ORIGINAL PAPER

Micropropagation of Pueraria tuberosa (Roxb. Ex Willd.)and determination of puerarin content in different tissues

M. S. Rathore Æ N. S. Shekhawat

Received: 1 March 2009 / Accepted: 6 September 2009 / Published online: 21 September 2009

� Springer Science+Business Media B.V. 2009

Abstract Pueraria tuberosa, a medicinally important

leguminous plant, yielding various isoflavanones including

puerarin, is threatened, thus requiring conservation. In this

study, fresh shoot sprouts of P. tuberosa, produced by

tubers, were used as explants for in vitro micropropagation.

Surface-sterilized nodal shoots were incubated on

Murashige and Skoog (MS) medium supplemented with

8.88 lM benzyladenine (BA), 50 mg l-1 ascorbic acid, and

25 mg l-1 of each of citric acid and adenine sulphate. Cut

ends of nodal stem segments rapidly turned brown, and

cultures failed to establish. When 100 mg l-1 ascorbic acid

(ABA) and 25.0 mg l-1 polyvinyl pyrrolidone (PVP) were

added to the medium, explants remained healthy, and cul-

tures were established. Bud-breaking of nodal stem explants

resulted in multiple shoot formation. Shoots proliferated

(35–40 shoots per culture vessel) on MS medium as

described above, but supplemented with 4.44 lM BA and

0.57 lM indole acetic acid (IAA) and additives. After 4–5

passages, proliferating shoots exhibited tip-browning and

decline in growth and multiplication. However, when

shoots were transferred to fresh shoot proliferation medium

supplemented with 2.32 lM kinetin (Kn), sustained growth

and high rate of shoot proliferation (50–60 shoots per cul-

ture vessel) was observed. Shoots rooted when transferred

to medium consisting of half- strength MS medium with

9.84 lM indole butyric acid (IBA) and 0.02% activated

charcoal. Alternatively, individual shoots were pulsed with

984.0 lM IBA and transferred to glass bottles containing

sterile and moistened soilrite. These shoots rooted ex-vitro

and were acclimatized in the greenhouse. Plants were then

analyzed for puerarin content using HPLC, and leaves

showed maximum accumulation of purerarin.

Keywords Hardening � Micropropagation �Pueraria tuberosa � Puerarin

Abbreviations

BA 6-Benzyladenine

HPLC High performance liquid chromatography

IBA Indole-3-butyric acid

Kn Kinetin

MS Murashige and Skoog (1962) medium

NAA a-Naphthaleneacetic acid

NOA Naphthoxyacetic acid

PGR Plant growth regulator

PVP Polyvinyl pyrrolidone

SFP Spectral flux photon

Introduction

Pueraria tuberosa (Roxb. Ex Willd.) DC., a leguminous

plant with medicinal properties, is native to Aravalli ranges

of India. P. tuberosa is commonly known as Indian kudzu,

viddarikand, or patal khola in Hindi. Stems are twinning,

woody, and climb over trees. P. tuberosa produces sweet

and starchy root tubers (Lindley 1985) that are eaten raw

by wild animals and tribes inhabiting the region. Roots of

this plant are highly nodulated, and it is reported that

Pueraria spp. enrich soils through biological nitrogen fix-

ation (Selvakumar et al. 2008).

Pueraria tuberosa yields various isoflavonoids of high

antioxidant properties including daidzin, genistin, tectori-

din, and puerarin, (Kim et al. 2003; Pandey et al. 2007;

M. S. Rathore (&) � N. S. Shekhawat

Plant Biotechnology Unit, Department of Botany, Jai Narain

Vyas University, Jodhpur, Rajasthan 342033, India

e-mail: mahendersr@gmail.com

123

Plant Cell Tiss Organ Cult (2009) 99:327–334

DOI 10.1007/s11240-009-9608-9

Goyal and Ramawat 2008a, b). Puerarin, highly abundant

in P. tuberosa, has hypothermic, spasmolytic, hypotensive,

and anti arrhymatic activities (Kintzios et al. 2004). A

therapeutic effect of puerarin on diabetic nephropathy has

been reported (Mao and Gu 2005). Crude extracts of P.

tuberosa have contraceptive effects and induce uterine

changes in rats (Prakash et al. 1985). Plant derivatives also

demonstrate hypocholesterolemic effects (Zheng et al.

2002). This is also important in the treatment of alcohol

dependency due to inhibition of alcohol transport across

the gut membrane (Rezvani et al. 2003). This plant pos-

sesses lupinoside which can prevent damage of insulin

activity by free fatty acid (Dey et al. 2007).

