Callus-Mediated Organogenesis and Effect of Growth Regulators

ORIGINAL PAPER

Callus-mediated organogenesis and effect of growth regulatorson production of different valepotriates in Indian valerian(Valeriana jatamansi Jones.)

Jayashankar Das • Ashiho A. Mao •

Pratap J. Handique

Received: 11 February 2012 / Revised: 29 May 2012 / Accepted: 19 June 2012 / Published online: 3 July 2012

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012

Abstract A reproducible and efficient callus-mediated

shoot regeneration system was developed for the large-scale

production of Valeriana jatamansi Jones., a highly medic-

inal plant species of global pharmaceutical importance.

Effect of Murashige and Skoog (MS) medium supple-

mented with different concentrations of 2,4-dichlorophe-

noxyacetic acid (2,4-D), a-naphthaleneacetic acid (NAA)

and indole-3-butyric acid (IBA) on callus induction and

production of valepotriates accumulation was studied by

using different explants. In V. jatamansi, the degree of

callus induction varied significantly depending on explants

type and the growth regulators used. Among different

explants used, rhizomes have the highest callus induction

potential followed by leaf. The callus induction frequency

was found to be optimum in rhizome explants on media

supplemented with 0.5 mg/l 2,4-D. The regenerative ability

of proliferated compact calli was studied by the application

of cytokinins alone and in combination with auxin. MS

medium fortified with 0.75 mg/l thidiazuron in combination

with 0.5 mg/l NAA showed the highest regeneration

frequency (88.6 %) and produced the maximum number of

shoot buds (15.20 ± 0.20) capable of growing into single

plants. Vigorous callus obtained from MS medium sup-

plemented with different concentrations of 2,4-D, NAA and

IBA were used for industrially important valepotriates

[acevaltrate (ACE), valtrate (VAL) and didrovaltrate

(DID)] analysis. High performance liquid chromatography

analysis of callus revealed that medium with 2,4-D (1 mg/l)

was found responsible for increasing ACE and DID yield,

whereas VAL production was higher in case of medium

supplemented with NAA (1 mg/l). However, the accumu-

lation of valepotriates in callus decreased in logarithmic

phase after 8 weeks. IBA was not beneficial for the va-

lepotriate synthesis, as it helped to accumulate significantly

lower concentration of ACE, VAL and DID. Micropropa-

gated plantlets with well-developed root system were suc-

cessfully acclimatized in greenhouse condition, in root

trainers containing garden soil with a survival frequency of

100 %. As Indian valerian is a highly traded medicinal plant

due to extensive use of its industrially important secondary

metabolites, the present system can be utilized to obtain

mass multiplication of the species as well as for the stable

biomass and continuous valepotriate production for the

pharmaceutical industries.

Keywords Indian valerian � Callus � Regeneration �Valepotriates

Introduction

Valeriana jatamansi Jones. syn. V. wallichi (Fam: Valeri-

anaceae) commonly known as Indian valerian is a peren-

nial herb, distributed in Himalayas from Kashmir to Bhutan

and Khasi hills of Northeast India at an altitude

Communicated by J. Van Huylenbroeck.

J. Das � A. A. Mao

Plant Tissue Culture Laboratory, Botanical Survey of India,

ERC, Shillong 793 003, India

J. Das � P. J. Handique

Department of Biotechnology, Gauhati University,

Guwahati 781084, India

Present Address:J. Das (&)

Plant Bioresources Division, Regional Centre of IBSD,

Tadong, Gangtok 737102, India

e-mail: [email protected]

123

Acta Physiol Plant (2013) 35:55–63

DOI 10.1007/s11738-012-1047-2

1,800–3,500 m (Bos et al. 1997). V. jatamansi has been

used in Ayurvedic, Unani and modern system of medicine

due to its high medicinal and aromatic values. The essential

oil components of Indian valerian make it one of the most

demanded plant in the drug industry. Major constituents of

its volatile oil are patchouli alcohol and bornyl isovaltrate

(Bos et al. 1997). Essential oil from rhizomes exhibited

antifungal and antibacterial activities (Girgune et al. 1980).

