In vitro propagation from rhizomes, molecular evaluation and podophyllotoxin production in Himalayan May Apple (Sinopodophyllum hexandrum Royle T.S. Ying): an endangered medicinal plant

Abstract

The present study reports an optimized single step protocol for high frequency in vitro propagation through rhizome buds, molecular analysis and podophyllotoxin production in Sinopodophyllum hexandrum Royle T.S. Ying. Maximum in vitro establishment of buds (96%) was achieved on 0.2 mg/L BA and 0.1 mg/L GA3. Highest multiplication rate (1:4) was achieved on MS medium fortified with 2.0 mg/L BA and 0.2 mg/L NAA after 5th sub-culture. Shoots with swollen rhizomes when transferred for hardening to pots containing sand: soil: FYM (1:1:1) showed 100% survival after 1 month. Molecular analysis using SCoT markers resulted in 148 amplicons of 100–3000 bp and depicted genetic diversity between experimental plants procured from Kullu, Kinnaur and Lahaul-Spiti, whereas showed 99–100% similarity between mother plants and their tissue culture raised progeny. For podophyllotoxin production, callus was induced from in vivo leaves and petioles as well as in vitro roots and leaves of different experimental sites. Highest callus induction from in vivo and in vitro explants was achieved on MS and B5 medium under dark incubation. HPLC analysis revealed maximum podophyllotoxin production from in vitro roots (3.123% w/w) and its calli (0.267% w/w), initiated from experimental plants of district Kinnaur with 3% glucose in production medium. Elicitation of in vitro root calli of Kullu district with methyl jasmonate resulted in enhancing podophyllotoxin production (0.33% w/w).

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Vol.:(0123456789)
1 3
Plant Cell, Tissue and Organ Culture (PCTOC)
https://doi.org/10.1007/s11240-022-02288-3
ORIGINAL ARTICLE
In vitro propagation fromrhizomes, molecular evaluation
andpodophyllotoxin production inHimalayan May Apple
(Sinopodophyllum hexandrum Royle T.S. Ying): anendangered
medicinal plant
NehaSharma1· ManishaThakur1 · PoojaSharma1· YashPalSharma2· BhupenderDutt2
Received: 2 November 2021 / Accepted: 15 March 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022
Abstract
The present study reports an optimized single step protocol for high frequency invitro propagation through rhizome buds,
molecular analysis and podophyllotoxin production in Sinopodophyllum hexandrum Royle T.S. Ying. Maximum invitro
establishment of buds (96%) was achieved on 0.2mg/L BA and 0.1mg/L GA3. Highest multiplication rate (1:4) was achieved
on MS medium fortified with 2.0mg/L BA and 0.2mg/L NAA after 5th sub-culture. Shoots with swollen rhizomes when
transferred for hardening to pots containing sand: soil: FYM (1:1:1) showed 100% survival after 1 month. Molecular analysis
using SCoT markers resulted in 148 amplicons of 100–3000bp and depicted genetic diversity between experimental plants
procured from Kullu, Kinnaur and Lahaul-Spiti, whereas showed 99–100% similarity between mother plants and their tis-
sue culture raised progeny. For podophyllotoxin production, callus was induced from invivo leaves and petioles as well
as invitro roots and leaves of different experimental sites. Highest callus induction from invivo and invitro explants was
achieved on MS and B5 medium under dark incubation. HPLC analysis revealed maximum podophyllotoxin production
from invitro roots (3.123% w/w) and its calli (0.267% w/w), initiated from experimental plants of district Kinnaur with 3%
glucose in production medium. Elicitation of invitro root calli of Kullu district with methyl jasmonate resulted in enhancing
podophyllotoxin production (0.33% w/w).
Key message
The present study reports single step invitro propagation protocol, molecular diversity and fidelity of mother plants and
regenerants and podophyllotoxin production in Sinopodophyllum hexandrum Royle T.S. Ying.
Keywords In vitro propagation· Sinopodophyllum hexandrum· Podophyllotoxin· SCoT markers· Callus· Suspension
cultures
Introduction
Sinopodophyllum hexandrum Royle T.S. Ying (Himalayan
May Apple; Bankakri) previously known as Podophyllum
hexandrum Royle (http:// www. world flora online. org/), an
endangered perennial herb is one of the highly valued
medicinal plant belonging to the family Berberidaceae.
It is an important Himalayan herb distributed in China,
North India, Nepal, Bhutan, Pakistan and Afghanistan. In
India, it is mostly found in alpine Himalayas of Jammu and
Kashmir (3500–4000m), Himachal Pradesh, Uttarakhand,
Sikkim and Arunachal Pradesh (Qazi etal. 2011). The rhi-
zome and roots of S. hexandrum contains predominating
Communicated by Christophe Hano.
* Manisha Thakur
drmanisha72@yahoo.com
1 Department ofBiotechnology, Dr YS Parmar University
ofHorticulture andForestry, Nauni-Solan, H.P173230,
India
2 Department ofForest Products, Dr YS Parmar University
ofHorticulture andForestry, Nauni-Solan, H.P173230,
India

