Industrial Crops & Products 121 (2018) 313–319

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Industrial Crops & Products

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In vitro propagation of Digitalis lanata Ehrh. through direct shoot T

regeneration – A source of cardiotonic glycosides
⁎
Bhushan P. Bhusarea, C.K. Johnb, V.P. Bhattc, T.D. Nikama,
a
Department of Botany, Savitribai Phule Pune University, Pune, 411007, Maharashtra, India
b
Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Pune, 411008, Maharashtra, India
c
Herbal Research and Development Institute (HRDI), Mandal-Gopeshwar, 246401, Chamoli, Uttarakhand, India

A R T I C LE I N FO A B S T R A C T

Keywords: An efficient method for the in vitro propagation of Digitalis lanata Ehrh. through direct organogenesis from leaf
Digitoxin and petiole explants has been standardised. MS basal medium supplemented with cytokinins BAP, KIN, TDZ
Digoxin either alone or along with auxins IAA and NAA at different concentrations were tried. TDZ at 4.54 and
Direct organogenesis 6.81 μmol/l were optimum for direct regeneration of shoots from leaf (4.4 ± 0.6 shoots/explant), and petiole
(3.0 ± 0.8 shoots/explant) explants respectively. Among the various concentrations of auxins IAA, IBA and
NAA tried for rooting, the best response occurred on MS basal medium supplemented with 17.13 μmol/l IAA. On
greenhouse transfer about 60% of the plantlets survived. In vitro raised plantlets were morphologically similar to
mother plants. Cardiotonic glycosides digoxin and digitoxin were extracted by modified methods and estimated
by HPLC. There were no significant differences in digoxin and digitoxin content in leaves of naturally grown and
in vitro raised plants. The method for in vitro propagation of D. lanata through direct organogenesis from leaf and
petiole explants reported here will be of great use for the rapid and large scale clonal propagation, production of
biomass for extraction of cardiotonic glycosides, ex situ conservation, and improvement through conventional
plant breeding and transgenic methods.

1. Introduction
Digitalis lanata Ehrh. (“Woolly Foxglove”/“Grecian Foxglove”, family:
Plantaginaceae) is a biennial or perennial herb widely distributed in
temperate parts of Hungary, Romania and Balkan Peninsula in Europe,
North Africa and West Asia. It is cultivated in the Netherlands (Li et al.,
2014), in some parts of the United States (Hollman, 1985) and the
Himalayan region of India (Negi et al., 2012). The leaves of the plant
are exclusively used for the isolation of important cardiotonic glyco-
sides digoxin and digitoxin which are used in the treatment of con-
gestive heart failure (CHF) and atrial arrhythmia (Somberg et al., 1986;
Rahimtoola and Tak, 1996; Ehle et al., 2011). For oral use, Digitalis
glycoside is still the only safe drug treatment for improving hemody-
namics in patients through balancing the cardiac activity (Schwinger
et al., 2003; Pérez-Alonso et al., 2009; Patel, 2016; Kreis, 2017). As per
recent research findings Digitalis glycosides digoxin, digitoxin and la-
natoside C are effective in treating viral infections (Hoffmann et al.,
2008; Cheung et al., 2014; Zhyvoloup et al., 2017) and different types
of cancer (Prassas and Diamandis, 2008; Newman et al., 2008; Sharma
and Purkait, 2012; Wu et al., 2012a). Chemical synthesis of these gly-
cosides is possible, but too expensive to be cost effective (Clemente
et al., 2011). Although the medicinal glycoside content is low, the
pharmaceutical industry still relies on natural source. D. lanata is pre-
ferred over D. purpurea and other species of Digitalis as the content of
cardiotonic glycosides is higher (Bown, 1995; Duke, 2002). Traditional
method of propagation of D. lanata is through seeds. However, D. lanata
being native of temperate regions, seeds need to be stored at 4 °C. In
warmer climates, low seed germination, and rapid loss of seed viability,
when stored at room temperatures, pose limitations on the use of seeds
for large scale planting (Verma et al., 2016). At present there is de-
structive over-exploitation of D. lanata from wild to meet the ever-in-
creasing demands of both allopathic and traditional medicine in-
dustries. Collection of plant material before seeding results in rapid
disappearance of natural populations. Although there were attempts to
produce D. lanata varieties having high cardenolide contents through
conventional breeding methods (i.e. in-breeding and crossing), but
offsprings were not stable (Verma et al., 2012). To fix the character,
cumbersome long-term breeding programmes are required which can