Pueraria tuberosa is naturally propagated through seed

and tuber. However, wide-spread destruction of its natural

habitat and indiscriminate use of tubers have restricted its

reproduction and regeneration. As a result, this species is

threatened in its habitat, and has become rarely available.

Pueraria species exhibit high levels of genotypic diversity

(Pappert et al. 2000). Therefore, it is important to pursue a

concerted effort for germplasm conservation and develop

an efficient propagation system for selected clones to meet

market demand. Plant tissue culture has been used for off-

site conservation and micropropagation (Edson et al. 1997;

Arya et al. 2003). Thiem (2003) has developed a micro-

propagation system for P. lobata; while, Thanonkeo and

Panichajakul (2006) have reported on successful micro-

propagation of P. candollei var. mirifica. Moreover, Kin-

tzios et al. (2004) have reported on production of puerarin

from hairy root cultures induced from leaf explants of

phaseoloides transformed with Agrobacterium rhizogenes

using air lift bioreactors.

In this study, a micropropagation system for P. tuberosa

is established, and puerarin accumulation in roots, tubers,

and leaves of propagated plants was determined using

HPLC.

Materials and methods

Plant material

Plants and root tubers of P. tuberosa were collected from

Panerwa and Jhadol villages of Udaipur division (The

Aravalli Range) of Rajasthan (India) during the months of

July and August. These were transplanted to clay pots and

maintained in a greenhouse at Jai Narain Vyas University.

Fresh shoot sprouts from tubers were harvested, and

washed with sterile water. Nodal stem segments, 4.0–

5.0 cm in length, were pretreated with 0.1% (w/v) of

Bavistin (BASF India Limited, Mumbai, India) and 0.1%

(w/v) streptomycin (HiMedia Laboratories Private Limited,

Mumbai, India) solution for 15–20 min, surface-sterilized

with 0.1% HgCl2 for 3.0 min, rinsed 6–8 times with sterile

water, and kept in sterile and cold 0.1% of each of citric

acid and ascorbic acid for 15.0 min.

Explants were incubated on Murashige and Skoog

(MS) (1962) medium containing 8.0 g l-1 agar (Bacterio-

logical grade, Qualigens Fine Chemicals, Mumbai, India),

50 mg l-1 ascorbic acid (ABA), and 25 mg l-1 each of

citric acid and adenine sulphate, and supplemented with

(2.22, 4.44, 8.88, 13.32 and 17.76 lM) 6-benzyladenine

(BA) and (2.32, 4.65, 9.82, 13.92, and 18.56 lM) kinetin

(Kn). Single nodal explants was placed in each culture

tube. Cultures were maintained under 12 h photoperiod

of 30–40 lmol m-2 s-1 light intensity and 28 ± 2�C

temperature.

Following establishment of shoot cultures, shoots were

subcultured onto MS medium, as described above, but

supplemented with different concentrations of BA (1.11,

2.22, 4.44, 6.67, and 8.88 lM) and Kn (1.16, 2.32, 4.65,

6.69 and 9.28 lM). The medium also contained 100 mg l-1

of ascorbic acid (ABA), 25.0 mg l-1 polyvinyl pyrrolidone

(PVP), and 0.01–0.02% activated charcoal.

A total of one explant per tube was used, with ten

explants per treatment, and these were replicated three

times in a completely randomized design. All cultures were

subcultured to fresh medium once every 3 weeks.

Proliferating shoot cultures were further subcultured

onto fresh MS medium, as described above, but supple-

mented with 4.44 lM BA and (1.16, 2.32, 4.65, 6.69 and

9.28 lM) Kn, and 0.57 lM indole acetic acid (IAA). A

total of ten shoots per treatment, replicated thrice, were

used in a completely randomized design. Cultures were

sub-cultured once every 3–1/2 weeks. Data on number of

shoots per single explant and length of shoots (in mm) were

recorded after 3 weeks of culture.

Rooting

Shoots were rooted both in vitro and ex vitro. For in vitro

rooting, shoots of 4.0–5.0 cm in length were transferred to

rooting medium consisting of either full-, half-, and one-

fourth strength MS salts, 0.01–0.02% activated charcoal,

and varying concentrations of either indole-3-butyric acid

(IBA) (1.23, 2.46, 4.92, 9.84, 14.76 and 24.60 lM) or b-

naphthoxyacetic acid (NOA) (1.24, 2.47, 4.95, 9.89, 14.84

and 24.73 lM). A total of one shoot per treatment was

used, and this was replicated three times in a completely

randomized design.