Plant Cell, Tissue and Organ Culture (PCTOC)
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active metabolite called podophyllotoxin which belongs
to diarylnapthelene group of lignans and serve as a start-
ing precursor for the synthesis of anti-tumourdrugs, eti-
poside (VP-16-213) and teniposide (VM-26) used for
the treatment of lung and testicular cancer by inhibiting
topoisomerase II (Issell etal. 1984; Jackson and Dewick
1984; Canel etal. 2000; Farkya etal. 2004; Chaurasia
etal. 2012). Podophyllotoxin inhibits the microtubule
assembly formation during cell division leading to cyto-
toxicity (Holthuis 1988). Podophyllotoxin derivatives are
also known to exhibit various pharmacological properties
such as antiviral, antimicrobial, antidote, antihelminthic,
purgative, radioprotective and help in the treatment of
rheumatoid arthritis and leukemia (Castro etal. 2004).
Podophyllotoxin is in high demand around the world due
to its many desirable properties and low availability from
natural sources which limits the pharamaceutical firms to
produce beneficial drugs (Canel etal. 2000).
Under natural conditions, S. hexandrum may regenerate
from seeds and rhizomes but only the summer months are
congenial for its growth.Apart from this, blooming and fruit-
ing are irregular and seed germination is inconsistent, often
taking few months to several years for producing seedlings.
Plant grows slowly and removal of its roots and rhizomes for
podophyllotoxin extraction does not allow regeneration of
plants in situ. Besides this, agricultural propagation would
only yield a commercially viable harvest once every five to
six years. Therefore, the plant parts tend to be collected from
wild stands. Poor regeneration and unscrupulous harvesting
of S. hexandrum has resulted in making this plant scarce,
consequently it has been affirmed as endangered medicinal
plant by IUCN (IUCN 2020) and listed in Red Data Book as
well as CITES (2017) requiring good natural regeneration as
well as rapid and effective conservation.
Advances in plant tissue culture have opened a new plat-
form for quick and efficient way to produce large number
of clonally uniform plants in a limited amount of time and
space. Cell and organ culture also provides an alternative
and practical option for the large scale production of specific
phytochemicals in large quantities regardless of fluctuating
environmental conditions (Lila2005), making biotechnolog-
ical podophyllotoxin production from S. hexandrum using
plant cell culture a viable option.
Though invitro propagation of S. hexandrum has been
reported through seeds (Chattopadhyay etal. 2002; Li etal.
2009; Deb etal. 2018; Ahmad etal. 2021), callus (Heyenga
etal. 1990; Sultan etal. 2006) and somatic embryos (Rajesh
etal. 2014a, b) but there are few reports on organogenesis
from rhizome explants (Chakraborty etal. 2010) involving
an additional passage for invitro rooting. Keeping in view
the above facts, we report an effective single-step invitro
propagation protocol from rhizome buds, analysis of regen-
erants at the molecular level and podophyllotoxin production
in cell lines of S. hexandrum induced from different altitudi-
nal locations of Himachal Pradesh (India).
Materials andmethods
Experimental plant material
Plant material of S. hexandrum was collected from three
different locations of Himachal Pradesh with altitudinal
variations i.e.Kullu (Manali-Gulaba), Kinnaur (Chotkanda-
Nichar) and Lahaul-Spiti (Lahaul-Kardang) (Fig.1). The
identification was done in herbarium section of Department
of Forest Products, Dr YS Parmar University of Horticulture
and Forestry, Nauni, Solan (H.P.) with reference number
13,901. They were stored inside the glasshouse in earthen
pots containing garden soil. Leaves, petioles and sprouting
buds obtained from the stored rhizomes were used for estab-
lishment of invitro shoot and callus cultures.
Surface sterilization andculture conditions
The rhizome buds were treated with 0.2% carbendazim
(Bavistin BASF) for 30min followed by treatment with
mercuric chloride (0.2%) for 3min and 3–4 washings in
sterilized distilled water. pH of the medium was adjusted to
5.8 ± 0.2 before autoclaving at 15 lbs/inch2 for 15–20min.
The cultures were incubated at a temperature of 25 ± 2°C
with 16h photoperiod at a light intensity of 50–60 µmol/(m
s) provided by 40W cool white fluorescent tubes (Philips,
India). Cultures were sub-cultured after 4 weeks to fresh
medium and observations were recorded.
In vitro shoot induction andmultiplication
Sterilized rhizome buds were cultured on MS medium
(Murashige and Skoog 1962) with different concentration
and combination of growth regulators viz., BA, GA3 and
IAA for shoot proliferation and sub-cultured further on MS
medium containing BA, KIN, GA3, IAA and NAA for mul-
tiplication. The rate of multiplication, length and quality of
shoots were recorded at an interval of 4 weeks to determine
the best growth regulator combination for shoot growth and
rhizome development.
Hardening ofinvitro plantlets
Shoots with swollen rhizome were carefully separated from
shoot clumps, removed from culture vessels, gently rinsed
under running tap water to remove the traces of medium
adhering to them and further treated with 0.5% carbendazim
for 30min. Transplanting was done in small plastic pots
filled with soil: sand: FYM (1:1:1). High relative humidity