Abbreviations: BAP, N6-benzyladenine; HPLC, high performance liquid chromatography; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; KIN, kinetin; MS basal medium,
Murashige and Skoog basal medium; NAA, α-naphthalene-acetic acid; PGRs, plant growth regulators; TDZ, 1-phenyl-3-(1 2 3-thiadiazol-5-yl)-urea
⁎
Corresponding author.
E-mail address: tdnikam@unipune.ac.in (T.D. Nikam).

https://doi.org/10.1016/j.indcrop.2018.05.019
Received 25 January 2018; Received in revised form 4 May 2018; Accepted 7 May 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
B.P. Bhusare et al.
be prohibitively expensive. Hence, there is an urgent need for an effi-
cient in vitro propagation method which can be of use in improving D.
lanata through selection/transgenic methods. During the past three
decades there have been many reports on biotechnological approaches
for in vitro propagation and detection of cardiac glycosides in wild as
well as cultivated Digitalis species (D. cariensis, D. davisiana, D. ferru-
ginea, D. lamarcki, D. minor, D. obscura, D. purpurea, D. thapsi and D.
trojana) as well as production of cardiac glycosides in tissue and cell
culture (Patil et al., 2013; Verma et al., 2016). An efficient method for
in vitro propagation through direct organogenesis is a prerequisite for
the rapid and large-scale production of planting stocks, quick bulking of
any high glycoside containing variants, and for improvement through
genetic transformation. Hence it was decided to study the effects of
various concentrations and combinations of PGRs on shoot regeneration
from leaf and petiole explants of D. lanata, as well as rooting of these
shoots to produce plantlets. The micropropagation method reported
here will be of great use in rapid and large-scale propagation, and its
introduction and exploitation in many places outside its native range
without any climate/seasonality constraint and genetic improvement of
this industrially important medicinal plant through conventional (se-
lection) and modern (transgenic) methods.
2. Materials and methods
2.1. Source of explants
Seeds of D. lanata were obtained from Herbal Research and
Development Institute, Mandal-Gopeshwar (coordinates: 30.4095°N,
79.3198°E), Chamoli, Uttarakhand, India. Seeds were washed under
running tap water for 5 min followed by rinsing with sterile distilled
water in a laminar air-flow cabinet. Then the seeds were surface ster-
ilized with 0.1% (w/v) HgCl2 solution for 5 min and washed five times
with sterile distilled water. The surface sterilized seeds were inoculated
on MS (Murashige and Skoog, 1962) basal medium with 30 g l−1 su-
crose and 8.0 g l−1 agar. The pH of the medium was adjusted to 5.8
using 1 N NaOH or 1 N HCl prior to autoclaving at 121 °C, 103 kPa for
20 min. All cultures were incubated at 25 ± 2 °C for 8 h photoperiod
with 36 μmol m−2 s−1 light intensity provided by cool white, fluor-
escent tubes (Philips, India). After germination, seedlings were trans-
ferred on fresh MS basal medium at four weeks interval. Apical buds,
axillary buds, leaf and petioles from 12 week old seedlings were excised
and used as a source of explants.
2.2. Direct shoot regeneration
MS medium supplemented with various concentrations of N6-ben-
zyladenine (BAP), 6-furfurylaminopurine (KIN) and 1-phenyl-3-(1, 2, 3-
thiadiazol-5-yl)-urea (TDZ) and combinations of TDZ with indole-3-
acetic acid (IAA) and α-naphthaleneacetic acid (NAA) were used for
shoot multiplication. These plant growth regulators (PGRs) were pro-
cured from Sigma-Aldrich, USA. Four types of explants were used in-
itially i.e. apical buds (1 cm top segments containing apical buds), ax-
illary buds (1.5–2 cm segments containing one axillary bud in the
centre), leaf (1 cm squares) and petiole (1 cm long segments). Since the
results obtained with leaf and petiole explants with higher number of
shoots (3.0-4.4 shoots/explant), work was continued with these two
types of explants. Results obtained with apical and axillary bud explants
(1.0–2.0 shoots/explant), are not discussed in this communication.
The explants were inoculated in horizontal and vertical positions on
different culture media. All cultures were incubated at 25 ± 2 °C for
8 h photoperiod with 36 μmol m−2 s−1 light intensity. After four weeks
of culture, the explants with regenerated shoots were sub-cultured on