For ex vitro rooting, shoots were pulsed with 492, 984,

1,476, 1,968, and 2,460 lM IBA or 495, 989, 1,484, 1,978,

and 2,473 lM NOA. Treated shoots were grown on soilrite

(Keltech Energies Limited, Karnataka, India) moistened

with �- strength MS macro-salts solution in capped glass

bottles (135 9 170 mm), and maintained under greenhouse

328 Plant Cell Tiss Organ Cult (2009) 99:327–334

123

conditions. Data on number of rooted shoots were recorded

after 21 days following treatment.

Acclimatization

In vitro-rooted shoots were removed from culture vessels

and washed with sterile water to remove culture medium to

avoid bacterial or fungal growth. These were transferred to

soilrite in glass bottles and maintained in the greenhouse.

All plantlets rooted in vitro and ex vitro, were acclima-

tized by gradually opening and finally removing plastic caps

off glass bottles over a period of 2 weeks. Acclimatized

plants were transferred to black polybags containing sand,

black soil, and vermin-compost in 3:1:1(w/w/w) ratio.

Puerarin extraction and HPLC analysis

Quantitative analysis of puerarin (7-Hydroxy-3-(4-

hydroxyphenyl)-1-benzopyran-4-one 8-(b-D-glucopyrano-

side); C21H20O9) content was conducted using high-per-

formance liquid chromatography (HPLC) system (Waters

1525, Milford, Massachusetts, USA) as described by Kin-

tzios et al. (2004). Unknown samples were prepared by

harvesting different tissues of mature donor plants prior to

flowering.

Leaves and tubers were collected from plants derived

from in vitro shoot proliferation and roots were harvested

form plantlets under in vitro rooting stage. Extracts were

prepared with 80% (v/v) methanol (5.0 ml per 500 mg dry

weight) at 25�C. The extract was then filtered through a

syringe filter (0.45 lm). The extraction procedure was

repeated three times. Combined filtrates were concentrated

by drying in waterbath, and residue was dissolved in 2.0 ml

methanol, before analysis. A total of 20 ll of sample

was injected into a dual wavelength absorbance detector

(Waters-2487) equipped with a Waters-1525 pump. Puera-

rin was analyzed using 5 lm ODS2 4.6 9 250 mm ana-

lytical column (Waters spherisorb�) eluted with methanol/

water (85:15 v/v) at a flow rate of 1.0 ml min-1. All hard-

ware was controlled and managed through ‘‘Breeze’’ (ver-

sion 3.20) software. Data obtained were assessed using

this software to estimate the concentration of puerarin in

unknown samples by comparing with standard. For quanti-

tative analysis, the system was calibrated with pure puerarin,

P 5555, 80% HPLC; Molecular Weight of 416.38 (Sigma–

Aldrich Chemie, Steinheim, Germany). A standard curve

was established using concentrations of 0.500–10,000 ng

per 20 ll.

Data analysis

Data were analyzed using single factor ANOVA (Gomez

and Gomez 1984), and mean comparisons were conducted

using LSD at 5% level of probability. HPLC estimation of

puerarin standard curves was fit using linear regression. All

results were averaged over two separate analyses from two

different culture vessels for determination of puerarin

content. For each tissue analyzed, eight replications were

used for puerarin determination.

Results and discussion

Culture establishment

Freshly-harvested shoots collected off tubers from green-

house-grown plants were found to serve as suitable sources

of explants for culture establishment. Initially, cut ends of

nodal stem segments, and subsequently whole explants

turned brown in color, and released phenolic compounds

into the medium which adversely affected culture estab-

lishment. To overcome this, nodal stem explants were

treated with chilled antioxidant solution consisting of citric

acid and ascorbic acid.

When explants were incubated on MS with 8.88 lM

BA, 95% of explants exhibited bud break with 2–4 shoots

per node after 10–15 days following culture (Table 1;

Fig. 1a). At lower concentrations (2.22–4.44 lM) of BA,

frequency of bud break dropped dramatically, and pro-

duced short shoots; while, on higher BA concentrations

(13.32–17.76 lM), frequency of bud break remained high,

but callus formation was observed along base of explants.

When explants were incubated on medium with Kn, fre-

quency of budbreak ranged between 65 and 90%; however,

mean number of shoots was lower (1–1.35) than that

obtained with BA per explant (Table 1). BA in the culture

medium significantly increased the shoot number. These

findings are similar to those reported by others (Aitken-

Christie and Connett 1992; Amoo et al. 2009: Rathore et al.

2007; Shekhawat et al. 1993).

Shoot proliferation

Browning of the shoots and the culture medium occurred,

limiting both the growth and multiplication of the shoots.

