A high efficiency in vitro regeneration protocol and clonaluniformity analysis in Hypericum hookerianum Wight & Arn.,a lesser known plant of ethnomedicinal and economic importance

Reji Joseph Varghese1 • Anusha Bayyapureddy1 • Selvadoss Pradeep Pushparaj1 •

Sooriamuthu Seeni1

Received: 23 April 2015 / Accepted: 6 August 2015

� Botanical Society of Sao Paulo 2015

Abstract Hypericum hookerianum (Hypericaceae), a

critically endangered plant of Western Ghats, India, has

acquired significant importance due to its medicinal

implications and ornamental flowers. This species is under

severe anthropogenic pressure due to urbanization, tourism,

and plantation activities taking place in their natural

habitat. A clonal propagation strategy is standardized in

this species which offers an opportunity for a stable pro-

duction of active metabolites from this species as well as

their conservation. The study deals with the optimization of

axillary bud proliferation using nodal explants followed by

genetic stability analysis of regenerants. Maximum number

of shoots (3.66) was observed on the Murashige and Skoog

(MS) medium supplemented with Kinetin (2.325 lM) with

85 % shoot multiplication frequency. In vitro grown shoots

were rooted best in 1/2 MS medium supplemented with

indole-3-butyric acid (2.45 lM) with an average of

6.8 ± 0.79 roots/shoot and 95.5 % rooting frequency.

Plantlets were acclimatized best (90 %) in a mixture of

sterile sand and farmyard manure (3:1). Micropropagated

plants were subjected to random amplified polymorphic

DNA analysis and phytochemical analysis to confirm their

clonal stability. In RAPD analysis, 1032 amplicons were

collectively generated which were monomorphic and sim-

ilar to the mother plant. The comparable major phyto-

chemical constituents in regenerants and mother plants

together with genetic uniformity data obtained from RAPD

analysis confirmed clonal fidelity of the regenerants.

Findings in this study are the first report on micropropa-

gation and assessment of genetic stability of micropropa-

gated plantlets in H. hookerianum which suggests that

in vitro axillary shoot proliferation can effectively be used

as a tool for propagation and conservation of H.

hookerianum.

Keywords Clonal uniformity � Hypericum hookerianum �Micropropagation � Random amplified polymorphic DNA

Introduction

The genus Hypericum (Hypericaceae) consists of 484

species occurring as temperate and tropical highland herbs,

shrubs, and infrequent trees throughout the world (Crockett

and Robson 2011). Many of them are ethnomedicinal

plants offer opportunities for discovering drug molecules as

demonstrated in successful prospecting of anti-plasmodial

compounds in stem bark extracts of Hypericum lanceola-

tum Lam. in Cameroon (Zofou et al. 2011). Hypericum

perforatum Linn. (St. John’s Wort), a native herb of Eur-

asia recognized by the pharmaceutical industry as a source

of hypericin, hyperforin, and flavonoids, and hence become

a sought-after species in Europe as an effective herbal

alternative to classic synthetic antidepressants to treat mild

to moderate depression. Economic evaluation of St. John’s

Wort suggests it to be a cost-effective alternative to generic

antidepressants with reduced side effects and improved

outcomes (Solomon et al. 2013). As a top selling herbal

dietary supplement, its sale value in the USA is calculated

as 8.2 million dollars and it represented nearly 13 % of all

European herbal product sales in 2004, valued at more than

€ 70 million in Germany alone (Backer et al. 2006).

& Reji Joseph Varghese

jvreji@yahoo.com

1 Department of Biotechnology, Centre for Bioresource

Research and Development, Sathyabama University,

Chennai 600 119, India

123

Braz. J. Bot

DOI 10.1007/s40415-015-0201-7

An important factor in the pharmaceutical application of

a plant is the stable content of its active principles. Vari-

ations in medicinally bioactive ingredients within culti-

vated H. perforatum and batch to batch variations of the

products are not uncommon and are a cause of concern for

the pharmaceutical industry (Bagdonaite et al. 2010).

Variability of the species is attributed to several factors

including genotype, developmental stage of the donor

plant, local weather conditions, method used for plant

production (Zobayed and Saxena 2004), pollutants, and

insect infestations which invariably compromise product

quality. Controlled production of plants free from con-

taminating agents is essential for uniform preparations to

meet the increasingly stringent safety requirements of the

regulatory agencies (Bruni and Sacchetti 2009). All such

problems would be overcome and uniformity of the plant

products better achieved if shoot cultures (Pasqua et al.