fresh media. The effect of different PGRs on shoot multiplication was
recorded as number of explants responding (percentage), number of
shoots per explant, and length of regenerated shoots.
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Industrial Crops & Products 121 (2018) 313–319
2.3. Root induction and acclimatization
In vitro developed healthy shoots (2.0–2.5 cm long) were excised
and placed on MS media supplemented with various concentrations of
auxins such as IAA (1.42–34.26 μmol/l), IBA (1.22–14.70 μmol/l) and
NAA (1.34–16.11 μmol/l). The effect of different auxin treatments were
recorded as number of shoots responding as percentage, number of
roots per shoot and length of roots after eight weeks. The rooted
plantlets were carefully removed from the medium, gently washed
under tap water to remove the adhering media, and transferred to
plastic pots containing an autoclaved mixture of soil and sand (3:1).
The potted plantlets were irrigated with half strength MS medium (only
major and minor salts). Initially these pots were maintained in the
culture room having 25 ± 2 °C temperature, 80–90% relative hu-
midity, and 8 h photoperiod with 36 μmol m−2 s−1 light intensity for
two weeks. Subsequently the plantlets were transferred to a greenhouse
with 27 ± 2 °C temperature, 60–80% relative humidity, and natural
light (60 μmol m2 s−1) and photoperiod (10–12 h). Percent plant sur-
vival, plant morphology, and cardiotonic glycoside content were re-
corded three months after greenhouse transfer.
2.4. Extraction and HPLC analysis of digoxin and digitoxin from leaves of
D. lanata
To assess the effect of in vitro regeneration on secondary metabolite
production, HPLC-DAD method was used to quantify the digoxin and
digitoxin from leaves of in vitro regenerated plants of D. lanata and
compared with those in leaves of naturally grown plants. A modified
Pellati et al. (2009) protocol was used for cardenolides (digoxin and
digitoxin) extraction. By the modified method digoxin and digitoxin
were eluted in short time span (10 min/sample). In brief, basal leaves of
D. lanata were excised from naturally grown and in vitro raised plantlets
and dried at 40 °C in a hot air oven till constant weight. The dried leaves
were powdered and 0.5 g powder was added to 20 ml of 75% (v/v)
methanol in conical flask, shake extracted by placing on a rotary shaker
for 12 h at 75 rpm, followed by ultrasonication for 15 min at 60 °C,
cooling to room temperature, and centrifuging at 4000 rpm for 15 min.
The supernatants were filtered using Whatman (grade 1) filter paper.
The filtrates were concentrated under vacuum using a rotary evaporator
at 30 °C. The residue was dissolved in 2 ml of 75% (v/v) methanol and
filtered using 0.22 μm cellulose acetate filters. The filtrates were placed
in HPLC vials and stored at −20 °C till use.
HPLC analyses for digoxin and digitoxin were carried out using an
HPLC-1200 infinity series system (Agilent Technologies, Waldbronn,
Germany), on a Symmetry C18 column (4.6 × 250 mm I.D., 5 μm,
Waters, Dublin, Ireland). Digoxin and digitoxin content were calculated
separately using calibration curves of pure standards (Sigma-Aldrich
Chemie, Steinheim, Germany) of jointly estimated digoxin and digi-
toxin. For the calibration curves, concentrations of (12.5, 25, 50, 100,
200, 300 and 400 μg/ml) were used (n = 3 for each concentration, R
values were 0.999 and 0.998 for digoxin and digitoxin respectively).
These cardenolides were eluted with an isocratic system of acet-
onitrile (ACN): water (H2O): methanol (CH3OH) at the proportion of
66:07:27 (flow rate: 0.4 ml/min, column temperature: 20 °C, sample
injection volume: 10 μl, detector set at: 220 nm, and total running time:
10 min). Three HPLC runs were performed for each sample.
2.5. Experimental design and statistical analysis
All the experiments were organized in completely random design
(CRD) and conducted three times. Data from each experiment of four-
teen replicate (one explant per replicate) were analyzed by using
ANOVA in SPSS software. The percentage data was arcsine transformed
before subjecting to statistical analyses. The homogeneity of means/
comparison of means was done using Duncan’s Multiple Range Test
(DMRT) at 5% probability level. Similarly, HPLC data were analysed
B.P. Bhusare et al. Industrial Crops & Products 121 (2018) 313–319