Incorporation of 25.0 mg l-1 PVP, 100.0 mg l-1 ascorbic

acid and 0.02% activated charcoal in culture medium

prevented browning and deterioration of cultures (Thomas

2008). On this medium containing 4.44 lM of BA,

39.80 ± 1.98 shoots of length 7.42 ± 0.27 cm were pro-

duced. On medium containing 4.65 lM of Kn fewer shoots

were produced (Table 2). On MS ? 4.44 lM of BA or

4.65 lM of Kn, after 4–5 cycles, shoots exhibited tip

burning, declined growth and also hyper hydration. Shoots

multiplication was also achieved by subculturing the shoot

clumps on MS with 4.44 lM BA, 1.16 lM Kn, 0.57 lM

Plant Cell Tiss Organ Cult (2009) 99:327–334 329

123

IAA and additives (Fig. 1b). On this amended culture

medium 56 ± 2.79 shoots of length 7.83 ± 0.21 cm dif-

ferentiated per culture vessel (Table 3). Shoots regenerated

were healthy and strong. Effect of BA was significantly

higher over Kn. With the combination of these several

factors, considerably high rate of shoot multiplication was

achieved for P. tuberosa. Krikorian (1994) suggested that

0.05–1.0% of activated charcoal can be incorporated in

culture medium. Activated charcoal adsorbs growth regu-

lators, but it also adsorbs substances presumed to be del-

eterious like phenolics, oxidized phenolics and gases like

ethylene and methane (Thomas 2008). These are inhibitory

substances that should be avoided or eliminated from in

vitro environment. It is assumed that darkening of cultures

and the culture medium is due to polyphenol oxidase

activity. Because of this, many such agents are used to

counter the darkening (Krikorian 1994).

Delay in subculture resulted in leaf fall, yellowing and

drying of shoots, therefore cultures have to be subcultured

Table 1 Effects of BA and Kn

on multiple shoot induction

from nodal stem segments of P.tuberosa grown on MS medium

with additives

PGR concentration (lM) Frequency of bud

break induction (%)

Mean number of

shoot/explant ± SD

Mean shoot

length ± SD (cm)

Control 0.00 40 0.40 ± 0.51 0.50 ± 0.66

BA 2.22 80 1.30 ± 0.48 1.25 ± 0.26

4.44 85 1.80 ± 0.42 2.01 ± 0.39

8.88 95 2.70 ± 0.48 2.98 ± 0.41

13.32 95 2.40 ± 0.51 2.45 ± 0.49

17.76 90 2.20 ± 0.42 2.15 ± 0.24

Kn 2.32 50 0.60 ± 0.69 1.0 ± 0.57

4.65 65 1.30 ± 0.48 1.33 ± 0.29

9.28 80 1.50 ± 0.52 1.75 ± 0.26

13.92 90 1.40 ± 0.51 1.60 ± 0.31

18.56 90 1.30 ± 0.48 1.35 ± 0.33

Fig. 1 Micropropagation of P. tuberosa. a Bud break from nodal

segments grown in MS with 8.88 lM BA and additives, b shoot

proliferation of shoots grown on MS with 4.44 lM BA, 1.16 lM Kn,

and 0.57 lM IAA along with additives, c in vitro rooting of shoots

grown on half-strength MS medium along with 9.84 lM IBA and

0.02% activated charcoal, d ex vitro rooted shoot along with tuber

formation (arrow indicates tuber), e excised shoot from a proliferating

culture showing in vitro tuber formation, f acclimatized plantlets of P.tuberosa in nursery, g germination of in vitro- derived tuber on PGR-

free MS medium (bar = 5.0 mm), and h ex vitro germination of

tubers formed in vitro on soilrite (bar = 5.0 mm)

Table 2 Effects of cytokinins on shoot proliferation of P. tuberosaincubated on MS supplemented with 0.57 lM IAA and additives

PGR concentration (lM) Mean number of

shoots/explant ± SD

Mean shoot

length ± SD

(cm)

Control 0.00 13.0 ± 1.56 3.12 ± 0.25

BA 1.11 23.60 ± 1.17 4.02 ± 0.32

2.22 28.50 ± 1.43 5.24 ± 0.26

4.44 39.80 ± 1.98 7.42 ± 0.27

6.67 35.70 ± 1.70 6.50 ± 0.25

8.88 32.30 ± 1.63 4.79 ± 0.28

Kn 1.16 20.80 ± 1.68 3.47 ± 0.19

2.32 23.80 ± 0.78 4.38 ± 0.24

4.65 25.60 ± 1.71 4.94 ± 0.22

6.69 26.80 ± 1.98 5.48 ± 0.26

9.28 24.30 ± 0.82 4.96 ± 0.12

330 Plant Cell Tiss Organ Cult (2009) 99:327–334

123

after a regular interval. It is suggested that once axillary

meristem is activated by treatment of cytokinins, these are

conditioned and thus require low cytokinins for prolifera-

tion. Repeated transfer of explants has been reported to be

useful for cloning (Franclet and Boulay 1989; Deora and

Shekhawat 1995). Rate of shoot multiplication achieved in

present study is high for any such plant. It was observed

that cytokinins (BA and Kn) along with auxin (IAA) have

significant effects on shoot proliferation (Rubio et al.