2003; Kirakosyan et al. 2004; Karppinen 2010) and

micropropagated plants (Bacila et al. 2010) are used. In

fact, micropropagation allows controlled production of

uniform and pathogen-free plants favoring studies on

hypericin production (Savio et al. 2012).

A few genera such as Hypericum, Rhododendron,

Thalictrum etc., that inhabit sparsely in the peak of certain

mountain ranges ([2500 m) of Western Ghats and Hima-

layas with apparently no distribution in the intervening

plains are now depleting in an alarming rate due to

increased anthropogenic activities particularly in Munnar,

Nilgiris, and Palani hill ranges in south Western Ghats

(Radha et al. 2013). Furthermore, over harvesting of

Hypericum hookerianum Wight & Arn. from the wild, due

to the presence of their high-value active principles also

may be a reason for their reduction in population density in

their natural habitat. Perusal of literature reveals that

among an estimated 27 species of Hypericum reported

from India, Hypericum patulum Thunb. (Baruah et al.

2001) and H. perforatum (Goel et al. 2009) of the Hima-

layas and Hypericum mysorense Heyne. (Shilpashree and

Rai 2009) of southern India have been successfully

micropropagated. H. hookerianum, the Hooker’s Wort is a

lesser known round topped, evergreen shrub distributed in

Sikkim Himalaya, Khasi, and Jaintia hills of the Eastern

Himalayas and Palni and Nilgiri hills of the Western Ghats

in Southern India. Its distribution extends to pockets of

Bangladesh, Myanmar, China, and Thailand as well. Shoot

extracts of this species are used by the Toda tribe of the

Nilgiri hills and communities in the Himalayan region as

an antimicrobial agent to ward of skin infections and to

treat wounds, burns, and conditions like anxiety and

inflammation (The Wealth of India 1997; Mukherjee and

Suresh 2000; Vijayan et al. 2004). Pharmacological

screening revealed antimicrobial (Mukherjee et al. 2001),

antitumor (Dongre et al. 2008), antiviral (Vijayan et al.

2004), and antioxidant (Chandrashekhar et al. 2009)

properties of the species. Many of the earlier workers had

used horticultural collections for their investigations due to

non-availability of this species in natural forests. Micro-

propagation through plant tissue culture offers opportuni-

ties for the conservation of endemic, endangered, and

difficult to propagate plant taxa which are explained by

several studies (Wochok 1992). Hence, in this paper, we

have described for the first time a successful clonal mul-

tiplication of H. hookerianum using axillary shoot prolif-

eration in nodal explant cultures of a selected wild plant

and also have tested clonal fidelity among the plantlets and

with the mother plant using biochemical and molecular

tools.

Materials and methods

Plant material

Pale red colored terminal shoot cuttings were collected

from a healthy flowering plant of H. hookerianum growing

by the side of Pambar river in Pambar Shola forest segment

(510 MSL) Palni hills of the Western Ghats in June, 2014.

Culture initiation

Defoliated 6–8 cm shoot cuttings were first thoroughly

washed under running tap water for 15–20 min and then

treated with liquid detergent (Tween-20) for 5–10 min.

Later, these explants were washed with double distilled

water for 5 min. Subsequently, explants immersed in 6 %

(v/v) sodium hypochlorite solution for 5–15 min and

washed with sterile double distilled water for 3–4 times.

Eventually, the explants were treated with aqueous solution

of 0.1 % (w/v) HgCl2 for 5–10 min and rinsed with sterile

double distilled water for 3–5 times to remove traces of

mercuric chloride. The sterilized explants of 1.0–1.5 cm

were transferred (four nodes/flasks) on to 50 ml of MS

(Murashige and Skoog 1962) medium supplemented with

varying concentrations of individual cytokinins viz. kinetin

(KIN), 6-benzylaminopurine (BAP), thidiazuron (TDZ) or

combinations of auxins viz. 3-indoleacetic acid (IAA)/a-naphthaleneacetic acid (NAA) and cytokinin (Table 1) and

adjusted to pH 5.8 before solidification using 0.15 %

Gelzan. All plant growth regulators (PGRs) and GelzanTM

CM were procured from Sigma-Aldrich, St.Louis, USA

where as all other nutrients of the media were of analytical

reagent grade procured from Merck, Darmstadt, Germany.

After dispensing 50 ml aliquots into culture flask, the

medium was autoclaved at 121 �C and 1.1 kg/cm pressure

for 18 min. All cultures were incubated in a culture room

maintained at 24 ± 2 �C and 12 h photoperiod, and

J. V. Reji et al.

123

illumination at 50–60 lEm-2s-1 was provided by a bank

of daylight fluorescent tubes (Philips India Ltd, Mumbai).