Table 1 marked reduction in shoot number. There was no significant change in

Effect of TDZ on direct shoot regeneration from leaf explants of D. lanata after the number of shoots produced when optimal concentration of TDZ
four weeks of culture. (4.54 μmol/l) was used in combination with different concentrations of
TDZ Percentage Shoot number ± S.E. Shoot length (cm) ± S.E. IAA or NAA.
(μmol/ response for direct
l) shoot bud 3.2. Rooting
formation (%)

00.0 0.0 ± 0.0 c 0.0 ± 0.0 b 0.0 ± 0.0 c When healthy regenerated shoots were transferred to MS basal
2.27 14.2 ± 8.2 bc 1.0 ± 0.5 ab 2.3 ± 1.3 abc medium supplemented with different auxins (IAA, IBA and NAA) at
4.54 19.0 ± 12.5 bc 3.9 ± 2.6 a 2.1 ± 1.0 abc various concentrations, the percentage of rooting differed significantly
6.81 57.1 ± 0.0 a 4.4 ± 0.6 a 4.3 ± 0.8 a
depending upon the concentrations of auxins used viz. IAA, IBA and
9.08 33.3 ± 9.5 b 2.0 ± 0.4 ab 3.8 ± 0.9 ab
11.35 23.8 ± 4.7 bc 1.8 ± 0.1 ab 3.6 ± 0.4 ab
NAA. Whereas control medium (not containing auxin) did not show any
13.62 4.7 ± 4.7 c 0.8 ± 0.8 ab 1.0 ± 1.0 bc root formation (Table 3). Root emergence was observed at cut ends of
shoots after three weeks and after eight weeks of culture a well de-
Data represent mean ± SE of three repeated experiments, each with fourteen veloped root system was observed (Fig. 1e). Rooting was observed in all
explants. The values followed by different letters for each variable are sig- treatments but the percentage response varied. Among the auxins (IAA,
nificantly different at 5% probability level according to Duncan’s multiple IBA and NAA) tested for root induction, IAA was more effective than
range test (DMRT).
IBA and NAA. Rooting increased (2.3 ± 0.2–13.9 ± 1.1) with the
increase in concentration (1.42–17.13 μmol/l) of IAA. The best re-
with ‘t-test’ and expressed as mean values ± standard deviation (SD).
sponse for rooting was recorded on 17.13 μmol/l IAA, in which
81.6 ± 1.6% of regenerated shoots formed roots, with 13.9 ± 1.1
3. Results number of roots per shoot and 2.1 ± 0.2 cm average length of roots
(Fig. 1e). Further increase in concentration of IAA (22.84–34.26 μmol/
3.1. Influence of PGRs and source of explants on direct organogenesis from l), resulted in decrease in percentage of shoots producing roots and
leaf and petiole explant number of roots per shoot (Table 3). Use of NAA (1.34–16.11 μmol/l)
and IBA (1.22–14.70 μmol/l) resulted in lower number of roots
All concentrations of TDZ (2.27–13.62 μmol/l) resulted in re- (3.8 ± 0.3 and 4.8 ± 1.0) per shoot. The roots formed in medium
generation of shoot buds in leaf explant. Explants inoculated on control supplemented with IAA were thick and long, while those formed on
medium (no PGR) and media supplemented with other PGRs didn’t media supplemented with IBA and NAA were slender and weak.
show any signs of shoot regeneration, and became necrotic after one The acclimatized plantlets were successfully established in soil
week (data not shown). Within a week after transfer on TDZ containing where more than 60% of them survived. All plants were healthy and
media there was swelling and slight increase in the size of vertically grew vigorously. Leaf morphology (oblanceolate with acute apex,
placed leaf explants; and after two weeks, formation of adventitious broadly dentate margin, ciliate at the base) of both, in vitro regenerated
shoot buds and shoots were observed. With increase in the concentra- plantlets and acclimatised plantlets were same (Fig. 1f).
tion of TDZ from 2.27 μmol/l to 6.81 μmol/l, there was gradual increase
in the percentage of explant forming multiple shoots (15–57.1%), 3.3. Quantification of digoxin and digitoxin from leaf of D. lanata
average number of shoots per explant (1–4.4) and average shoot length