6-Methylapigenin and hesperidin isolated from rhizome of

Indian valerian showed anxiolytic and sedative activities

(Wasowski et al. 2002). The sedative and tranquilizing

properties of the plant are also due to the presence of

nonglycosidic iridoid esters known as valepotriates. The

major valepotriates are valtrate (VAL), acevaltrate (ACE)

and didrovaltrate (DID) due to which V. jatamansi pos-

sesses antispasmodic, anticonvulsive and antidepressant

properties (Gupta et al. 1986). DID, VAL and their

degraded product baldrinal were found to be cytotoxic in

rat hepatoma cells (Bounthanh et al. 1981). In addition, the

antitumor activity of DID was demonstrated in vivo on

female mice KREBS II ascetic tumors (Marder et al. 2003).

Due to extensive use in the modern pharma and perfumery

industries, V. jatamansi is listed as one of the most

exploited plant of Himalayan range. However, it is not yet

cultivated anywhere in India for the large-scale production

and all demands for its domestic and foreign trade are met

from its wild population (Gupta et al. 2006). Over the years

its indiscriminate collection has led to its large-scale

depletion in the wild and has necessitated its replenishment

and cultivation.

During the last several years, there is an increase

demand of in vitro culture techniques which offer a feasible

tool for rapid clonal propagation and germplasm conser-

vation of rare, endangered and threatened medicinal and

aromatic plants (Abraham et al. 2010). Cultured plant cells

synthesize, accumulate and sometimes exude many classes

of secondary metabolites. Numerous alkaloids, saponins,

cardenolides, anthraquinones, polyphenols and terpenes

have been reported from in vitro cultures (Verpoorte et al.

2002; Vanisree and Tsay 2004). In dedifferentiated cells,

some biosynthetic potential typical for the developed

organs from which they were initiated can be conserved. In

Pueraria lobata callus cultures, the bioactive isoflavonoid

content depended on the source organ, reflecting relations

in the mature plant (Matkowski 2004). A range of envi-

ronmental and nutritional factors are known to influence

the biosynthetic pathways of secondary metabolites

(Stintzing and Carle 2004). Plant growth regulators have

been widely used in promoting the biosynthesis of both

inducible and constitutive secondary metabolites, including

medicinal compounds such as anticancer alkaloids

(Vanisree and Tsay 2004; Verpoorte et al. 2002).

The continuous monitoring of a chosen metabolite is a

prerequisite for the successful development of production

technology. Also, the super-efficient clones of cultured cell

or tissues can be selected by monitoring the level of the

compound of interest, or can be complemented by a

selecting agent facilitating the process.

Though an earlier fragmentary report was published on

micropropagation of V. jatamansi (Kaur et al. 1999), the

result was scanty towards standardization of the protocol

for the commercial scale. The present research work is

based on twofold objectives, viz., to develop an efficient

and rapid propagation protocol of V. jatamansi for the

fulfillment of market demand and to quantify the indus-

trially important valepotriates (VAL, ACE and DID)

accumulated in the calli of different ages. This is a first

attempt to study the effect of different growth regulators on

in vitro accumulation of valepotriates in the callus of

V. jatamansi.

Materials and methods

Callus induction

Leaf (ca. 1 9 1 cm), petiole (ca. 0.5–0.7 cm) and rhizomes

(ca. 1 9 1 cm) explants were taken from a 12-month-old

single genotype of V. jatamansi maintained in the green-

house, BSI, ERC, Shillong, for callus induction studies. The

explant cuttings of 2.0–2.5 cm long were rinsed in running

tap water three times and washed in a 2 % (v/v) Tween 20

detergent solution for 15 min. Then the plant materials were

surface-sterilized in a solution of 10 % (v/v) sodium

hypochlorite for 5 min followed by 0.1 % (w/v) mercuric

chloride for 1 min. Finally, the explants were rinsed 3 times

with sterilized distilled water. The explants were estab-

lished in Murashige and Skoog’s (1962) basal medium

supplemented with 3 % (w/v) sucrose and 0.8 % (w/v)

agar-agar (Hi-media, Mumbai, India). The pH of media was

adjusted to 5.8 before autoclaving at 15 psi and 121 �C for

20 min. All the explants were cultured on MS medium

supplemented with different concentrations of 2,4-D, NAA

and IBA (0.25, 0.5, 1.0, 2.0, 3.0 mg/l). Callus cultures were

subcultured at 4-week intervals on respective media.