Observations of percentage contamination, shoot initiation,

and shoot characters were recorded at weekly intervals.

Shoot multiplication

After 8 weeks of culture initiation, nodes of 0.8–1.0 cm

length dissected from the shoots initiated in presence of

KIN (2.325 lM) were subcultured for multiplication in

medium supplemented with KIN (2.325 lM) and combi-

nations of KIN (2.325 lM) with other auxins as the case

may be. Hormone-free basal medium served as control.

Caulogenic efficiency of the explants was determined from

the number of shoot-forming explants against the total

number of nodal explants inoculated after 6 weeks of

subculture. The number of shoots produced per node,

length of the shoots, and branching of the shoots, if any,

were assessed at weekly intervals, and optimal type and

concentration/combination of PGR(s) required for shoot

multiplication and growth were assessed. Repeated sub-

culture of the nodes was continued at 6-week intervals

through at least six cycles to observe any decline in the

shoot multiplication rate and any abnormality associated

with multiplication.

Rooting and ex vitro establishment of plantlets

Terminal shoot cuttings (3.5–4.5 cm) with and without

axillary branches were excised from the multiplied shoot

cultures and used for root initiation in half-strength MS

Gelzan (0.15 %) basal medium and medium supplemented

with individual concentrations of IAA (0.571–17.12 lM),

0.492–14.76 lM indole-3-butyric acid (IBA), and NAA

(0.537–16.11 lM). Four shoots implanted into 50 ml

medium in 250 ml culture flask were tested for number of

shoots initiated, and the increase in shoot and root length

were recorded at weekly intervals.

Well-developed plants with a minimum of three roots

were taken out carefully from the culture flasks and washed

in running tap water to remove agar from the roots. Those

plantlets were planted in root trainers containing a hard-

ening mixture consisting of autoclaved locally available

river sand and farmyard manure (3:1) and kept in a green

house for 6–8 weeks with controlled humidity (70 ± 5 %)

and temperature (25 ± 2 �C).

Table 1 Shoot initiation after 6 weeks in single nodal explants of field-grown flowering plants of Hypericum hookerianum raised in MS medium

supplemented with different concentrations of cytokinins, combinations of cytokinin (KIN) and auxins (IAA, NAA)

S. no Treatments (lM) Number of nodes

responded (%)

Number of

shoots/node

Shoot length

(cm)

Number of

nodes/shoot

Root/callus

1 0.0 24 (53.33) 1.75g 1.75h 4.12d –

2 KIN (0.465) 27 (60.00) 2.33f 2.22fg 4.44cd –

3 KIN (1.86) 24 (53.33) 3.00bcde 2.50def 4.75bc –

4 KIN (2.325) 27 (60.00) 3.66a 3.33a 5.55a –

5 KIN (2.79) 27 (60.00) 3.44ab 3.00abc 5.11ab –

6 KIN (4.65) 21 (46.66) 3.14bc 2.14fg 4.28cd –

7 KIN (9.30) 24 (53.33) 2.62def 1.25i 3.37e –

8 BAP (0.444) 21 (46.66) 2.57ef 1.85gh 3.57e –

9 BAP (1.776) 24 (53.33) 2.87cde 1.50hi 3.50e –

10 BAP (2.22) 24 (53.33) 3.00bcde 1.50hi 3.37e –

11 BAP (4.44) 21 (46.66) 2.85cde 1.14i 3.33e –

12 BAP (8.88) 21 (46.66) 2.28f 0.71j 3.04e –

13 TDZ (0.090) 24 (53.33) 2.75cdef 2.75cde 4.75bc –

14 TDZ (0.227) 24 (53.33) 3.12bcd 2.37ef 5.12ab –

15 TDZ (0.3178) 24 (53.33) 2.50ef 1.25i 3.45e –

16 TDZ (0.454) 27 (60.00) 1.77g 0.55j 3.37e –

17 KIN (2.325) ? IAA (0.943) 29 (64.44) 2.57ef 3.20ab 5.50a ?R

18 KIN (2.325) ? IAA (2.829) 21 (46.66) 2.28f 3.12abc 4.75bc ??R

19 KIN (2.325) ? NAA (1.074) 27 (60.00) 2.33f 2.85bcd 5.11ab ?C

20 KIN (2.325) ? NAA (2.148) 18 (40.00) 1.25g 1.35i 3.37e ???C

Number of ? signs indicates the degree of callusing (C) and rooting (R); - sign indicates no response

Mean values followed by the same letter are not significantly different (P B 0.05) according to ANOVA and Tukey’s multiple range test

A high efficiency in vitro regeneration protocol and clonal uniformity analysis in Hypericum…

123

Analysis and comparison of phytochemicals

in the regenerated plants

Hypericin synthesis in regenerated shoots as well as natural

plants was assessed by extracting and quantifying the same

by the UV spectrophotometry method described by Kop-

erdakova et al. (2007) using pure hypericin as standard

(Sigma-Aldrich, St. Louis, USA).