(2.3–4.3 cm) (Table 1). Further increasing the concentration of TDZ to HPLC chromatogram of methanolic extracts of leaves is shown in
9.08 μmol/l or 13.62 μmol/l resulted in decrease in the percentage of (Fig. 2a) for standard peaks. The peak eluted at 6.6 tR is for digoxin and
explants forming multiple shoots (33.3–4.7%), the average number of 8.4 tR is for digitoxin. At the same retention time elution of both car-
shoots per explant (2–0.8) and average shoot length (3.8–1.0 cm). denolides were observed in leaves of in vitro grown plants (Fig. 2b) and
Among various concentrations (2.27, 4.54, 6.81, 9.08, 11.35 and leaves of naturally grown plants (Fig. 2c). Digoxin (36.1 ± 0.1 μg/gm
13.62 μmol/l) of TDZ tested, 6.81 μmol/l TDZ was the best for leaf of D.W and 35.8 ± 0.0 μg/gm of D.W) was found more significant than
explant, and resulted in 57.1% of explants responding to shoot induc- digitoxin (10.0 ± 0.0 μg/gm of D.W and 8.7 ± 0.0 μg/gm of D.W)
tion, 4.4 ± 0.6 number of shoots with 4.3 ± 0.8 cm shoot length from leaf samples of naturally grown and in vitro regenerated plants
(Fig. 1a and b). (Table 4). There was no significant difference between cardenolide
Effect of various PGRs on shoot regeneration from petiole explant content in the leaves of naturally grown and in vitro regenerated plants.
are presented in Table 2. In the present study, only distal end of petiole
explant showed regeneration when placed vertically on the medium. 4. Discussions
Proximal end as well as horizontal positioning of petiole explants did
not show any regeneration. Despite, types and concentrations of PGRs As per the method reported here, multiple shoots were induced
in the medium, the first change in cultured petiole explants was swel- directly from leaf and petiole explants in D. lanata without a callus
ling within a week and formation of shoot bud by the second week after phase. Plants produced by direct organogenesis exhibit greater genetic
transfer to shoot induction medium. The petiole explants produced up fidelity than those regenerated through indirect organogenesis (with an
to three shoots on 4.54 μmol/l TDZ containing medium after eight intermediate callus phase) (Karam and Al-Majathoub, 2000; Khan et al.,
weeks of culture initiation (Fig. 1c and d). Explants inoculated on 2009; Skała et al., 2015; Pérez-Alonso et al., 2018). To our knowledge,
control medium (not containing PGRs) did not show shoot induction. this is the first report of an efficient in vitro propagation method in D.
Among the various PGRs used, TDZ was found to be the most effective lanata through direct organogenesis from leaf and petiole explants. In
at 4.54 μmol/l concentration where 73.8 ± 8.5% response, average the present study, for in vitro induction of shoots, two different explants
3.0 ± 0.8 shoots and 2.8 ± 0.4 cm shoot length were observed (leaf and petiole) and three cytokinins (BAP, KIN and TDZ) alone or in
(Table 2). Use of KIN or BAP instead of TDZ resulted in relatively lower combination with auxins IAA and NAA were used. Among the two ex-
frequencies of shoot proliferation (49.9 ± 10.9 and 59.5 ± 10.3) and plants tried, response of petiole was better than leaf. Among the three
fewer shoots (1.8 ± 0.1 and 1.5 ± 0.0) per explants. There was a cytokinins tried TDZ gave the best response for adventitious shoot re-
gradual increase in the number of shoots (2.0 ± 0.0–3.0 ± 0.1) with generation with leaf as well as petiole explant. Similar results were
increasing the concentrations of TDZ from 2.27 to 4.54 μmol/l. How- reported in different plant species (Kumar et al., 2010; Kumar and
ever, higher concentrations (9.08 and 13.6 μmol/l) of TDZ, resulted in a Reddy, 2012; Wu et al., 2012b; Padmanabhan et al., 2015; Rathore