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was maintained by covering the plantlets with glass-jars
and survival as well as growth of plants was recorded every
week. After 1 month, the hardened plants were transplanted
to bigger pots containing garden soil.
Molecular analysis
DNA isolation andquantification
Genomic DNA was isolated from 200mg tender leaves as
described by Doyle and Doyle (1987) with modifications
and DNA pellet was dissolved in TE (Tris-EDTA) buffer.
DNA samples were digested with RNase to remove RNA
impurities. Quantification of DNA was done in nanodrop
(Eppendorf) and quality was assessed on 0.8% agarose gel
stained with ethidium bromide.
PCR amplification
Amplification of DNA was done using thermal cycler (Bio-
RAD) with 36 SCoT primers in 10 µL aliquot containing
1U/µL of Taq DNA polymerase, 10 mM dNTPs, 10X PCR
buffer containing 1.5 mM MgCl2, 13 pmol primer (Banga-
lore GeNei™) and template DNA (25 ng). PCR amplifica-
tion was performed under following temperature profiles:
Initial denaturation (94°C for 3min), 40 cycles of denatura-
tion (94°C for 1min), annealing (varied with Tm ± 5°Cof
primer) for 1min and extension (72°C for 2min). Final
extension was done at 72°C (5min) and a hold at 4°C.
Electrophoresis ofamplified DNA
Amplified DNA products were analyzed on 1.5% agarose gel
containing 3 µL/100 mL ethidium bromide using 1X TAE
as gel and tray buffer. 3 µL of ×6 loading dye was added to
each PCR tube and 13 µL of sample was loaded in each well.
Medium range ruler was loaded along with PCR amplified
products to assess the size of amplified products. Imaging
of amplified products was done through gel documentation
system (Biovis).
Banding profile analysis
Amplicons produced from SCoT markers were evaluated
for similarities/dissimilarities. Gel images were trans-
formed into binary matrix considering clear and reproduc-
ible bands. Polymorphic information content (PIC value)
for each primer was calculated as described by Anderson
etal. (1993) using the formula PIC = 1 –
∑pi
2, where piis
the frequency of the ith allele for marker (Smith etal. 1997).
The power of each primer to differentiate between the geno-
types was evaluated by resolving power (Rp) (Prevost and
Wilkinson, 1999) using formula Rp=
∑Ib
, where Ib is band
informativeness that has value of : 1−[2 × (0.5-p)], where p
Fig. 1 Sites for collection of experimental material of Sinopodophyllum hexandrum Royle

Plant Cell, Tissue and Organ Culture (PCTOC)
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is the proportion of Sinopodophyllum genotypes containing
the band. Effective multiplex ratio (EMR) was determined
using formula; EMR= nβ, where ‘n’ is the multiplex ratio
measured as the average number of DNA fragments ampli-
fied per genotype and ‘β’ is the fraction of polymorphic
markers and is estimated as β = PB/(PB + MB), indicating
‘PB’ as no. of polymorphic loci and ‘MB’ as monomorphic
loci. Marker index (MI) was also calculated as a product of
PIC and EMR (Varshney etal. 2007). A pair wise similarity
index was constructed by Jaccard coefficients (Jaccard 1908)
which were further consigned to UPGMA cluster analysis
and a dendrogram was constructed using NTSYS-pc soft-
ware (Numerical taxonomy system, applied biostatistics,
Inc., New York, USA) ver. 2.21a (Rohlf 1998).
Podophyllotoxin production
Callus induction andmultiplication
Leaf and petioles from invivo plants and roots and leaves
from invitro plants were used as explants for callus initia-
tion. The invivo explants were surface sterilized with 0.1%
HgCl2 for 2min followed by 3–4 washings with sterile dis-
tilled water. The explants were inoculated on MS and B5
(Gamborg etal. 1968) nutrient media supplemented with dif-
ferent concentration and combination of growth regulators
viz., BA, KIN, NAA, 2,4-D and IAA and incubated under
dark and light conditions inside the culture room for callus
induction and multiplication.
Production ofpodophyllotoxin
Two stage culture system was adopted for the production of
podophyllotoxin in the medium. Friable calli derived from
different explants was cultured on MS and B5 broth con-
taining similar concentration of growth regulators used for
callus maintenance. 500mg callus was inoculated in 150
mL conical flasks containing 70 mL of MS and B5 broth
and incubated under dark at 100rpm on gyratory shaker
at 22 ± 2°C. Cells from exponential phase were thereafter
inoculated at 10% inoculum density in 150 mL conical flasks
containing 40 mL of production medium (Half strength MS
and B5 devoid of sucrose containing half the concentra-
tion of growth regulators used for callus maintenance) and
incubated on a shaker at 22 ± 2°C under dark conditions.
The fresh weight was recorded by harvesting the cells at an
interval of 7 days.
Effect ofcarbon source
The effect of different carbon sources i.e. sucrose, fructose
and glucose (3.0%) on podophyllotoxin production was
studied by incorporating them into the MS broth containing
half the concentration of growth regulators used for callus
maintenance. Incubation was done at 22 ± 2°C on orbital
shaker incubator at 100rpm under dark and their effect on
podophyllotoxin production was recorded. Callus inoculated
on production broth was taken as control and all the experi-
ments were repeated thrice.
HPLC analysis
Well dried cell mass (500mg) was powdered and extraction
was carried out through reflux method by heating over water
bath with methanol (50 mL) at 60°C for 30min, with con-
stant stirring which helped in leaching out the lignans from
the cells. Well dried extracted samples were then diluted
with mobile phase (9 mL methanol: 1 mL water), centrifuged
at 3500rpm followed by filtration through 0.2μm membrane
prior to injecting (20µL) into HPLC column Sunfire C-18
(4.6 × 250mm, 5μm). Analysis was performed at 280nm
by using methanol : water (62:38mL ) as mobile phase, at a
flow rate of 0.9mL/min for 22min at 28°C (Sharma etal.
2018). Commercially available podophyllotoxin (0.1mg/L,
Sigma-Aldrich Lot No. SLBV-2323) was used as standard
for calculating the podophyllotoxin content in the sample
based on the total area of the peak obtained from an integra-
tor and estimated through regression equation.
Effect ofelicitors
For studying the effect of elicitors on podophyllotoxin pro-
duction, callus was inoculated on production medium con-
taining methyl jasmonate and chitin (0.5–1.5mM) followed
by incubation under similar culture conditions.
Statistical analysis
The data for the various parameters was subjected to com-
pletely randomized design (CRD) (Cochran and Cox 1963;
Gomez and Gomez 1984). The analysis of variance approach
was used for CRD statistical analysis based on mean values
per treatment.
Results
In vitro propagation
Establishment ofrhizome buds
In vitro shoot proliferation through direct organogene-
sis was observed when surface sterilized rhizome buds
were inoculated on MS medium supplemented with dif-
ferent concentrations of BA, GA3 and IAA in culture
tubes. Maximum bud proliferation (96%) was observed