Total phenols, anthocyanins, and flavonoids were

extracted by homogenizing 500 mg fresh tender shoots of

wild plants and five randomly selected regenerants indi-

vidually in a glass mortar and pestle using 0.01 % acidified

methanol and 80 % aqueous methanol, respectively, and

individual extracts filtered through 200 lm nylon screen.

The residues were re-extracted once with the respective

solvent and the combined extracts of each sample were

centrifuged at 4000 RPM in a Kubota 6200 high-speed

centrifuge (Kubota Corporation, Tokyo, Japan). Super-

natants were collected and made up to 20 ml each with

respective solvent before measuring absorbance at 506,

510, and 700 nm for anthocyanins and 510 nm for flavo-

noids. Folin–Ciocalteu method was used to determine total

phenolic content of the sample (0.1 ml) as described by

Singleton and Rossi (1965). Calibration curve was pre-

pared at 765 nm using gallic acid at concentrations of

0.4–1.6 mM. Anthocyanins in acidified methanol extract

were estimated following the method of Wrolstad et al.

(1990). Flavonoids in the methanol extracts were estimated

by aluminum chloride method described by Zhishen et al.

(1999). All the values were expressed on fresh weight basis

and each value represents mean ± SD of five different

determinations.

Assessment of genetic uniformity using RAPD

Twenty-four DNA samples from random-selected regen-

erated plants along with the mother plant were used for

RAPD analysis. Total genomic DNA was extracted from

leaves of each individual using CTAB method (Murray and

Thompson 1980). Totally, 20 arbitrary 10-mer RAPD pri-

mers were used for the RAPD analysis. RAPD amplifica-

tions were performed in 0.025 cm3 reaction mixture

containing 0.2 mM dNTP’s, 10 mM Tris–HCl, 1.5 mM

MgCl2, 50 mM KCl, 0.1 % Triton X-100, 1.0 U Taq DNA

polymerase (Finnzymes, Helsinki, Finland), 15 pmol pri-

mers (IDT, Coralville, USA), and 50 ng of genomic DNA

in a thermal cycler (MastercyclerR-ep, Eppendorf AG,

Hamburg, Germany) following the sequence of cycles

followed for Mucuna pruriens (Padmesh et al. 2006).

Amplified products were resolved in 1.2 % agarose gel

(19TBE) followed by EtBr staining. The gel was visual-

ized on a UV transilluminator and documented by Biorad

Gel documentation system (Biorad, USA). Scorable bands

were recorded and based on band data the genetic distance

matrix was formulated by Nei’s genetic distance analysis

method (Nei 1973) and the phenogram constructed using

UPGMA with Popgene version 1.32.

Statistical analysis

Each experiment on culture initiation and multiplication

consisted of five replicates with four explants per flask and

repeated thrice. A single factor analysis of variance

(ANOVA) was performed whenever there were sufficient

amounts of data to justify it using MS Excel software

(version 2007). For a multiple comparison of means,

Tukey’s test was used at the 5 % significance level.

Results

Culture initiation

The immediate response of the isolated nodes was the

disappearance of the pale red color within 2 days of culture

in all the media tried. However, there was recurrence of the

red color with varying frequencies in regenerated shoots

that raised especially in media supplemented with BAP and

TDZ during the course of culture. Explants were free from

exudates but fungal infection first noticed from inside tis-

sue at cut basal ends of the nodes in 3–8 days was not

controllable as it spreads all over the explants and the

nutrient medium in 15 days. Up to 47 % of the cultures

were infected and lost as attempts to disinfect the explants

with repeated washings in 6 % (v/v) sodium hypochlorite

and 0.1 % (w/v) HgCl2 and rinsing in sterile distilled water

failed. Remaining nodes cultured in MS basal and hor-

mone-supplemented media responded with 1–2 bud for-

mation within a week more or less at the same frequency

but with differences in the number of harvestable shoots

and rate of growth of the shoots (Table 1). Invariably, an

average 1.8 shoots of 1.7 cm length were formed from each

node in 6 weeks in the basal medium itself. However,

supplementation of the medium with cytokinins stimulated

multiple shoot formation. Among cytokinins, KIN sup-

plemented at an optimal concentration of 2.325 lM was

the best to induce formation of up to 3.66 ± 0.48 callus-

free healthy shoots of 3.33 ± 0.34 cm length in 6 weeks.