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B.P. Bhusare et al.
Fig. 1. In vitro multiplication of D. lanata via direct shoot organogenesis from leaf and petiole explants. a, b – Regeneration of multiple shoots from leaf explants; c, d –
Regeneration of multiple shoots from petiole explants; e – Rooting of regenerated shoots; f – Acclimatized plantlets growing in a pot containing sterilized soil and
sand (3:1) in the greenhouse.
et al., 2016). In many woody plant species, TDZ has proved the best
cytokinin for shoot regeneration (Huetteman and Preece, 1993). In the
present study, combinations of TDZ with IAA and NAA were only as
effective as TDZ alone. According to Prakash et al. (2004) and Liu et al.
(2006), in Rhodiola spp. and Curcuma amada, use of TDZ in combination
with auxin promoted shoot regeneration. However, Magnusson et al.
(2008) got opposite effect when they used TDZ with auxin in Withania
coagulans. Using TDZ with auxin may result in excessive callus forma-
tion in D. trojana Ivanina, and may lead to higher rates of somaclonal
variation (Cingoz and Gurel, 2016). In the present study TDZ at low
concentrations such as 4.54 μmol/l and 6.81 μmol/l resulted in direct
regeneration of adventitious shoots from leaf and petiole explants.
Lower concentration of TDZ promoted shoot regeneration and higher
concentrations promoted formation of somatic embryos in Saintpaulia
ionantha Wendl (Mithila et al., 2003). In the present investigation, at
higher concentrations of TDZ (9.08 and 13.6 μmol/l) a reduction in the
frequency of shoot regeneration from leaf and petiole explant was ob-
served. This may be due to sensitivity of shoots to vitrification and
cresting (Huetteman and Preece, 1993). Furthermore, TDZ is known to
stimulate purine synthesis and accumulation, and inhibit cytokinin
activity by increasing the level of endogenous cytokinin/s which alters
cytokinin metabolism (Murthy et al., 1998). Petiole explant responding
better than leaf explants, may be because of meristematic activity in
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Industrial Crops & Products 121 (2018) 313–319
younger leaves was initiated on the entire surface or in older explants
was restricted to the basal ends respectively, as reported by Ghimire
et al. (2016). Huang and Dai (2011) have also reported similar results.
Higher compactness of xylem and phloem tissue and the level of en-
dogenous hormones might be the reason for regeneration of shoot
mostly from base of petiole and cut edges of the midrib (Karam and Al-
Majathoub, 2000). Leaf apex reaches maturity first and, afterward
moves to base, due to this the cells present at base are sensitive in
nature (Yepes and Aldwinckle, 1994). Low frequency of shoot re-
generation from leaf surface in contact with the medium may be due to
poor respiration (Lo, 1997). A deficiency of oxygen may result in poor
supply of free energy for induction of shoot and tissue differentiation
(Thorpe, 1988). In the present study the vertically placed explants
showed high regeneration capacity and more shoot buds as compared
to horizontally placed explants. However, horizontal placing of explants
on the medium was found better than vertical placing, may be because
of constant contact of explant and medium (Arockiasamy et al., 2002;
Sharma and Wakhlu, 2001). Lee-Stadelmann et al. (1991) also reported
similar results for regeneration of shoot from leaf and petiole explants
in Populus. In the present study leaf explant showed better results than
petioles for number of regenerated shoots and their length. Hedayat
et al. (2009) and Ghimire et al. (2012) reported lower frequency for
shoot regeneration from petiole explants
B.P. Bhusare et al. Industrial Crops & Products 121 (2018) 313–319

Table 2
Effect of different concentrations and combinations of plant growth regulators (BAP, KIN, TDZ, IAA and NAA) on shoot induction and proliferation from petiole
explants of D. lanata after four weeks of culture.
PGRs Concentrations (μmol/l) Percentage response for direct shoot bud formation (%) Shoot number ± S.E. Shoot length ± S.E. (cm)
Control 0.0 ± 0.0 f 0.0 ± 0.0 f 0.0 ± 0.0 f