2009). Plant growth is directly affected with mineral

availability and to control this plants have evolved complex

regulatory mechanisms. Recent advances suggest PGRs

participate in control mechanism through cross-talk (Kup-

pusamy et al. 2009; Shimizu-Sato et al. 2009). It is now

evident that PGR hardly ever acts alone, but their pathways

are interlinked (Dettmer et al. 2009). On the contrary,

mineral nutrient uptake influences internal PGR biosyn-

thesis, this further justifies equilibrium between PGR syn-

thesis and nutrient uptake (Amoo et al. 2009; Rubio et al.

2009). In vitro tuber formation in cultures was obtained

when subculturing was delayed (Fig. 1e). Formation of

these tubers is presumed to be associated with nutrient

availability. Tubers when placed on PGR-free MS medium

gave rise to plantlets (Fig. 1g).

Rooting and acclimatization

The in vitro produced shoots were rooted by in vitro as well

as ex vitro approaches. Ninety-five percent of cloned

shoots rooted in vitro on � MS salts with 9.84 lM IBA

and 0.02% activated charcoal (Fig. 1c). Lateral root initi-

ation and primordium growth is promoted by auxin (Fukaki

and Tasaka 2009). Induction of rooting is affected by

several intrinsic and extrinsic factors (Wilson and Van

Staden 1990; Schiefelbein and Benfey 1991; Martin 2002).

The concentration of IBA and way of its treatment also

influences root induction (Van der Krieken et al. 1993).

The roots (2.60 ± 0.51 roots of length 4.53 ± 0.28 cm)

produced on this composition were healthy and strong as

compared to the roots (2.00 ± 0.47 roots of length

3.02 ± 0.35 cm) produced on medium containing higher

concentration (14.84 lM) of NOA (Table 4). On lower

(less than 9.84 lM) concentrations of IBA, shoots showed

delayed and poor response. On higher (24.60 lM) con-

centration of IBA the number (2.20 ± 0.63) of roots and

length (3.52 ± 0.21) was reduced.

Approximately 100% of shoots rooted ex vitro when

transferred to soilrite and grown under greenhouse condi-

tions after treatment with 984 lM IBA for 5.0 min

(Fig. 1d). Pulse treatment with NOA again showed delayed

and poor response as compared to IBA (Table 5). Effect of

IBA was found significant in inducing rooting as compared

to NOA (Rathore et al. 2007). Ex vitro tuber formation was

also observed at the base of rooted shoot (Fig. 1d). These

tubers produced plants on soilrite (Fig. 1h) under green

house in the months of September to November, though the

Table 3 Effects of concentrations of kinetin (Kn) on multiplication

of shoots of Pueraria tuberosa on MS with 4.44 lM BA, 0.57 lM

IAA, and additives

PGR concentration (lM) Mean shoot

number ± SD

Mean shoot

length ± SD

(cm)

Control 0.0 13.0 ± 1.56 3.12 ± 0.25

Kn 1.16 42.00 ± 2.44 7.12 ± 0.19

2.32 56.30 ± 2.79 7.83 ± 0.21

4.65 53.30 ± 1.88 7.07 ± 0.29

6.69 49.20 ± 1.81 6.54 ± 0.23

9.28 46.30 ± 1.63 5.04 ± 0.24

Table 4 Effects of type and

concentration of auxin on in

vitro rooting of shoots of

P. tuberosa grown on

half-strength MS with 0.02%

activated charcoal

PGR concentration (lM) Frequency

of rooting (%)

Mean root

number/explant ± SD

Mean root

length ± SD (cm)

Control 0.00 40.0 0.40 ± 0.69 0.45 ± 0.73

IBA 1.23 85.0 1.40 ± 0.51 1.57 ± 0.21

2.46 90.0 1.60 ± 0.51 2.00 ± 0.27

4.92 95.0 1.90 ± 0.56 3.31 ± 0.32

9.84 98.0 2.60 ± 0.51 4.53 ± 0.28

14.76 88.0 2.40 ± 0.51 3.83 ± 0.19

24.60 80.0 2.20 ± 0.63 3.52 ± 0.21

NOA 1.24 50.0 0.90 ± 0.31 1.23 ± 0.47

2.47 60.0 1.20 ± 0.42 1.85 ± 0.15

4.95 70.0 1.50 ± 0.52 2.28 ± 0.30

9.89 75.0 1.70 ± 0.48 2.44 ± 0.28

14.84 85.0 2.00 ± 0.47 3.02 ± 0.35

24.73 85.0 1.60 ± 0.51 2.10 ± 0.30

Plant Cell Tiss Organ Cult (2009) 99:327–334 331

123

percent of the tubers producing plants was low (20.0–

25.0%).