Higher and lower concentrations of KIN induced less

number of shoots which were invariably short with 3–4

nodes at higher concentrations (4.65 and 9.30 lM). Both

BAP (2.22 lM) and TDZ (0.227 lM) induced the forma-

tion of up to 3.00 ± 0.82 and 3.12 ± 0.58 shoots,

respectively, and the shoots formed were shorter than those

induced by KIN. Shoots were less than 1.5 cm long at

concentrations of BAP exceeding 1.776 lM, and at

J. V. Reji et al.

123

4.44–8.88 lM and TDZ at 0.318–0.454 lM stunted shoots

(0.6–1.3 cm) with closely arranged nodes (3.0–3.5) and

remarkably condensed internodes were formed (Fig. 1).

BAP appeared to inhibit shoot elongation more than most

of the concentrations of TDZ. Though short, these shoots

were free from hyperhydricity, deformation, and other

morphological defects. Besides, there were only minor

differences in the number of shoots initiated between the

same concentration of KIN and BAP in the range of

1.776–4.44 lM and comparable concentrations of TDZ at

0.09–0.318 lM. Fewer and shorter shoots were formed in

the highest concentration (0.454 lM) of TDZ, however.

The cytokinins differed in their action on pigment syn-

thesis in the regenerated shoots also. All the axillary shoots

initiated in basal medium and medium supplemented with

various concentrations of KIN and TDZ were green. BAP

at concentrations exceeding 2.22 lM invariably induced

somewhat pale shoots. Combinations of KIN

(2.325–4.65 lM) and auxins (IAA/NAA) tested for shoot

initiation was not good as fewer shoots (2.3 ± 0.67) were

formed often accompanied by root or callus formation

(Table 1). Combinations of KIN and NAA induced cal-

lusing accompanied by 1–3 bud formation, and enhanced

bud/callus formation depended on the relative concentra-

tion of KIN and NAA used. High concentration of NAA

(2.148–5.37 lM) in a combination induced more of cal-

lusing, and vice versa with KIN (0.93–9.3 lM) led to bud

formation. Callus formation usually occurred at cut basal

end of the node and to some extent in the newly formed

buds. There was no symptom of bud formation when high

concentration (5.37 lM) of NAA was used in combination

with low (0.93 lM) KIN. Though the shoots produced in

presence of KIN were green in color, both the buds and

callus that proliferated upon the explants in combinations

of KIN and NAA were red pigmented to varied extent.

Multiplication

Since auxins were not required for shoot initiation, individ-

ual nodes of in vitro raised shoots were used for shoot mul-

tiplication using selected concentrations (2.325–9.3 lM) of

KIN and BAP and TDZ (0.09–0.454 lM). Six weeks of

subculture resulted in more shoots emanating from the sub-

cultured nodes than during culture initiation from primary

node cultures. Better performance in terms of early bud break

within a week followed by quick growth of comparatively

larger number of shoots noticed in all the subculture treat-

ments was characteristic of the multiplication process. Dif-

ferences between the cytokinin types in their ability to

multiply shoots were also prominent during multiplication.

Though there was a marginal increase in the number of

shoots (1.75 ± 0.45) produced per node even in the basal

medium, and BAP at 2.22–4.44 lM produced

4.4–4.6 shoots/node, the harvestable number of nodes

Fig. 1 Shoots of Hypericum

hookerianum formed from field-

derived nodes after 6 weeks of

culturing in MS basal and MS

media containing varying

concentrations of cytokinins

A high efficiency in vitro regeneration protocol and clonal uniformity analysis in Hypericum…

123

produced were very less (1–2) in compared to the number of

harvestable shoots obtained with KIN. Moreover, all the

shoots multiplied through subsequent subculture in presence

of BAP were stunted with condensed nodes and reduced

leaves. Shoots multiplied using different concentrations of

TDZ also showed these abnormal features. Highest number

(5.50 ± 0.42) of healthy shoots/node with long internodes

and fully expanded leaves was recorded at 2.325 lM KIN

which was optimum for shoot multiplication (Table 2).

Shoot multiplication was free from callusing in all the tested

concentrations of the cytokinins except in the case of

4.44 lM BAP which invariably induced the formation of a

few condensed shoots scattered over massive greenish

brown callus.

