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

DOI 10.1007/s11240-009-9608-9

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
Ó Springer Science+Business Media B.V. 2009

Abstract Pueraria tuberosa, a medicinally important analyzed for puerarin content using HPLC, and leaves
leguminous plant, yielding various isoflavanones including showed maximum accumulation of purerarin.
puerarin, is threatened, thus requiring conservation. In this
study, fresh shoot sprouts of P. tuberosa, produced by Keywords Hardening
Micropropagation

tubers, were used as explants for in vitro micropropagation. Pueraria tuberosa
Puerarin
Surface-sterilized nodal shoots were incubated on
Murashige and Skoog (MS) medium supplemented with
Abbreviations
8.88 lM benzyladenine (BA), 50 mg l-1 ascorbic acid, and
BA 6-Benzyladenine
25 mg l-1 of each of citric acid and adenine sulphate. Cut
HPLC High performance liquid chromatography
ends of nodal stem segments rapidly turned brown, and
IBA Indole-3-butyric acid
cultures failed to establish. When 100 mg l-1 ascorbic acid
Kn Kinetin
(ABA) and 25.0 mg l-1 polyvinyl pyrrolidone (PVP) were
MS Murashige and Skoog (1962) medium
added to the medium, explants remained healthy, and cul-
NAA a-Naphthaleneacetic acid
tures were established. Bud-breaking of nodal stem explants
NOA Naphthoxyacetic acid
resulted in multiple shoot formation. Shoots proliferated
PGR Plant growth regulator
(35–40 shoots per culture vessel) on MS medium as
PVP Polyvinyl pyrrolidone
described above, but supplemented with 4.44 lM BA and
SFP Spectral flux photon
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 Introduction
supplemented with 2.32 lM kinetin (Kn), sustained growth
and high rate of shoot proliferation (50–60 shoots per cul- Pueraria tuberosa (Roxb. Ex Willd.) DC., a leguminous
ture vessel) was observed. Shoots rooted when transferred plant with medicinal properties, is native to Aravalli ranges
to medium consisting of half- strength MS medium with of India. P. tuberosa is commonly known as Indian kudzu,
9.84 lM indole butyric acid (IBA) and 0.02% activated viddarikand, or patal khola in Hindi. Stems are twinning,
charcoal. Alternatively, individual shoots were pulsed with woody, and climb over trees. P. tuberosa produces sweet
984.0 lM IBA and transferred to glass bottles containing and starchy root tubers (Lindley 1985) that are eaten raw
sterile and moistened soilrite. These shoots rooted ex-vitro by wild animals and tribes inhabiting the region. Roots of
and were acclimatized in the greenhouse. Plants were then this plant are highly nodulated, and it is reported that
Pueraria spp. enrich soils through biological nitrogen fix-
ation (Selvakumar et al. 2008).
M. S. Rathore (&)
N. S. Shekhawat
Pueraria tuberosa yields various isoflavonoids of high
Plant Biotechnology Unit, Department of Botany, Jai Narain
Vyas University, Jodhpur, Rajasthan 342033, India antioxidant properties including daidzin, genistin, tectori-
e-mail: mahendersr@gmail.com din, and puerarin, (Kim et al. 2003; Pandey et al. 2007;

123
328
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
123
Plant Cell Tiss Organ Cult (2009) 99:327–334
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
Plant Cell Tiss Organ Cult (2009) 99:327–334
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 spherisorbÒ) 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
329
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
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330 Plant Cell Tiss Organ Cult (2009) 99:327–334

Table 1 Effects of BA and Kn

PGR concentration (lM) Frequency of bud Mean number of Mean shoot
on multiple shoot induction
break induction (%) shoot/explant ± SD length ± SD (cm)
from nodal stem segments of P.
tuberosa grown on MS medium Control 0.00 40 0.40 ± 0.51 0.50 ± 0.66
with additives
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

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 2 Effects of cytokinins on shoot proliferation of P. tuberosa

incubated on MS supplemented with 0.57 lM IAA and additives
PGR concentration (lM) Mean number of Mean shoot
shoots/explant ± SD 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
Fig. 1 Micropropagation of P. tuberosa. a Bud break from nodal 4.44 39.80 ± 1.98 7.42 ± 0.27
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, 6.67 35.70 ± 1.70 6.50 ± 0.25
and 0.57 lM IAA along with additives, c in vitro rooting of shoots 8.88 32.30 ± 1.63 4.79 ± 0.28
grown on half-strength MS medium along with 9.84 lM IBA and Kn 1.16 20.80 ± 1.68 3.47 ± 0.19
0.02% activated charcoal, d ex vitro rooted shoot along with tuber
2.32 23.80 ± 0.78 4.38 ± 0.24
formation (arrow indicates tuber), e excised shoot from a proliferating
culture showing in vitro tuber formation, f acclimatized plantlets of P. 4.65 25.60 ± 1.71 4.94 ± 0.22
tuberosa in nursery, g germination of in vitro- derived tuber on PGR- 6.69 26.80 ± 1.98 5.48 ± 0.26
free MS medium (bar = 5.0 mm), and h ex vitro germination of 9.28 24.30 ± 0.82 4.96 ± 0.12
tubers formed in vitro on soilrite (bar = 5.0 mm)

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

Table 3 Effects of concentrations of kinetin (Kn) on multiplication these tubers is presumed to be associated with nutrient
of shoots of Pueraria tuberosa on MS with 4.44 lM BA, 0.57 lM availability. Tubers when placed on PGR-free MS medium
IAA, and additives
gave rise to plantlets (Fig. 1g).
PGR concentration (lM) Mean shoot Mean shoot
number ± SD length ± SD Rooting and acclimatization
(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
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
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 4 Effects of type and

PGR concentration (lM) Frequency Mean root Mean root
concentration of auxin on in
of rooting (%) number/explant ± SD length ± SD (cm)
vitro rooting of shoots of
P. tuberosa grown on Control 0.00 40.0 0.40 ± 0.69 0.45 ± 0.73
half-strength MS with 0.02%
activated charcoal 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

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

Table 5 Effect of auxin

PGR concentration (lM) Frequency of Mean number of Mean root
treatment of shoots of
rooting (%) roots/shoot ± SD length ± SD (cm)
P. tuberosa on ex-vitro root
induction 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

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).

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