Both in vitro and ex vitro rooted plantlets were trans-

ferred to bottles containing autoclaved soilrite, which were

moistened with one-fourth strength MS liquid medium.

These were capped with polycarbonate and placed near pad

section in a greenhouse. After induction of roots from the

shoots the caps of bottles were gradually loosened and

finally removed. Plantlets were exposed to green house

conditions after 15–20 days of rooting. More than 85% of

the micropropagated plants were hardened productively

after a period of 45–50 days. The hardened and acclima-

tized plantlets were then successfully transferred to black

polybags (Fig. 1f).

Table 5 Effect of auxin

treatment of shoots of

P. tuberosa on ex-vitro root

induction

PGR concentration (lM) Frequency of

rooting (%)

Mean number of

roots/shoot ± SD

Mean root

length ± SD (cm)

Control 0 30.0 0.20 ± 0.42 0.28 ± 0.59

IBA 492 95.0 3.40 ± 1.07 2.40 ± 0.13

984 100.0 6.80 ± 0.91 4.41 ± 0.32

1,476 95.0 5.50 ± 0.52 4.08 ± 0.18

1,968 95.0 5.20 ± 0.63 3.69 ± 0.13

2,460 90.0 4.60 ± 0.51 3.52 ± 0.36

NOA 495 30.0 0.90 ± 0.56 1.06 ± 0.58

989 60.0 1.30 ± 0.48 1.75 ± 0.15

1,484 65.0 1.80 ± 0.63 1.99 ± 0.21

1,978 75.0 3.90 ± 0.56 2.59 ± 0.28

2,473 70.0 3.00 ± 0.66 2.24 ± 0.13

Fig. 2 HPLC analysis of puerarin content of in vitro derived tubers, leaves, and roots of P. tuberosa

332 Plant Cell Tiss Organ Cult (2009) 99:327–334

123

Puerarin content in different tissues of micropropagated

plants

Puerarin accumulation in the in vitro produced roots, tubers

and leaves was determined using HPLC. Roots, tubers and

leaves all accumulated puerarin (Fig. 2). Puerarin accu-

mulation was found to be the highest in leaf tissues

(696.73 lg g-1 dry wt.) followed by roots (413.37 lg g-1

dry wt.) and in vitro formed tubers with 149.12 lg g-1 dry

wt. of puerarin, respectively. The accumulation of puerarin

in organs of in vitro regenerated plants was found to be

higher as compared to mother plant. Puerarin accumula-

tion in mother plant was also higher in leaf tissue

(421.35 lg g-1 dry wt.) followed by roots (342.17 lg g-1

dry wt.) and then tuber with 126.74 lg g-1 dry wt.,

respectively (Table 6). The reason for increase production

in vitro can be due to culture conditions and the role of

different PGRs in promoting biosynthesis of active com-

pounds. Goyal and Ramawat (2008a) reported several fold

increase in levels of isoflavonoids with the incorporation of

two cytokinins together. Thanonkeo and Panichajakul

(2006) depicted the role of temperature in the production of

isoflavone. Incorporation of additives and activated char-

coal in the culture medium may have favored production

(Thomas 2008). Plants in ex vitro conditions are exposed to

several kinds of biotic and abiotic stresses, which can

affect the secondary metabolism of the plant. One can thus

clearly find seasonal and diurnal variations in concentra-

tions in plants. Beside these developmental stages, exoge-

nous and endogenous signals, regulation of metabolic

pathways either by genes or enzymes, compartmentation

and their transport play an important role (Verpoorte and

Alfermann 2000).

A successful and efficient micropropagation protocol

has been reported for the first time for P. tuberosa. High

rate of shoot multiplication with uniform growth has been

achieved. Plantlets were hardened successfully by ex vitro

approaches. This reduces need of in vitro root induction

and is more economical. The protocol developed can be

applied for large scale multiplication of P. tuberosa and for

study of secondary metabolites.