Regeneration of multiple shoot bud from callus

Randomly selected compact calli were transformed to

growth regulator-free MS basal medium to overcome the

carryover effect of auxins. To evaluate the effect of growth

regulators on the callus potential for shoot regeneration,

calli were excised, divided into small pieces (0.5 9

0.5 cm) transferred to the regeneration medium for shoot

induction. MS medium supplemented with different con-

centrations of TDZ (0.5, 0.75 mg/l) and kinetin (Kn; 2.0,

56 Acta Physiol Plant (2013) 35:55–63

123

3.0 mg/l) alone and in combination with NAA (0.5 mg/l)

was used for shoot proliferations from callus in 150-ml

culture flasks (Borosil, India). Callus along with the initi-

ated multiple shoot buds were subcultured on respective

regeneration medium after 4 weeks interval to obtain

healthy shoots.

Culture conditions

The cultures were maintained at 24 ± 2 �C and relative

humidity (RH) of 50 ± 5 % under 16 h photoperiod with

30 lmol m-2s-1 photosynthetic photon flux density

(PPFD) provided by cool-white fluorescent light tubes

(Philips, India).

Extraction and HPLC analysis

Vigorous callus obtained from rhizome explants supple-

mented with 2,4-D (0.5, 1.0 mg/l), NAA (0.5, 1.0 mg/l)

and IBA (1 mg/l) were used for valepotriates (ACE, VAL

and DID) analysis. The calli were dried and subjected to

dichloromethane extraction 3 times at room temperature

(25 ± 2 �C). The extract was dissolved in methanol to

obtain a concentration of 1 mg/ml, filtered through a

membrane filter (0.22 lm pore size, Merck), analyzed by

HPLC and compared with the reference compounds (Sigma

Aldrich, USA).

HPLC analysis of valepotriates was performed in a

Shimadzu LC-10A gradient HPLC coupled with 2 LC-

10AD pumps, 10A UV detector and manual injector with a

20 ll sample loop. The separation conditions were: Waters

Nova pack C18 column (25 9 4.6 mm i.d.); mobile phase

acetonitrile: water; 80:20 (v/v); flow rate 1 ml/min;

detector wavelength 254 nm and sensitivity 0.04 Aufs.

Rooting and acclimatization

Healthy shoots of 4–5 cm were excised and cultured for

rooting on MS medium supplemented with different con-

centrations of auxins viz. indole-3-acetic acid (IAA), NAA

and IBA (0.05, 0.10, 0.20 mg/l) in 150-ml culture flasks

(Borosil, India). MS medium without growth regulators

was used as control. Plantlets with well-developed roots

were removed from the culture medium, washed gently

under running tap water, and transferred to root trainers

containing garden soil and acclimatized under greenhouse

condition (24 ± 2 �C temp. and 80 ± 5 % RH) without

use of any organic fertilizers.

Data analysis

The callus induction, shoot regeneration and rooting

experiments were repeated three times with minimum 24

explants each. All the valepotriates were analyzed with 3

replicates. To analyze the effect of various treatments, the

data were subjected to analysis of variance (ANOVA) and

statistical significance between mean values was assessed by

Duncan’s multiple range test at P \ 0.05 by using statistical

software SPSS ver. 15 (SPSS Inc., Chicago, USA).

Results and discussion

Callus induction

The disinfection treatment used was efficient for in vitro

establishment with approximately 94 % of the explants

remaining aseptic. In V. jatamansi callus induction varied

significantly depending on the explant type. Among dif-

ferent explants used, rhizomes have the highest callus

induction potential followed by leaf. Rhizome explants

started swelling within 6–8 days, whereas leaf and petiole

materials started within 10–12 days of inoculation. MS

basal medium without growth regulators exhibited no cal-

lus proliferation. All the calli in the growth regulators-

supplemented media were observed to be initiated from the

cutting edge of the explants. The induced calli were fast

growing, yellowish green and compact (Fig. 1a, b). The

callus induction frequency was found optimum in rhizome

explants (Table 1) on media supplemented with 0.5 mg/l

2,4-D (86 %) followed by 0.5 mg/l NAA (75.8 %). In our

study, 0.5 mg/l 2,4-D was the best auxin responsible for

obtaining the vigorous and compact callus from rhizome

(0.23 ± 0.06 g), leaf (0.17 ± 0.06 g) and petiole

(0.13 ± 0.03 g) explants. Effect of different explants and

growth regulators on callus induction of Allium chinense

was observed by Yan et al. (2009). Different tissues may

have different levels of endogenous hormones, and there-

fore, the type of explants source would have a critical

impact on the callus induction and its regeneration success.