Combinations of 2.325 lM KIN and 1.074–2.685 lMIAA/NAA multiplied less number of shoots than 2.325 lMKIN and the multiplication was accompanied by various

callusogenic and rhizogenic responses. Nodal explants of

normal shoots that emanated from a single mature nodal

explant, multiplied in presence of 2.325 lM KIN were

dissected out and repeatedly subcultured through at least

six cycles of 6 weeks each in the same medium to multiply

(Fig. 2) and raise a stock of 3752 shoots (5.14 ± 0.90 cm)

free from morphological defects in 36 weeks.

Rooting and ex vitro establishment of plantlets

Shoot cuttings inoculated onto half-strength MS basal

medium rooted at 15 % efficiency showing formation of a

few (1–2), short (0.3–0.5 cm) and fragile roots after

40–50 days. Supplementation of the medium with auxins

(IAA, IBA) at 2.45–4.9 lM advanced rooting at 40–90 %

rate (Fig. 3). Specific requirement of IBA at 2.45 lM was

essential for inducing maximum number (6.8 ± 0.79) and

percentage (94 %) of rooting in 3 weeks as further increase

in the concentration (4.9 lM) resulted in significant

reduction in rooting (40 %). Among other auxins, the roots

so formed were off-white and hardy. At the concentrations

tested, IAA induced the formation of up to 3.1 ± 0.57

roots which were pale brown colored. During root initia-

tion, the shoots did not show any significant longitudinal

growth in any of the treatments and were free from

branching. The green house established plantlets showed a

survival rate of 90 % during ex vitro hardening. Fully

acclimatized plants were transferred to their natural habitat

and are surviving in a healthy condition.

Analysis and comparison of phytochemicals

in the regenerated plants

All the phytochemicals analyzed in mother plants and

in vitro raised plants did not vary significantly (Table 3).

The results obtained in total phenols, flavonoids,

Table 2 Multiplication of

Hypericum hookeniarum shoots

after 6 weeks of sub-culturing

shoot culture derived

0.8–1.0 cm nodal explants in

MS medium with selected

concentrations of cytokinins

Sl. no Treatments (lM) Mean number of shoots/node Mean length of shoots/node (cm)

1 0.0 2.00ef 2.68cd

2 KIN (2.325) 5.50a 3.53a

3 KIN (4.650) 3.75cd 3.21bc

4 KIN (9.300) 2.58e 2.19d

5 BAP (2.220) 4.45bc 2.12d

6 BAP (4.440) 4.62ab 0.83e

7 BAP (8.880) 3.59d 0.39e

8 TDZ (0.090) 2.43ef 3.69b

9 TDZ (0.227) 2.19ef 3.56b

10 TDZ (0.454) 1.84f 2.15d

Mean values followed by the same letter in each column are not significantly different (P B 0.05)

according to ANOVA and Tukey’s multiple range tests

Fig. 2 Hypericum hookeniarum. In vitro multiplied shoots after

4 weeks of subculture in MS medium supplemented with KIN

(2.325 lM)

J. V. Reji et al.

123

anthocyanins pigments, and even with the major active

principle and the marker compound in the genus as whole,

hypericin, also were in a comparable range. As there was

no significant differences observed in the analyzed phyto-

molecules between wild plant and tissue-cultured plants,

micropropagated plants or the in vitro cultured shoots as

such can be used for the production of those compounds

with pharmacological interest by maintaining a similar

pattern from those of wild plant.

Assessment of genetic uniformity using RAPD

Of the 20 primers initially screened with genomic DNA of

themother plant, only 15were used for final characterization

based on the findings of the initial screening. In the RAPD

profile, the number of scorable bands for each primer varied

from 1 (OPC 04) to 12 (OPC 11). These primers produced 83

distinct scorable band classes with an average of 5.5 bands/

primer. A total number of 1992 bands (number of samples

analyzed 9 number of scorable bands with all 15 primers)

were generated. None of the primers revealed polymorphism

in any of the sample used for the assay. A typical RAPD

profile generated with Primer OPC 02 is shown in Fig. 4.

Banding patterns of all the 23 randomly selected micro-

propagated plants for a particular primer were identical to

the mother plant indicating absence of variation among the

micropropagated plants. This was further confirmed by

similarity matrix based on Nei’s coefficient revealing pair-

wise value between the mother plant and the plantlets to be1,

indicating 100 % similarity.

Discussion

Shoot regenerants of seedling tissues of an open-pollinated

species may not be genetically uniform and multiplication

through adventitious organogenesis or indirect organogen-

esis may add to the variation, thereby compromising genetic

uniformity of the plants raised through tissue culture. Pub-

lished reports indicate multiplication of Hypericum species

mostly using seedling explants (Cellarova et al. 1992;

Zobayed and Saxena 2004; Cirak et al. 2007; Ayan and

Cirak 2008; Oluk and Orhan 2009; Oluk et al. 2010; Namli

et al. 2010; Reji and Seeni 2013; Seeni et al. 2013), adult

explants devoid of resident meristems (Pretto and Santarem

2000; Ayan et al. 2005; Goel et al. 2009), and rarely from

explants having resident meristems (Baruah et al. 2001;

Eliane and Leandro 2003; Shilpashree and Rai 2009).