Acknowledgments We gratefully acknowledge financial supports

provided to the Department of Botany by University Grants Com-

missions (UGC) of India and the Department of Science and Tech-

nology (DST), Govt. of India under SAP (Special Assistance

Programme) and FIST (Infrastructure Development in Science and

Technology) schemes, respectively. The basic laboratory and green-

house infrastructure used for research work have been established as

Regional Micropropagation Unit for Arid regions with major funds of

Department of Biotechnology (DBT), Govt. of India under Net-

working programmes on micropropagation.

References

Aitken-Christie J, Connett M (1992) Micropropagation of forest trees.

In: Kurata K, Kozai T (eds) Transplant production systems.

Kluwer, The Netherlands, pp 163–194

Amoo SO, Finnie JF, Van Staden JV (2009) In vitro propagation of

Huernia hystrix: an endangered medicinal and ornamental

succulent. Plant Cell Tiss Organ Cult 96:273–278. doi:10.1007/

s11240-008-9484-8

Arya V, Shekhawat NS, Singh RP (2003) Micropropagation of

Leptadenia reticulata—a medicinal plant. In vitro Cell Dev Biol

Plant 39:180–185. doi:10.1079/IVP2002394

Deora NS, Shekhawat NS (1995) Micropropagation of Capparisdecidua (Forsk.) Edgew-a tree of arid horticulture. Plant Cell

Rep 15:278–281. doi:10.1007/BF00193736

Dettmer J, Elo A, Helariutta Y (2009) Hormone interactions during

vascular development. Plant Mol Biol 69:347–360. doi:10.1007/

s11103-008-9374-9

Dey D, Pal BC, Biswas T, Roy SS, Bandyopadhyay A, Mandal SK,

Giri BB, Bhattacharya S (2007) A lupinoside prevented fatty

acid induced inhibition of insulin sensitivity in 3T3 L1

adipocytes. Mol Cell Biochem 300:149–157. doi:10.1007/

s11010-006-9378-1

Edson JL, Wenny DL, Leege-Brusven AD, Everett RL (1997) Using

micropropagation to conserve threatened rare species in sustain-

able forests. J Sustain Forest 5:279–291

Franclet A, Boulay M (1989) Rejuvenation and clonal silviculture for

Eucalyptus and forest species harvested through short rotation.

In: Pereira JS, Lederberg JJ (eds) Biomass production by fast-

growing trees. Kluwer, The Netherlands, pp 267–274

Fukaki H, Tasaka M (2009) Hormone interactions during lateral root

formation. Plant Mol Biol 69:437–449. doi:10.1007/s11103-

008-9417-2

Gomez KA, Gomez AA (1984) Statistical procedure for agricultural

research. Wiley, New York

Goyal S, Ramawat KG (2008a) Synergistic effect of morphactin on

cytokinin-induced production of isoflavonoids in cell cultures of

Pueraria tuberosa (Roxb. Ex. Willd.) DC. Plant Growth Regul

55:175–181. doi:10.1007/s10725-008-9271-x

Goyal S, Ramawat KG (2008b) Ethrel treatment enhanced iso-

flavonoidds accumulation in cell suspension cultures of Puerariatuberosa, a woody legume. Acta Physiol Plant 30:849–853. doi:

10.1007/s11738-008-0190-2

Kim C, Shin S, Ha H, Kim JM (2003) Study of substance changes in

flowers of Pueraria thunbergiana Benth. during storage. Arch

Pharm Res 26:210–213. doi:10.1007/BF02976832

Kintzios S, Makri O, Pistola E, Matakiadis T, Shi HP, Economou A

(2004) Scale-up production of puerarin from hairy roots of

Pueraria phaseoloides in an airlift bioreactor. Biotechnol Lett

26:1057–1059. doi:10.1023/B:BILE.0000032963.41208.e8

Krikorian AD (1994) In vitro culture of plantation crops. In: Vasil IK,

Thorpe TA (eds) Plant cell and tissue culture. Kluwer, Dordr-

echt, pp 497–537

Table 6 Puerarin contents in various tissues of analyzed through

HPLC

Source of tissue Tissue Puerarin content

(lg g-1 dry wt.)

