0.6
50.12 ± 0.6
40.84 ± 0.8
33.79 ± 0.4
24.10 ± 0.2
17.46 ± 0.1

Conclusion
DSO
TPC

The present study demonstrates an effective in vitro

11.01 ± 0.8
6.58 ± 0.3
6.03 ± 0.4
3.13 ± 0.3
3.02 ± 0.5
22.55 ± 0.9
14.03 ± 0.7
12.53 ± 0.3
7.5 ± 0.3
4.6 ± 0.1

propagation protocol via callus mediated and direct shoot

organogenesis for N. jatamansi, a critically endangered
TTC

medicinal plant of alpine Himalayas. The regeneration

frequency is reported to be significantly higher in CMO
20.11 ± 0.3
15.76 ± 0.5
14.10 ± 0.2
10.33 ± 0.7
7.35 ± 0.5
36.76 ± 0.2
24.22 ± 0.5
22.44 ± 0.3
17.43 ± 0.2
12.25 ± 0.3
gm DW: alkaloids: mg AE/gm DW: tannins: mg TAE/gm DW)

derived plantlets than that of DSO derived plants with

superior production of secondary metabolites and sig-
TAC

nificantly higher antioxidant potential. Low temperature

and meta-topolin have profound effect on callus growth,
26.23 ± 0.4
20.45 ± 0.6
19.40 ± 0.6
12.32 ± 0.8
8.78 ± 0.2
30.14 ± 0.4
23.55 ± 0.2
20.12 ± 0.3
13.24 ± 0.2
9.22 ± 0.1

shoot regeneration and improving photosynthetic pig-

ment contents in regenerated plantlets which revealed a
TFC

definite advantage over the previously published report

Donor plant

on this threatened taxa. In vitro derived plants were

28.59 ± 0.7
22.0 ± 0.5
20.14 ± 0.3
13.10 ± 0.2
8.54 ± 0.6
45.59 ± 0.7
36.12 ± 0.5
30.43 ± 0.3
22.26 ± 0.2
15.87 ± 0.2

genetically analysed by molecular markers and flow

TPC

cytometry and were found to be genetically stable under

in vitro conditions. Different regeneration pathways,
plant parts, solvent systems and PGRs greatly influenced
Acetonitrile

Acetonitrile
Chloroform

Chloroform
Methanol

Methanol
Solvent

Acetone

Acetone

the in vitro phytochemical production and antioxidant

Water

Water

capacity of N. jatamansi. The developed protocol can be

utilized for the improvement of bioactive constituents by
Plant part

genetic manipulations and commercial propagation of

Rootstock

this high value critically endangered, medicinal plant of

Leaf

alpine Himalayas.

123
 Plant Cell Tiss Organ Cult

Fig. 6 Antioxidant activity of N. jatamansi in donor plant as well as b Metal chelating activity (DSO direct shoot organogenesis; CMO
in vitro-derived plants using different solvent systems. a Comparative callus mediated organogenesis)
IC50 values of DPPH activity with respect to standard ascorbic acid,

Table 7 Effect of different cytokinins at their optimum concentration on photosynthetic pigment content of N. jatamansi shoots during the
multiplication phase
Cytokinin Chlorophyll a Chlorophyll b Chlorophyll Total chlorophyll Total carotenoid Total
treatment (mg/g FW) (mg/g FW) a/b ratio (mg/g FW) (mg/g FW) chlorophyll/carotenoid
ratio

Mother plant 0.44 ± 0.05d 0.17 ± 0.02c 2.58 ± 0.3e 0.61 ± 0.07d 0.145 ± 0.01e 4.20 ± 0.1c
In vitro derived plants
BA-1 mg/l 0.45 ± 0.04d 0.17 ± 0.01c 2.64 ± 0.2d 0.62 ± 0.04d 0.153 ± 0.03d 4.05 ± 0.2b
TDZ-1 mg/l 0.50 ± 0.06c 0.18 ± 0.04c 2.77 ± 0.6c 0.68 ± 0.07c 0.160 ± 0.02c 4.25 ± 0.4b
b b b b b
2,iP-1 mg/l 0.62 ± 0.04 0.22 ± 0.06 2.81 ± 0.3 0.84 ± 0.03 0.175 ± 0.06 4.80 ± 0.2a
a a a a a
mT-1 mg/l 0.71 ± 0.06 0.25 ± 0.06 2.84 ± 0.2 0.96 ± 0.06 0.197 ± 0.04 4.87 ± 0.4a
Values represent mean ± SE of 5 replicates/treatment and all the experiments were repeated three times. Means followed by the same letter in
the column are not significantly different as indicated by Tukey–Kramer HSD (P B 0.05)

Acknowledgments Financial supports received from DBT research
grant no. BT/PR-10533/BCE/08/622/2008 and other DBT funded
projects are gratefully acknowledged. The authors would like to thank
the Bioinformatics Centre, NEHU and Prof. A. Chatterjee, Dr.
N. Ghosal from Department of Biotechnology and Bioinformatics,
NEHU for providing the facilities carried out during the course of study.
Authors’ contributions statement BB carried out the experiments,
analyzed the data and drafted the manuscript. All the authors were
involved in the designing of the experiments. SK and PT supervised
the work. SK edited the manuscript. All authors read and approved
the final version of this manuscript.
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