BAP 2.22 33.3 ± 4.7 de 1.3 ± 0.0 e 1.2 ± 0.1cde

4.44 52.3 ± 8.5 abcde 1.8 ± 0.1 bcde 1.5 ± 0.0 bcde
6.66 59.5 ± 10.3 abcd 1.5 ± 0.0 de 1.2 ± 0.0 cde
8.88 35.7 ± 8.2 de 1.3 ± 0.1 e 1.1 ± 0.1 cde

KIN 2.32 49.9 ± 10.9 abcde 1.8 ± 0.1 bcde 1.3 ± 0.1 bcde
4.65 47.6 ± 8.5 abcde 1.6 ± 0.1 cde 1.4 ± 0.2 bcde
9.3 38.0 ± 8.5 cde 1.8 ± 0.1 bcde 1.7 ± 0.4 bcd
13.95 35.7 ± 12.3 de 1.5 ± 0.2 de 1.4 ± 0.2 bcde

TDZ 2.27 47.6 ± 8.5 abcde 2.0 ± 0.0 bcd 1.7 ± 0.2 bcd
4.54 73.8 ± 8.5 a 3.0 ± 0.1 a 2.8 ± 0.2 a
9.08 57.1 ± 8.2 abcd 2.1 ± 0.2 b 1.9 ± 0.4 b
13.6 28.5 ± 4.1 e 2.1 ± 0.2 bc 1.8 ± 0.2 bc

TDZ + IAA 4.54 + 2.85 57.1 ± 10.9 abcd 1.6 ± 0.1 cde 1.2 ± 0.1 cde
4.54 + 5.71 57.1 ± 4.1 abcd 1.6 ± 0.2 cde 0.9 ± 0.1 e
4.54 + 11.42 64.2 ± 7.1 abc 1.7 ± 0.2 bcde 1.2 ± 0.1 cde
4.54 + 17.13 42.8 ± 4.1 bcde 1.5 ± 0.2 de 1.0 ± 0.1 de

TDZ + NAA 4.54 + 2.68 66.7 ± 10.2 ab 1.7 ± 0.0 bcde 1.2 ± 0.0 cde
4.54 + 5.37 57.1 ± 8.2 abcd 1.4 ± 0.1 e 1.4 ± 0.0 bcde
4.54 + 10.74 42.8 ± 10.9 bcde 1.6 ± 0.1 cde 1.1 ± 0.2 cde
4.54 + 16.11 33.3 ± 4.7 de 1.4 ± 0.1 e 1.0 ± 0.2 de

Data represent mean ± SE of three repeated experiments, each with fourteen explants. The values followed by different letters for each variable are significantly
different at 5% probability level according to Duncan’s multiple range test (DMRT).

Table 3
Effect of different concentration of auxins (NAA, IBA and IAA) on in vitro rooting of shoots of D. lanata after eight weeks of culture.
PGRs Concentrations (μmol/l) Percentage response for root induction Root number ± S.E. Root length (cm) ± S.E.
Control. 0.0 ± 0.0 i 0.0 ± 0.0 i 0.0 ± 0.0 e

NAA 1.34 40.0 ± 5.7 def 3.8 ± 0.3def 1.5 ± 0.0 abc
2.68 35.0 ± 5.5 def 3.2 ± 0.2 defg 1.7 ± 0.2 ab
5.37 8.0 ± 8.0 hi 0.7 ± 0.7 hi 0.4 ± 0.4 de
10.74 13.3 ± 13.3 ghi 1.4 ± 1.4 ghi 0.6 ± 0.6 cde
16.11 13.3 ± 7.0 ghi 1.4 ± 0.7 ghi 1.2 ± 0.8 bcd

IBA 1.22 35.0 ± 13.2 def 3.4 ± 1.1 def 2.2 ± 0.4 ab
2.45 38.3 ± 4.4 def 3.7 ± 0.2 def 1.9 ± 0.2 ab
4.90 51.6 ± 11.6 cde 4.8 ± 1.0 cde 2.0 ± 0.1 ab
9.80 28.3 ± 3.3 fg 3.9 ± 0.3 def 1.8 ± 0.3 ab
14.70 11.6 ± 1.6 ghi 1.8 ± 0.2 ghi 2.4 ± 0.3 a