Mother plant Leaf 696.73 ± 0.85

Root 413.37 ± 0.65

Tuber 149.12 ± 0.69

In vitro-grown plantlets Leaf 421.35 ± 0.42

Root 342.17 ± 0.37

Tuber 126.74 ± 0.26

Plant Cell Tiss Organ Cult (2009) 99:327–334 333

123

Kuppusamy KT, Walcher CL, Nemhauser JL (2009) Cross-regulatory

mechanisms in hormone signaling. Plant Mol Biol 69:375–381.

doi:10.1007/s11103-008-9389-2

Lindley J (1985) Flora Medica: a botanical account of all the more

important plants used in medicine. Ajay Book Service, New

Delhi, p 243

Mao CP, Gu Z-L (2005) Puerarin reduces increased c-fos, c-jun, and

type IV collagen expression caused by high glucose in glomer-

ular mesangial cells. Acta Pharmacol Sin 26:982–986. doi:

10.1111/j.1745-7254.2005.00133.x

Martin KP (2002) Rapid micropropagation of Holostemma ada-kodienSchult., a rare medicinal plant, through axillary bud multiplica-

tion and indirect organogenesis. Plant Cell Rep 21:112–117. doi:

10.1007/s00299-002-0483-7

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bioassays with tobacco tissue cultures. Physiol Plant 15:473–497.

doi:10.1111/j.1399-3054.1962.tb08052.x

Pandey N, Chaurasia JK, Tiwari OP, Triphati YB (2007) Antioxidant

properties of different fractions of tubers from Pueraria tuberosaLinn. Food Chem 105:219–222. doi:10.1016/j.foodchem.2007.

03.072

Pappert RA, Hamrick JL, Donovan LA (2000) Genetic variation in

Pueraria lobata (Fabaceae), an introduced, clonal, invasive plant

of the southeastern United State. Am J Bot 87:1240–1245

Prakash AO, Saxena V, Shukla S, Mathur R (1985) Contraceptive

potency of Pueraria tuberosa D.C. and its hormonal status. Acta

Eur Fertil 16:59–65

Rathore JS, Rathore MS, Singh M, Singh RP, Shekhawat NS (2007)

Micropropagation of mature tree of Citrus limon. Indian J

Biotechnol 6:239–244

Rezvani AH, David HO, Marina P, Massi M (2003) Plant derivatives

in the treatment of alcohol dependency. Pharmacol Biochem

Behav 75:593–606. doi:10.1016/S0091-3057(03)00124-2

Rubio V, Bustos R, Irigoyen ML, Cardona-Lopez X, Rojas-Triana M,

Paz-Ares J (2009) Plant hormones and nurient signaling. Plant

Mol Biol 69:361–373. doi:10.1007/s1110-008-9380-y

Schiefelbein JW, Benfey PN (1991) The development of plant roots:

new approaches to underground problems. Plant Cell 3:1147–

1154. doi:10.1105/tpc.3.11.1147

Selvakumar G, Kundu S, Gupta AD, Shouche YS, Gupta HS (2008)

Isolation and characterization of nonrhizobial plant growth

promoting bacteria from nodules of Kudzu (Pueraria thunbergi-ana) and there effect on wheat seedling growth. Curr Microbiol

56:134–139. doi:10.1007/s00284-007-9062-z

Shekhawat NS, Rathore TS, Singh RP, Deora NS, Rao SR (1993)

Factors affecting in vitro clonal propagation of Prosopis ciner-aria. Plant Growth Regul 12:273–280. doi:10.1007/BF00027208

Shimizu-Sato S, Tanaka M, Mori H (2009) Auxin-cytokinin interac-

tions in the control of shoot branching. Plant Mol Biol 69:429–

435. doi:10.1007/s11103-008-9416-3

Thanonkeo S, Panichajakul S (2006) Production of isoflavones,

daidzein and genistein in callus cultures of Pueraria candolleiWall. ex Benth. var. mirifica. Songklanakarin J Sci Technol 28:

45–53

Theim B (2003) In vitro propagation of isoflavone-producing

Pueraria lobata (Willd.) Ohwi. Plant Sci 165:1123–1128. doi:

10.1016/S0168-9452(03)00320-0

Thomas TD (2008) The role of activated charcoal in plant tissue

culture. Biotechnol Adv 26:618–631. doi:10.1016/j.biotechadv.

2008.08.003

Van der Krieken WM, Breteler H, Visser MHM, Mavridou D (1993)

The role of the conversion of IBA into IAA, on root regeneration

in apple: introduction of a test system. Plant Cell Rep 12:203–

206. doi:10.1007/BF00237054

Verpoorte R, Alfermann AW (2000) Metabolic engineering of plant

secondary metabolism. Kluwer, The Netherlands

Wilson PJ, Van Staden J (1990) Rhizocaline, rooting co-factors, and

the concept of promoters and inhibitors of adventitious rooting-a

review. Ann Bot 66:479–490

Zheng G, Zhang X, Zheng J, Gong W, Zheng X, Chen A (2002)

Hypocholesterolemic effect of total isoflavones from Puerarialobata in ovariectomized rats. Zhong Yao Cai 25:273–275

334 Plant Cell Tiss Organ Cult (2009) 99:327–334

123