Therefore, mass multiplication of hitherto lesser known H.

hookerianum achieved through repeated axillary meristem

multiplication devoid of callusing and morphological

abnormalities in half-strength MS medium containing

2.325 lMKIN, easy rooting of the shoots at 96 % rate using

2.45 lM IBA and confirmation of genetic uniformity of the

rooted plants through RAPD profiling makes the single node

culture suited for mass cloning of the species. Utility of

axillary meristem culture using nodal explants in clonal

multiplication and conservation of a species is recognized

(Hu and Wang 1983) and indispensability of clonal for

future planting of high yielding chemotypes especially from

a hotspot of biodiversity like the Western Ghats of India is

recommended (Krishnan et al. 2011).

Culture initiation process marked by severe fungal con-

tamination originating from inside tissues in cut ends of

nodal explants and loss of 45 % of the nodes indicated that

the source of infection is endophytic. Since the shoot extracts

of H. hookerianum exhibit wide ranging antibacterial

activities against both gram-negative and gram-positive

bacteria (Mukherjee et al. 2001) and are not antifungal to

arrest growth of Aspergillus flavus Raper & Fennell,

Alternaria solani (Ell & Mart.) L. R. Jones and Grout,

Fusarium moniliforme Sheldon, and Phytophthora sp

Fig. 3 Hypericum hookeniarum. In vitro rooted plantlets obtained

from MS medium supplemented with IBA (2.45 lM) ready for

hardening

Table 3 Comparison of phytochemicals produced in in vitro raised

Hypericum hookeniarum plants with their mother plant

Chemical constituents Wild plant Tissue-cultured plants

Hypericin 1.21 ± 0.12 0.98 ± 0.46

Flavonoids 7.96 ± 0.15 8.12 ± 0.32

Anthocyanins 2.85 ± 0.21 1.96 ± 0.34

Total phenols 9.56 ± 0.67 9.24 ± 0.35

Results are the mean values of three separate determinations with

randomly collected plantlets. Hypericin and total phenol values are

expressed in mg g-1 DW (dry weight) and flavonoids and antho-

cyanins in mg g-1 FW (fresh weight)

A high efficiency in vitro regeneration protocol and clonal uniformity analysis in Hypericum…

123

(unpublished results). Endophytic fungi colonizing living

internal tissues without causing overt negative effects within

the host and yet capable of synthesizing host plant metabo-

lites have been demonstrated in H. perforatum (Kusari et al.

2008, 2009) andTaxus species (Zhang et al. 2008).However,

infection free nodes were free from exudates and responded

readily with multiple shoot initiation (Table 1). The use of

node cultures in linear amplification of pre-existing axillary

buds during culture initiation in H. brasiliense has been

described by Cardoso and de Oliveira (1996). Initiation of

1.8 shoots in the basal medium itself similar to the obser-

vation of these authors indicated presence of endogenous

growth regulators to induce shoot organogenetic response.

However, exogenous supply of cytokinin was essential for

enhancedmultiplication. Kinetin supplemented at 2.325 lMinduced maximum formation of 3.66 ± 0.48 callus-free

healthy shoots of 3.3 ± 0.34 cm length (Table 1) in contrast

to BAP or BAP and auxin supplementation stimulated

optimal shooting responses reported in other Indian (Baruah

et al. 2001; Shilpashree and Rai 2009), European (Cellarova

et al. 1992; Ayan and Cirak 2006; Wojcik and Podstolski

2007; Namli et al. 2010), and Brazilian (Cardoso and de

Oliveira 1996; Santarem and Astarita 2003) species of

Hypericum. Although there is a precedence of BAP used for

both initiation and multiplication in H. perforatum (Pretto

and Santarem 2000; Ayan et al. 2005), KIN promoting

maximum shoot initiation and multiplication without a need

for separate elongation phase inH. hookerianum is new.As it

is unstable during autoclaving, KIN is often identified with

low shooting response (Amoo et al. 2011).