The selection of proper donor plants and organs should

already be considered when starting the culture, unless it

can be overcome by a suitable treatment. However, in most

circumstances the dedifferentiation apparently also

involves some of the biochemical properties of the cells.

The modification of relative biosynthesis to degradation

ratios of a desired product can also influence the final levels

of a desired compound in the culture (Stintzing and Carle

2004).

The degree of callus formation was varied within all the

explants as well as PGRs used. Within different concen-

trations of NAA and IBA, 0.5 mg/l NAA produced the

richest callus from rhizomes (0.21 ± 0.07 g) with callus

induction frequency 75.8 % followed by 1.0 mg/l NAA

(0.16 ± 0.01 g) with 68.2 %. No somatic embryos or

adventitious roots formed from any callus. 2,4-D was

Acta Physiol Plant (2013) 35:55–63 57

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considered as one of the most effective auxins for the callus

induction and found effective in case of Pennisetum

glaucum (Jha et al. 2009), Pinus caribaea (Akaneme and

Ene-Obong 2005), Tylophora indica (Thomas 2009) and

Juncus effusus (Xu et al. 2009). Medium fortified with

different concentrations of IBA produced significantly less

biomass than the other auxins used. However, the rate of

callus proliferation was inversely proportional to the con-

centrations of auxins in V. jatamansi (Table 1). Compact

calli are important in the in vitro cultures as they have

Fig. 1 Callus induction and regeneration of plantlets from callus in

V. jatamansi Jones. a 8-week-old vigorous callus (bar 10 mm)

induction from leaf explant on MS medium with 0.5 mg/l 2,4-D. b 8-

week-old vigorous callus induction (bar 10 mm) from rhizome

explant on MS medium with 0.5 mg/l 2,4-D. c, d Regenerated

plantlets (bar 15 mm) from callus on MS medium with 0.75 mg/l

TDZ and 0.5 mg/l NAA. e 8-week-old in vitro-raised plantlet with a

healthy root system. f Acclimatized plants in the greenhouse

condition and g 1-year-old established plants


123

ability for organogenesis. They are more efficient to

develop chlorophyll than friable calli from the same

explants; this might be due to the chloroplast development

and integrity favored by cell aggregation (George and

Sherrington 1984).

Shoot regeneration from callus

The regenerative ability of proliferated compact calli was

studied by the application of cytokinins alone and in

combination with auxin. After 2 weeks of culture, most of

the calli turned green in order to give response towards

regeneration of plantlets. Shoot primordia appeared after

2 weeks in the regeneration medium. MS medium sup-

plemented with 0.75 mg/l TDZ in combination with

0.5 mg/l NAA showed the highest regeneration frequency

(88.6 %) and produced the highest number of shoot buds

(15.20 ± 0.20) capable of growing into single plants

(Table 2). Medium fortified with equal concentration

(0.5 mg/l) of TDZ and NAA produced 12.30 ± 0.21 shoots

having 80.4 % regeneration potential. Addition of NAA at

low concentration with TDZ-supplemented medium

Table 1 Effect of different concentrations of auxins on callus induction from different explants of V. jatamansi

Plant growth regulators (mg/l) Callus induction frequency (%) Mean weight of the callus (gm)