IAA 1.42 25.0 ± 5.7 fgh 2.3 ± 0.2 efgh 1.8 ± 0.2 ab
2.85 51.6 ± 3.3 cde 3.6 ± 0.3 def 1.9 ± 0.1 ab
5.71 61.6 ± 3.3 bc 5.4 ± 0.3 cd 2.2 ± 0.0 ab
11.42 80.0 ± 5.0 ab 6.5 ± 0.2 bc 2.3 ± 0.0 ab
17.13 81.6 ± 1.6 a 13.9 ± 1.1 a 2.1 ± 0.2 ab
22.84 68.0 ± 0.0 abc 12.4 ± 0.9 a 2.3 ± 0.1 ab
28.55 53.3 ± 2.6 cd 12.0 ± 0.9 a 2.2 ± 0.2 ab
34.26 32.0 ± 4.0 efg 8.6 ± 1.3 b 2.2 ± 0.2 ab
28.55 53.3 ± 2.6 cd 12.0 ± 0.9 a 2.2 ± 0.2 ab
34.26 32.0 ± 4.0 efg 8.6 ± 1.3 b 2.2 ± 0.2 ab

Data represent mean ± SE of three repeated experiments, each with fourteen explants. The values followed by different letters for each variable are significantly
different at 5% probability level according to Duncan’s multiple range test (DMRT).

Auxins are reported to be helpful for initiation of lateral roots and
growth of root primordium (Fukaki and Tasaka, 2009). IAA, IBA and
NAA all successfully induce adventitious roots in present study. Among
the three auxins used for root induction, at higher concentrations IAA
was found to be more effective than IBA and NAA. Both IAA and IBA
gave somewhat similar results for root induction. The roots grew vig-
orously and consistently in the media containing IAA and IBA. Hence,
both IAA and IBA can be used for root induction in D. lanata. Similar
observation was made by Singh and Shetty (2012) in Jatropha curcas.
Plantlets produced in the present study were morphologically
identical to the mother plants from which the explants were collected,
as expected when the shoot regeneration is through direct
organogenesis. Our modification of Pellati et al. (2009) protocol was
found to be a simple and quick extraction procedure for elution of di-
goxin and digitoxin. In the present study we could detect and quantify
digoxin and digitoxin in D. lanata by modified method. Whereas
Yücesan et al. (2014) in Digitalis lamarckii and Mohammed et al. (2015)
in D. cariensis could not detect and quantify digoxin and digitoxin, may
be because low amount of plant material used for analysis or lack of
enzymatic regulation in the cardenolide formation in cultured leaves.
The modified method we have employed may be useful in other Digitalis
species as well.

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Fig. 2. HPLC-DAD chromatograms for detection and quantification of digoxin and digitoxin from leaves of in vitro grown plants in comparison with that from leaves
of naturally grown plant. a-Chromatogram for standard peaks; b-Chromatogram for leaves from in vitro grown plant; c-Chromatogram for leaves from naturally
grown plant.

Table 4 regeneration without callus formation has resulted in true to type

Quantification of digoxin and digitoxin in leaves from in vivo grown and in vitro plantlets and ruled out possibility of somaclonal variations. The method
grown plants of D. lanata using HPLC method. standardized will be of great use in mass propagation of D. lanata by
Source of leaf Total amount of Digoxin Total amount of Digitoxin Micropropagation industry, facilitating its large scale commercial cul-
material (μg/gm D.W.) (μg/gm D.W.) tivation without depending on seeds. This regeneration system can also
be used for the in vitro production of biomass from which pharmaceu-
In vivo grown plant 36.1 ± 0.1 8.7 ± 0.0
tically active biomolecules such as digitoxin and digoxin can be ex-
In vitro grown plant 35.8 ± 0.0 10.0 ± 0.0
tracted. The method will be useful in regenerating transgenic plants
Data represent mean ± SD of three replicates. from any transformation events.

5. Conclusions
Conflict of interest
Here we report an efficient in vitro propagation method for D. lanata
through direct organogenesis from leaf and petiole explants. Direct The authors declare that there are no conflicts of interest.

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B.P. Bhusare et al. Industrial Crops & Products 121 (2018) 313–319

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