The morphological abnormalities of shoots induced by

higher concentrations of BAP (8.88 lM) and TDZ

(0.454 lM) in the present system were free from hyper-

hydric and necrotic malformations, which are similarly

reported in shoot cultures of H. maculatum and H. hirsutum

raised using 1.816 lM TDZ (Coste et al. 2011). In fact,

even at 8.88 lM BAP where massive callus with shoots

spread upon was formed, the shoots were free from such

malformations. However, contrary to all published report

on Hypericum so far, BAP at concentrations exceeding

2.22 lM inhibited chlorophyll biosynthesis up to 30 %

without affecting the multiplication rate. During shoot

initiation stage itself, some of the newly formed shoots

were somewhat pale which increased to a higher degree

during multiplication. Despite its significant negative

influence on shoot elongation, TDZ differed from BAP in

not inhibiting chlorophyll synthesis. Though observations

such as abnormal shoots with rosette type leaves and cal-

loid outgrowths with stunted shoot formation at high con-

centrations of TDZ and BAP are common in many other

plant species, it is not general in Hypericum species.

Moreover, preferential use of BAP and TDZ for shoot

multiplication is also reported in Hypericum species such

as H. triquetrifolium and H. perforatum (Oluk and Orhan

2009; Banerjee et al. 2012). Combinations of KIN and

auxins (IAA, NAA) tested for culture initiation resulted in

rhizogenesis or callusogenesis and fewer shoot formation

and confirmed superiority of using 2.325 lM KIN for

caulogenesis (Table 1). However, NAA induced red pig-

mentation in buds and calluses, and was distinct and wor-

thy of further investigation.

Uniform multiplication of 5.50 shoots as against 3.6

shoots obtained during culture initiation indicated

improved acclimatization of the nodes and enhanced par-

ticipation of the axillary meristems in shoot multiplication

as reported in other woody species (Sudha et al. 1998;

Ajithkumar and Seeni 1998). Scale-up of as many as 3752

shoots by repeated subculture of single nodes without

decline using 2.324 lM KIN is a simple and rapid method

for multiplication of H. hookerianum. High frequency

(94 %) rooting with 8.3 long off-white hardy root forma-

tion recorded in 3 weeks augurs well with sustainable

production of the this depleted resource. For successful

commercial exploitation of a species, it is compulsory to

Fig. 4 RAPD profile of mother plant and the in vitro raised Hypericum hookeniarum plants with primer OPC 02 [Lane M-100 bp DNA Marker

(New England Biolabs, England, UK); Lane 1–23 micropropagated plants; Lane 24 mother plant]

J. V. Reji et al.

123

check clonal uniformity of the micropropagated plants

(Khawale et al. 2006) as evidenced from earlier reports in

several medicinal plants. However, the synthesis and con-

centration of bioactive molecules in the plants that raised

though tissue culture have always been an issue of debate

for pharmaceutical companies and ayurveda practitioners.

Hence, the production of genetically and chemically uni-

form plantlets is important for a plant like H. hookerianum

which is not yet commercially exploited. The RAPD profile

obtained with genomic DNA and random primers at an

average of 5.5 bands/primer and the banding pattern

revealed no variation among the tested clones. The true-to-

type nature of clones was further confirmed by similarity

matrix based on Nei’s coefficient and phenogram based on

UPGMA analysis. The remarkable genetic uniformity of

the clones evident from whatever fraction of the total

genome examined in such analyses together with the uni-

formity in production of secondary metabolites confirms

clonal fidelity of the micropropagated plants for conser-

vation and commercial uses.

Hypericum hookerianum is a folk medicine of Toda

tribe of the Nilgiris for treating burns, wounds, skin

infections, and conditions of anxiety and inflammation

(The Wealth of India 1997; Vijayan et al. 2004). Hypericin,

a naphthodianthrone is endowed with photodynamic ther-

apeutic applications against retroviruses (HSV, HIV) and

cancer (Karioti and Bilia 2010). Presence of hypericin has

been already reported in this plant (Reji and Seeni 2013;

Seeni et al. 2013). On an average, the calculated concen-

tration of hypericin and other major phytochemical con-

stituents in the mother plant and in vitro raised plantlets of

H. hookerianum which was more or less similar (Table 3)

indicates the similarity in mother plants with the tissue-

cultured ones. This is supported by the genetic fidelity

assay done by RAPD analysis which is a common method

to analyze genetic diversity among plant species. In con-

clusion, the successful development of an in vitro clonal

multiplication protocol for this ethanomedicinally and

commercially important Hypericum species using mature

nodal explants together with evaluation of quality assur-

ance and genetic fidelity of the micropropagated plants

provides an ample package for the conservation and sus-

tainable use of target species.

Acknowledgments The authors thank Jeppiaar Educational Trust,

Sathyabama University, Chennai for the financial support.

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