2,4-D NAA IBA Rhizome Petiole Leaf Rhizome Petiole Leaf

0.25 0.0 0.0 54.6 48.2 51.0 0.19 ± 0.09c 0.12 ± 0.02b 0.14 ± 0.07b

0.5 0.0 0.0 86.0 64.1 74.2 0.23 ± 0.06a 0.13 ± 0.03a 0.17 ± 0.06a

1.0 0.0 0.0 72.5 62.7 66.0 0.21 ± 0.04b 0.12 ± 0.03b 0.14 ± 0.06b

2.0 0.0 0.0 62.6 48.2 50.4 0.17 ± 0.08d 0.12 ± 0.04b 0.13 ± 0.06c

3.0 0.0 0.0 55.2 42.8 44.4 0.14 ± 0.01f 0.11 ± 0.05c 0.11 ± 0.06e

0.0 0.25 0.0 70.5 52.6 61.4 0.17 ± 0.01d 0.12 ± 0.02b 0.13 ± 0.03c

0.0 0.5 0.0 75.8 59.0 68.9 0.21 ± 0.07b 0.11 ± 0.06c 0.14 ± 0.09b

0.0 1.0 0.0 68.2 51.6 58.5 0.16 ± 0.01e 0.11 ± 0.06c 0.13 ± 0.08c

0.0 2.0 0.0 54.0 46.2 48.7 0.12 ± 0.01h 0.11 ± 0.06c 0.11 ± 0.07e

0.0 3.0 0.0 50.6 40.5 42.8 0.10 ± 0.01j 0.08 ± 0.09ef 0.09 ± 0.03fg

0.0 0.0 0.25 44.7 38.9 42.4 0.11 ± 0.01i 0.09 ± 0.04de 0.10 ± 0.05f

0.0 0.0 0.5 64.8 50.5 56.9 0.12 ± 0.01h 0.10 ± 0.04d 0.11 ± 0.05e

0.0 0.0 1.0 70.8 56.2 62.0 0.13 ± 0.07g 0.09 ± 0.04de 0.12 ± 0.07d

0.0 0.0 2.0 42.8 40.1 42.2 0.10 ± 0.05j 0.07 ± 0.04efg 0.08 ± 0.08gh

0.0 0.0 3.0 36.5 30.5 32.6 0.09 ± 0.04jk 0.07 ± 0.04efg 0.07 ± 0.04ghi

Values represent mean ± SE of three replicates with 24 explants each. Means within a column followed by different letters differ significantly at

P B 0.05 as compared by Duncan’s multiple range test

Table 2 Influence of different plant growth regulators on regeneration of shoot buds and elongation of proliferated shoots from callus

of V. jatamansi

Plant growth regulators (mg/l) Regeneration frequency (%) Mean number of shoots/callus Mean shoot length (cm)

TDZ Kn NAA

0.5 0.0 0.0 56.3 7.20 ± 0.24d 2.20 ± 0.17de

0.75 0.0 0.0 64.2 11.90 ± 0.31c 2.50 ± 0.08c

0.5 0.0 0.5 80.4 12.30 ± 0.21b 2.80 ± 0.07b

0.75 0.0 0.5 88.6 15.20 ± 0.20a 3.60 ± 0.07a

0.0 1.0 0.0 31.0 3.50 ± 0.09g 2.10 ± 0.11cd

0.0 2.0 0.0 34.8 3.70 ± 0.15g 2.30 ± 0.12cd

0.0 3.0 0.0 52.1 5.10 ± 0.17e 2.40 ± 0.11cd

0.0 1.0 0.5 53.0 5.30 ± 0.14e 2.10 ± 0.10cd

0.0 2.0 0.5 66.2 5.40 ± 0.30e 2.40 ± 0.12cd

0.0 3.0 0.5 48.2 4.70 ± 0.21ef 2.30 ± 0.06cd




123

enhanced the callus regeneration frequency with more

number of shoot response (Fig. 1c, d). The same effect was

observed in case of Kn supplemented media in combina-

tion with NAA. However, 2 mg/l Kn along with 0.5 mg/l

NAA showed 66.2 % regeneration frequency proliferating

5.40 ± 0.30 shoots (Table 2). Synergistic effect of auxin

and cytokinin on shoot regeneration from calli were

observed in Solanum tuberosum (Shirin et al. 2007),

Cynondon dactylon (Zhang et al. 2007), Ipomoea obscura

(Mungole et al. 2009), Juniperus excels (Shanjani 2003),

Aframomum corrorima (Tefera and Wannakrairoj 2006)

and Pennisetum glaucum (Jha et al. 2009). Huetteman and

Preece (1993) and Gyves et al. (2001) emphasized the

potential use of TDZ in the regulation of adventitious shoot

proliferation and hypothesized on the synergism existing

between TDZ and other endogenous and exogenous auxins.

However, significant decreased shoot regeneration found in

case of medium supplemented with cytokinin alone than in

combination with auxin. The efficiency of TDZ on shoot

regeneration in many medicinal plants was reported

(Thomas 2003, 2007; Mithila et al. 2003; Sanikhani et al.