Plant Cell, Tissue and Organ Culture (PCTOC)
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in 0.2mg/L BA and 0.1mg/L GA3 supplemented medium
after 2 weeks with an average of 5.1 ± 0.04 shoots having
6.3 ± 0.03cm shoot length (Table1; Fig.2). Chakraborty
etal. (2010) reported invitro regeneration of S. hexan-
drum from rhizome buds on medium containing BA and
NAA.
Shoot multiplication
Well proliferating shoots were cultured on MS medium
supplemented with different growth regulator combina-
tions for multiplication. It was observed that shoot multi-
plication rate varied in all the growth regulator combina-
tions tested. Highest rate of shoot multiplication (1:4) was
observed on MS medium supplemented with 2.0mg/L BA
and 0.2mg/L NAA after 5th sub-culture (Table2; Fig.2).
Fig. 2 In vitro propagation from
rhizomes a intact rhizome; b
excised buds; c invitro sprout-
ing (after 14 days) & d shoot
elongation (after 6 weeks) on
MS + BA(0.2mg/L) + GA3(0.1
mg/L); e shoot multiplication on
MS + BA(2.0mg/L) + NAA(0.2
mg/L); Hardened plants after f 3
days; g 1 month and h 3 months
of transfer
Table 1 Effect of different
concentration and combination
of growth regulators on
invitro shoot proliferation in S.
hexandrum Royle
Sr. no Concentration of
plant growth regu-
lators (mg/L)
No. of days required
for shoot initiation
Regeneration
frequency (%)
Average number
of shoots ± SE
Average shoot
length(cm) ± SE
BA GA3IAA
Control – – – – – –
1 0.1 0.1 – 16 89 2.7 ± 0.09 5.1 ± 0.04
2. 0.2 0.1 – 14 96 5.1 ± 0.04 6.3 ± 0.03
3. 0.3 0.1 – 18 72 3.2 ± 0.04 5.1 ± 0.04
4. 0.4 0.1 – 19 69 2.5 ± 0.08 5.1 ± 0.04
5. 0.5 0.1 – 20 67 3.3 ± 0.04 4.7 ± 0.05
6. 0.5 0.1 0.01 21 59 2.8 ± 0.12 5.1 ± 0.02
7. 0.5 0.2 0.02 17 57 4.0 ± 0.07 4.2 ± 0.03
8. 0.5 0.3 0.03 19 68 3.0 ± 0.07 5.1 ± 0.04
9. 0.5 0.4 0.04 16 62 2.6 ± 0.10 2.7 ± 0.04
10. 1.0 0.5 0.05 17 58 2.8 ± 0.04 3.1 ± 0.05
C.D 0.206 0.103
SE (m) 0.07 0.035
SE (d) 0.099 0.049
C.V 6.223 2.688