2006). According to George and Sherrington (1984) cell

differentiation and morphogenesis may be promoted by

insufficient oxygen environment such as in compact calli.

On the other hand, they reported that due to this factor

friability of callus is mostly associated with somatic

embryogenesis while compact callus can be readily used

for organogenesis. Also, in our case no somatic embryo-

genesis occurred during plant regeneration.

Effect of PGRs on valepotriates accumulation in callus

Callus derived from medium supplemented with different

concentrations of 2,4-D (0.5, 1.0 mg/l), NAA (0.5, 1.0 mg/l)

and IBA (1 mg/l) were subjected to HPLC analysis for

quantifying ACE, VAL and DID and the result revealed

that all treatments showed the presence of valepotriates.

Medium with 2,4-D (1 mg/l) was found to be responsible

for increasing ACE (Fig. 2a) and DID (Fig. 2c) yield,

whereas VAL (Fig. 2b) production was higher in case of

medium supplemented with NAA (1.0 mg/l). The

increased concentration of auxins like 2,4-D and NAA

could have caused a mild but chronic oxidative stress

response in undifferentiated mass of cells potentially

capable of inducing valepotriate accumulation. Reactive

oxygen species have been shown to trigger the production

of various secondary metabolites, including terpenes (Zhao

et al. 2005). 8-week-old cultures produced optimum bio-

mass and valepotriates yield (Fig. 2). The accumulation of

valepotriates in callus decreased in logarithmic phase after

8 weeks. Previously, the presence of valepotriates was

characterized in callus of Valeriana glechomifolia (Maur-

mann et al. 2009). In our study, ACE and DID were the

major valepotriates found in V. jatamansi callus.

IBA was not beneficial for the valepotriate production,

as it helped to accumulate significantly lower concentration

of ACE, VAL and DID than 2,4-D and NAA. The observed

impact of auxins on valepotriate metabolism was not nec-

essarily dependent on phytohormone-induced develop-

mental changes on callus (De Klerk et al. 1999). The

benefits of auxin exposure were apparently correlated to

auxin stability, since metabolically stable type of auxins,

such as IBA was not beneficial for valepotriate yield.

Differences in auxin stability and auxin-induced secondary

effects could also explain the concentration and auxin-type

dependence of valepotriate content responses in calli (Bello

0

0.2

0.4

0.6

0.8

1

1.2

0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBAAce

valtr

ate

cont

ents

(g%

dry

wei

ght)

Auxin type and concentration

4 wk

8wk

12wk

0

0.1

0.2

0.3

0.4

0.5

0.6

0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBA

Val

trat

e co

nten

ts (

g% d

ry w

eigh

t)


4 wk

8wk

12wk

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBADid

rova

ltria

te c

onte

nt (

g% d

ry w

eigh

t)


4 wk

8wk

12wk

a

b

c

Fig. 2 Time-course effect of different plant growth regulators on

valepotriate accumulation in callus of V. jatamansi from rhizome

explants: a influence of different efficient auxins on acevaltrate accu-

mulation, b influence of different efficient auxins on valtrate

accumulation, c effect of different efficient auxins on didrovaltrate

accumulation. Bar indicates mean of 3 replicates ±SE (n = 10)


123

de Carvalho et al. 2004). However, the comparative anal-

ysis of individual valepotriate accumulation within every

4-week interval showed significant variation among all the

treatments analyzed. Active secondary metabolite produc-

tion through callus culture is known to be an effective

process in pharmaceutical industries. Several industrially

important secondary metabolites, viz., anthocyanin from

Panax sikkimenesis (Mathur et al. 2010), cerpegin from

Ceropegia juncea (Nikam and Savant 2009), cynarin

from Cynara cardunculus (Silvia et al. 2006), rosmarinic

acid from Lavandula officinalis (Georgiev et al. 2006),

rutin from Hemidesmus indicus (Misra et al. 2005), crocin

from Crocus sativus (Chen et al. 2003), lithospermic acid

from Salvia miltiorrhiza (Morimoto et al. 1994) and

piceatannol from Arachis hypogaea (Ku et al. 2005) were

produced by in vitro callus culture.

Rooting and acclimatization

In vitro shoots, regenerated from shoot clusters proliferated

on multiple shoot induced medium and callus regeneration

medium were separated and used for the rooting experi-

ments. Rooting initiated after 2 weeks in all cultures

including control. But, the response of rooting (%), number

and length of roots were achieved significantly higher

when cultured with auxin-supplemented media. Among

different auxins used, NAA (0.05, 0.1 mg/l) and IAA

(0.1 mg/l) were effective in producing longer and healthy

roots with 100 % response (Table 3). IBA showed signif-

icantly lower root-inducing potential both in response to

root numbers and root length. MS medium fortified with

0.1 mg/l NAA produced the highest number of roots

(17.30 ± 2.01) followed by 0.05 mg/l NAA (12.40 ±

1.20). NAA when used as low concentrations was consid-

ered as an effective rooting hormone in many plant systems

(Mao et al. 1995; Sanches-Gras and Calvo 1996; Rout et al.

2000). Also, IAA was reported to enhance the root for-

mation in case of Hedeoma multiflorum (Koroch et al.

1997) and Woodfordia frusticosa (Krishnan and Seeni

1994). However, MS basal medium without auxins pro-

duced significantly lower number of roots and were not

found healthy for hardening in greenhouse. There are many

reports on the microshoots of various medicinal plants

rooted on only MS medium without the growth regulators

(Christine and Chan 2007; Mao et al. 1995). Explants

having a functional rooting system are more likely to sur-

vive transition to greenhouse. Roots were washed thor-

oughly before being transferred to root trainers.

Micropropagated plantlets with well-developed root system

were successfully acclimatized in greenhouse condition, in

root trainers containing garden soil with a survival

frequency of 100 % (Fig. 1f). The in vitro-derived plants

were phenotypically similar to the parental stock and no

morphological abnormalities have been observed in the

micropropagated plants.

Conclusion

A highly efficient and reproducible protocol was developed

for the large-scale production of V. jatamansi. The indirect

organogenesis system of Indian valerian can provide a

mass production of disease-free and genetically uniform

plant materials throughout the year for the fulfillment of

market demand globally and conservation of the species.

The standardized protocol can offer stable biomass and

continuous valepotriate production for the pharmaceutical

industries. Furthermore, using bioreactor, callus induction

protocol can be useful for continuous callus culture and

the extraction of valepotriate on a commercial scale.

Table 3 Effect of different concentrations of auxins on rooting of in vitro-raised elongated shoots in V. jatamansi

Auxins (mg/l) Response of rooting (%) Mean number of roots/explant Mean root length (cm)

NAA IBA IAA

0.0 0.0 0.0 34.2 3.30 ± 0.61e 4.10 ± 0.21e

0.05 0.0 0.0 100 12.40 ± 1.20b 8.30 ± 0.24b

0.10 0.0 0.0 100 17.30 ± 2.01a 9.30 ± 0.39a

0.20 0.0 0.0 64.6 7.20 ± 0.82d 7.90 ± 0.29bc

0.0 0.05 0.0 38.5 6.15 ± 0.98d 7.70 ± 0.21bc

0.0 0.10 0.0 60.8 10.80 ± 1.85bc 8.10 ± 0.21bc

0.0 0.20 0.0 42.0 5.20 ± 0.61d 6.60 ± 0.20d

0.0 0.0 0.05 75.2 7.80 ± 0.38d 7.90 ± 0.32bc

0.0 0.0 0.10 100 12.10 ± 1.29b 9.20 ± 0.39a

0.0 0.0 0.20 52.5 5.80 ± 0.61d 7.60 ± 0.20bc




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However, the valepotriate accumulation mainly depends on

the growth regulators used and age of the calli. As V. jat-

amansi is a highly traded medicinal plant due to extensive

use of its industrially important secondary metabolites, the

present micropropagation system can be utilized for further

research on metabolic pathways and transgenic approach to

build up the value added products and production of quality

material.

Author contribution J. Das performed the experiment,

analyzed the data and wrote the manuscript. A.A. Mao and

P.J. Handique designed the work, edited the manuscript

and supervised the entire research.

Acknowledgments The authors are thankful to the Joint Director,

BSI, Shillong, for providing the facilities and to the Department of

Biotechnology (DBT), Government of India, New Delhi, India, for

the award of research fellowship to J.D.

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Callus-Mediated Organogenesis and Effect of Growth Regulators

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