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IAA has been reported to be an important growth regulator
for invitro shoot multiplication of S. hexandrum alone or
in combination with BA (Arumugam 1990; Nadeem etal.
2000; Chakraborty etal. 2010). But in our studies, NAA
in combination with BA yielded better results.The regener-
ated shoots were healthy with swollen rhizomes. With each
shoot multiplication passage the pre-existing rhizomes at the
shoot-base increased in size with emergence of 1–2 roots,
hence the shoots were not subjected to additional invitro
rooting passage. On an average, four plants per rhizome were
produced which were further hardened. Contrary to our find-
ings, invitro shoots of P. hexandrum were transferred to
medium containing IBA or IAA for rooting (Chakraborty
etal. 2010; Guo etal. 2012).
Hardening
The hardening of invitro shoots was done gradually to accli-
matize them with outdoor environmental conditions.Well-
developed shoots separated from shoot clumps along with
rhizome planted in pots containing soil: sand: FYM (1:1:1)
showed 100% survival after 1 month. Fully hardened plants
were established in bigger pots containing garden soil after
2 months (Fig.2). Therefore, rhizomes can be used as a good
source material for fast propagation of S. hexandrum.
Molecular analysis
In the present study, gene targeted SCoT marker was used to
assess the genetic diversity among the S. hexandrum geno-
types collected from three different districts of Himachal
Pradesh and to evaluate genetic stability between mother
plants and their invitro raised progenies. SCoT is a simple
and reliable dominant marker linked with initiation codon
and based on the coding regions of the genome (Bhattacha-
ryya etal. 2014, 2017). High level of genetic polymorphism
and fidelity could be detected due to the multi-locus nature
and higher resolution power of this marker. Apart from prov-
ing highly effective in assessment of clonal fidelity between
mother plants and its tissue culture raised progeny, SCoT
markers indicated genetic variability in plant material of S.
hexandrum collected from different altitudes. Fourteen out
Table 2 Effect of growth regulators on invitro multiplication of shoots regenerated from rhizome buds
Sr. no Medium code MS medium supplemented with
(mg/L)
Shoot multiplica-
tion observed after
Multipli-
cation rate
Observations
BA Kin GA3 IAA NAA
1 Control – – – – – – – –
2 MM-1 0.5 – – – 0.1 – 1:1 No shoot multiplication
3 MM-2 1.0 – – – 0.1 – 1:1 No shoot multiplication
4 MM-3 1.5 – – – 0.1 – 1:1 No shoot multiplication
5 MM-4 2.0 – – – 0.1 6th subculture 1:2 Multiplication with healthy shoots and leaves
6 MM-5 0.5 – – – 0.2 6th subculture 1:2 Multiplication and healthy shoots and leaves
7 MM-6 1.0 – – – 0.2 6th subculture 1:3 Multiplication with healthy shoots and leaves
8 MM-7 1.5 – – – 0.2 6th subculture 1:2 Multiplication with healthy shoots and leaves
9 MM-8 2.0 – – – 0.2 5th subculture 1:4 Multiplication with healthy shoots and leaves
10 MM-9 0.5 – – 0.1 – 6th subculture 1:2 Multiplication with healthy shoots and leaves
11 MM-10 1.0 – – 0.1 – 6th subculture 1:2 Multiplication with healthy shoots and leaves
12 MM-11 1.5 – – 0.1 – 6th subculture 1:2 Multiplication with healthy shoots and leaves
13 MM-12 2.0 – – 0.1 – 6th subculture 1:2 Multiplication with healthy shoots and leaves
14 MM-13 – 0.5 – – 0.1 6th subculture 1:2 Multiplication with healthy shoots and leaves
15 MM-14 – 1.0 – – 0.1 6th subculture 1:3 Multiplication with healthy shoots and leaves
16 MM-15 – 1.5 – – 0.1 – 1:1 No shoot multiplication
17 MM-16 – 2.0 – – 0.1 – 1:1 No shoot multiplication
18 MM-17 – 0.5 – 0.1 – – 1:1 No shoot multiplication
19 MM-18 – 1.0 – 0.1 – – 1:! No shoot multiplication
20 MM-19 – 1.5 – 0.1 – – 1:1 No shoot multiplication
21 MM-20 – 2.0 – 0.1 – – 1:1 No shoot multiplication
22 MM-21 0.5 – 0.1 – 0.5 – 1:1 No shoot multiplication
23 MM22 1.0 – 0.1 – 1.0 – 1:! No shoot multiplication
24 MM-23 1.5 – 0.1 – 1.5 – 1:1 No shoot multiplication
25 MM-24 2.0 – 0.1 – 2.0 – 1:1 No shoot multiplication

Plant Cell, Tissue and Organ Culture (PCTOC)
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of 36 SCoT primers were able to amplify the genomic DNA
resulting in 148 clear and reproducible amplicons of size
range 100–3000bp (Fig.3). Highest PIC value (0.44) and
EMR (10.00) was recorded in SCoT-8 indicating equal dis-
tribution of this marker in a population and its suitability in
studying genetic relationship (Table3). The UPGMA den-
drogram based on Jaccard coefficient divided the tested sam-
ples into three clusters wherein, experimental material from
Kullu and Lahaul-Spiti showed similarity of 62% whereas,
plants from Kullu, Kinnaur and Lahaul-Spiti depicted 45%
similarity, thus showing variability between the accessions
of S. hexandrum collected from different altitudes. How-
ever, 99–100% similarity was recorded between mother
plants and its tissue culture raised progenies, thus advocat-
ing tissue culture propagation for the production of ‘true to
type’ planting material (Fig.4). SCoT markers have been
successfully applied in studying genetic diversity as well
as fidelity of medicinal orchids (Bhattacharyya etal. 2013)
Fig. 3 DNA fingerprinting pattern generated with SCoT primers a SCoT-5 and b SCoT-19. Medium range ladder (L); Mother plants (A,C & E)
and tissue culture propagated progeny (B, D & F) from Kullu, Kinnaur and Lahaul-Spiti, respectively
Fig. 4 Dendrogram obtained using Jaccard coefficient of mother plants (MP) and tissue culture propagated (TC) plants collected from three dif-
ferent altitudes of Himachal Pradesh

Plant Cell, Tissue and Organ Culture (PCTOC)
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and plants such as Giloe (Paliwal etal. 2013), Pittosporum
eriocarpum (Thakur etal. 2016), Bletilla striata (Guo etal.
2018), Bauhinia recemosa (Sharma etal. 2019), Rauwolfia
teteraphylla (Rohela etal. 2019) and Ginger (Sharma and
Thakur 2021).
Podophyllotoxin production
Callus induction
Leaf and petiole segments from invivo plants as well as
root and leaf segments from invitro shoots were used for
callus initiation. Out of different combination and concentra-
tion of growth regulators used, highest callus induction was
observed in invivo leaves (81.07%) and petioles (87.19%)
on MS medium supplemented with 0.1mg/L BA, 2.0mg/L
NAA and 0.1mg/L BA, 1.5mg/L NAA under dark incu-
bation, respectively. Similarly, highest callus induction in
invitro roots (90.67%) and leaves (89.20%) was observed
on B5 medium containing 0.5mg/L KIN, 0.1mg/L 2,4-D
and 0.5mg/L KIN, 2.0mg/L 2,4-D mg/L, respectively under
dark incubation (Table4; Fig.5). No callus was observed
in the explants incubated under light conditions. In their
studies on cell lines of P. hexandrum Anrini and Jha (2009)
initiated callus cultures from juvenile and mature explants
on half strength B5 and MS medium containing growth
regulatorsand characterized them for different morphologi-
cal and biochemical parameters including podophyllotoxin
content. Highest callus response has been recently reported
from leaves followed by stem on MS medium containing
NAA and BA (Zuhra etal. 2021) which is not in analogy
to our findings. Similar to our reports, Ahmad etal. (2007,
2021) initiated callus formation from seed embryos on BA
(2.5 µM) and NAA (0.5 µM) and reported their association
in formation of friable callus ideal for lignans production.
However, contrary to our findings, lower concentrations
of BA, 2,4-D and NAA have proved to yield good callus
induction after dark incubation in P. hexandrum (Sultan
etal. 2006; Chakraborty etal. 2010). Enhanced podophyl-
lotoxin production has also been reported from cell suspen-
sion cultures of P. hexandrum after feeding coniferyl alco-
hol as β-cyclodextrin complex (Woerdenbag etal. 1990),
treatment with tryptophan (Majumder 2012) as well as from
invitro roots and roots regenerated from calli (Sagar and
Zafar 2005) on B5 and MS media.
Cell suspension culture
Cell growth and production of podophyllotoxin in the cell
suspension medium was found to be inversely related hence,
the calli inoculated in the growth medium failed to produce
podophyllotoxin whereas, the production medium supported
its synthesis. Two-stage culture medium was adopted in the
Table 3 List of primers, their sequences, size of amplified products, PIC, Rp, EMR and MI values recorded by SCoT primers
Primers Sequence (5′–3′) Tm (°C) GC (%) Length (bp) Scorable
bands
Monomor-
phic bands
Polymor-
phic bands
Size (bp) No. of alleles PIC Rp EMR MI
SCoT-3 CAA CAA TGG CTA CCA CCG 62.7 55.5 18 8 2 6 300–2000 27 0.31 54 4.50 1.39
SCoT-5 CAA CAA TGG CTA CCA CGA 60.8 50 18 9 3 6 200–2500 31 0.28 62 4.00 1.12
SCoT-6 CAA CAA TGG CTA CCA CGC 62.8 55.5 18 10 4 6 200–1700 42 0.27 84 3.60 0.98
SCoT-8 CAA CAA TGG CTA CCA CGT 59.6 50 18 10 0 10 200–2500 32 0.44 64 10.00 4.40
SCoT-9 CAA CAA TGG CTA CCA GCA 60.9 50 18 11 1 10 100–1500 40 0.40 80 9.09 3.64
SCoT-10 CAA CAA TGG CTA CCA GCC 61.8 55.5 18 13 3 10 100–1500 48 0.34 96 7.69 2.62
SCoT-13 ACG ACA TGG CGA CCA TCG 68.6 61.1 18 12 3 9 250–300 50 0.33 100 6.75 2.22
SCoT-14 ACG ACA TGG CGA CCA CGC 71.1 66.6 18 9 4 5 200–3000 40 0.25 80 2.77 0.70
SCoT-19 ACC ATG GCT ACC ACC GGC 68.3 66.6 18 11 6 5 250–300 54 0.20 108 2.27 0.45
SCoT-20 ACC ATG GCT ACC ACC GCG 69.5 66.6 18 12 2 10 150–900 44 0.37 88 8.33 3.09
SCoT-21 ACG ACA TGG CGA CCC ACA 69.2 61.1 18 8 5 3 100–900 40 0.17 80 1.12 0.20
SCoT-22 AAC CAT GGC TAC CAC CAC 60.9 55.5 18 11 4 7 200–1500 52 0.33 104 4.45 1.47
SCoT-28 CCA TGG CTA CCA CCG CCA 70.6 66.6 18 13 4 9 100–1500 58 0.31 116 6.23 1.93
SCoT-32 CCA TGG CTA CCA CCG CAC 67.8 66.6 18 11 2 9 200–2000 42 0.36 84 7.36 2.70

Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
study as reported by Malik etal. (2008) for the production
of shikonin derivatives in Arnebia euchroma callus cultures.
Cell biomass formation in suspension cultures of P. hex-
andrum showed a quick growth after 7 days of inoculation
which was evaluated by taking fresh weight. A lag phase
of 7 days followed by log phase of 14 days and thereaf-
ter, a small stationary phase followed by deceleration was
recorded in the cell suspension cultures. The maximum aver-
age fresh weight 0.16g/week was recorded in invitro root
calli after 28 days of culture under dark incubation (Fig.6a).
Cells from exponential phase in growth medium were then
transferred to the production medium at 10% inoculum den-
sity and similar growth pattern was observed. There was
an increase in podophyllotoxin content till 28 days, but as
the culture period progressed, after a small stationary phase
decline in fresh weight was observed (Fig.6b). Appear-
ance of brown colour was observed in the broth after 14
days of incubation which further turned dark in subsequent
sub-cultures showing production of podophyllotoxin. Good
podophyllotoxin producing cultures are dark brown coloured
and when the colour changes to yellow green a complete loss
of podophyllotoxin production is reported (Uden etal. 1989;
Giri and Narasu 2000).
Standardization ofcarbon source
Carbon source plays a vital role in the synthesis of cell con-
stituents as a substrate and provide energy for cell growth
(Anbazhagan etal. 2010). Amongst three different carbon
sources tested i.e. sucrose, fructose and glucose at equal con-
centrations, it was observed that glucose was most suitable
for podophyllotoxin production. Maximum podophyllotoxin
content (0.251% w/w) was observed when 3% glucose was
incorporated in the production medium whereas its lowest
content was in suspension containing 3% fructose (Fig.7).
Glucose results in higher accumulation of podophyllotoxin
(Chattopadhyay etal. 2001, 2002, 2003) in comparison to
sucrose since sucrose is hydrolyzed to glucose and fructose
by cell wall invertase. Therefore, the slow consumption of
Fig. 5 Callus induction from
invivo a leaf; d leaf petiole and
invitro g leaf; j root; showing
initiation (b, e, h, k) and multi-
plication (c, f, i, l)

Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
sucrose may be because of low invertase activity (Martinez
etal. 1993).
HPLC analysis
In vitro roots of S. hexandrum cultures initiated from
experimental material of district Kinnaur depicted maxi-
mum (3.123% w/w) podophyllotoxin content in compari-
son to its induced calli (0.267% w/w). Similar trend was
observed in podophyllotoxin content in invitro roots of
Lahaul-Spiti (3.27% w/w) and Kullu (3.09% w/w) and
calli (0.258, 0.251% w/w) induced from them, respec-
tively (Table5; Fig.8). The content of podophyllotoxin in
invitro leaves, petioles and callus derived from them was
lower in comparison to roots. Podophyllotoxin (0.081%
w/w) was also observed in the root callus leached medium
initiated from plants of district Kinnaur, after fifth sub-
culture. Therefore, the present investigation reveals that
calli initiated from plants collected from higher altitudes
had higher podophyllotoxin content than from lower alti-
tudes, thus suggesting the effect of altitudinal variations in
its production. The levels of podophyllotoxin in P. hexan-
drum plants from higher altitudes were found to be higher
in comparison to its yield in plant material from lower
altitudes (Pandey etal. 2007, 2013; Sharma etal. 2019)
and its content is more in roots in comparison to leaves
(Pandey etal. 2007). The effect of altitude on metabolic
content has also been documented in various other medici-
nal plants (Demasi etal. 2018; Rana etal. 2020). High
podophyllotoxin accumulation has also been reported in
germinated somatic embryos and its calli of P. hexandrum
(Rajesh etal. 2014a, b).
Table 4 Effect of different concentration and combination of growth regulators on invitro callus induction in S. hexandrum using invitro root,
leaf and invivo leaf and petiole explants
Sr. no. MS + B5 medium supplemented with
(mg/L)
Regeneration frequency (%) Observations
BA Kin NAA 2,4D IAA MS medium B5 medium
In vivo leaf In vivo leaf
petiole
In vitro leaf In vitro root
Control – – – – – – – – – –
1 0.1 – 0.5 – – 65 65 70 70 White, nodular
2 0.1 – 1.0 – – 77 70 95 75 White, friable (used
for suspension
culture)
3 0.1 – 1.5 – – 75 75 90 75 White, compact
4 0.1 – 2.0 – – 75 70 95 80 White, comapct
5 0.1 – 2.5 – – 80 65 70 80 Creamy, compact
6 0.5 – – 0.5 – 85 95 75 95 Creamy, compact
7 0.5 – – 1.0 95 90 80 90 White, friable
8 1.0 – – 0.5 95 85 85 70 Creamy, fast growing
9 1.0 – – 1.0 – 80 70 80 80 Creamy, compact
10 1.0 – – – 0.5 65 70 85 85 White, nodular
11 0.5 – v – 1.0 70 80 80 85 White, nodular
12 1.0 – – – 0.5 75 85 85 80 Creamy, compact
13 1.0 – – – 1.0 76 80 75 80 White, nodular
14 – 0.5 – 0.1 0.1 75 85 70 85 Creamy, compact
15 – 0.5 – 0.2 0.1 77 80 75 80 White, nodular
16 – 0.5 – 0.5 0.1 70 75 70 85 Creamy, compact
17 – 0.5 – 1.0 0.1 75 70 70 90 White, compact
18 – 0.5 – 1.5 – 80 75 85 80 White, compact
19 – 0.5 – 2.0 – 80 75 80 85 Creamy, compact
20 – 0.5 – 2.5 – 75 70 85 80 White, compact
21 0.1 0.1 – – – 70 70 80 85 Creamy, compact
22 0.2 0.2 – – – 70 75 85 80 Creamy, compact
23 0.3 0.3 – – – 65 70 80 85 White, nodular
24 0.4 0.4 – – – 70 70 75 80 White, friable

Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
Effect ofelicitors
Elicitation is one of the strategies used in the plant cell
cultures to enhance the production of secondary metab-
olites (Namedo 2007). Elicitors activate plant natural
defense responses, thus increasing secondary metabolite
synthesis and accumulation. In the study, the effect of
methyl jasmonate and chitin was investigated in invitro
roots of S. hexandrum of district Kullu, as they showed
least podophyllotoxin content in comparison to other loca-
tions. So, exogenous elicitors were added to the production
medium for the enhancement of podophyllotoxin under
invitro conditions. Methyl jasmonate and chitin have been
reported to play an important role in signal transduction
processes and are involved in the regulation of defense
genes. In our study, for enhancing podophyllotoxin yield,
callus induced from invitro roots of S. hexandrum plants
of district Kullu were subjected to elicitation by incorpo-
rating different concentrations (0.5–1.5mM) of methyl
jasmonate and chitin in the production medium. Highest
podophyllotoxin content (0.33% w/w) was observed after
elicitation with 1mM methyl jasmonate followed by its
higher concentration of 1.5 mM (0.31% w/w). Chitin (1.5
mM) and methyl jasmonate (0.5mM) led to 0.29% w/w
podophyllotoxin accumulation (Fig.9) however, there
was no significant difference between these elicitors at
different concentrations. Methyl jasmonate has been pre-
viously used for enhancing podophyllotoxin accumula-
tion in Linum album and Podophyllum peltatum cell sus-
pension cultures (Furden etal. 2005; Anbazhagan etal.
2010). Bahabadi etal. (2011) also reported the enhanced
podophyllotoxin production in L. album cell cultures upon
elicitation with chitin and chitosan.
Fig. 6 Effect of days on growth of callus in growth (a) and production (b) medium

Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
Conclusions
In the present study, a single step protocol for invitro propa-
gation of S. hexandrum from rhizomes has been developed.
Molecular analysis of the regenerated plants predicted their
‘true to type’ nature whereas, genetic variation was observed
in experimental plants collected from Kullu, Kinnaur and
Lahaul-Spiti. Highest growth, biomass accumulation and
podophyllotoxin production was depicted in invitro roots
and its calli derived from plants procured from district Kin-
naur, thus signifying the importance of altitude in production
of this high valued metabolite.
0
0.05
0.1
0.15
0.2
0.25
0.3
Control
Sucrose 3.0
Fructose 3.0
Glucose 3.0
Control
Sucrose 3.0
Fructose 3.0
Glucose 3.0
Podophylltoxin (% w/w)
Carbon source (%)
Fig. 7 Effect of different carbon sources on percent podophyllotoxin
production after 35 days incubation
Fig. 8 Standard calibration curve of podophyllotoxin (a) and chromatograms of standard solution (b), invitro root calli of Kullu (c), Kinnaur
(d), Lahaul-Spiti (e)

Plant Cell, Tissue and Organ Culture (PCTOC)
1 3
Acknowledgements Authors are highly thankful to Chitralekha Bhard-
waj (analyst) Department of Forest Products, Dr YSP UHF, Nauni,
Solan (H.P) for HPLC assistance.
Author contributions NS, MT and PS were involved in designing
and execution of tissue culture and molecular research experiments,
contributed in writing and editing of the manuscript. YPS performed
HPLC analysis and BD helped in plant material procurement and
identification.
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
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