Int. J. Med. Arom.

Plants, ISSN 2249 4340

RESEARCH ARTICLE

Vol. 1, No. 3, pp. 306-312, December 2011

Development of Protocol for in vitro Propagation and Conservation of

Vitex negundo L.
M.M. RAHMAN*, S.K. BHADRA
Department of Botany, University of Chittagong, Chittagong, Bangladesh
Article History: Received 22nd October 2011, Revised 27th November 2011, Accepted 28th November 2011.

Abstract: Shoot apex, leaf and nodal segments of field grown plants of Vitex negundo L were aseptically cultured on MS
and B5 medium fortified with different concentrations of cytokinin (BAP and Kn) and auxin (NAA and IAA) at different
combinations. Nodal segments underwent direct organogenesis producing MSBs very efficiently. Maximum number of
MSBs (3.60.51) per explant was induced on MS medium supplemented with 2.0 mg/l BAP + 0.5 mg/l NAA. Shoot
apex though produced MSBs, the magnitude was meagre and the leaf segments failed to produce MSBs. Of the two basal
media, MS was found superior to B5 for promoting response of the explants. The highest elongation of MSBs
(2.170.17cm) took place in MS medium containing 2.0 mg/l BAP + 1.0 mg/l IAA. Elongated multiple shoot buds were
rooted on rooting media. The maximum number of roots (35.601.63) per shoot was observed in half strength MS medium supplemented with 1.0 mg/l NAA. In vitro developed complete seedlings were acclimatized in outer environment
with 82% survivability. Experiment on in vitro culture aided ex situ conservation was based on induced growth retardant
and for this purpose abscisic acid (ABA) at different concentrations and low nutrient conditions were used in MS medium to keep the growth of in vitro seedlings stunted. It was observed that the growth of seedlings was minimum
(0.710.10cm) in half strength MS medium containing 0.4mg/l ABA which is about three times less than that of full
strength MS medium and the seedlings can be stored more than eight months without any subculture.
Keywords: Conservation; In vitro; Propagation; Protocol; Vitex negundo.

Introduction
Vitex negundo L. (Bengali name: Nishinda)
is a widely used medicinal plant of the family
Verbenaceae. Leaves of this plant are antiparasitic and used as alterative, vermifuge and anodyne, effective against inflammatory swellings
of joints in rheumatic attack, relieve catarrh and
headache. Juice of fresh leaves removes foetid
discharges and worms from ulcers. Flowers are
astringent and cooling. Fruits are nervine stimulant, emmenagogue and vermifuge. Root is tonic, febrifuge, expectorant and diuretic. It regulates hormones and increases breast-milk production (Ghani 2003).
The availability of this plant species is reducing day by day due to indiscriminate collections by the local herbalists and habitat destruction. Still now there is no step for commercial
cultivation of this medicinal plant species. Under such a situation we need to develop technology for mass scale propagation and conservation
of this important medicinal plant species for
supporting the demand of the related industries.
*Corresponding author: (E-mail) mmrahman11@yahoo.com
2011 Open Access Science Research Publisher

In vitro culture based micropropagation technique has been successfully used for rapid and
mass propagation of many medicinal plants (Purohit and Dave 1996; Sudha and Seeni 1996;
Hassan and Roy 2005; Sultana and Handique
2005; Biswas et al. 2009; Baksha et al. 2007;
Roy 2008; Bhadra et al. 2009). In vitro regeneration of V. negundo has also been reported by
some authors (Sahoo and Chand 1998; Hiregoudar et al. 2006; Jawahar et al. 2008) In vitro culture technique has also been used in ex situ conservation of many plant species (Kartha et al.
1988; Bajaj 1988; Kondo et el. 1989). It is now
established that in vitro culture in conjunction
with cryopreservation or the use of growth retardants can be adopted for conservation of
plant species, which are at threatened or rare
state. The present study was therefore undertaken with the objectives to develop a more efficient and reliable protocol for rapid in vitro multiplication and ex situ conservation of this important medicinal plant species.

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Int. J. Med. Arom. Plants

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In vitro Propagation and Conservation of Vitex negundo

Materials and Methods

Results and Discussion

Shoot apex, leaf and nodal segments from

juvenile twigs of field grown plants were collected and thoroughly washed under running tap
water. The materials were then surface sterilized
with 1% savlon and liquid soap for 5 - 10 minutes with constant shaking. The materials were
then washed 3 - 4 times with distilled water for
complete removal of detergent and taken under
running laminar airflow cabinet and transferred
to 500 ml sterilized conical flask. After rinsing
with 70% ethanol for less than 60 seconds, they
were immersed in 0.1% HgCl2 for 5-7 minutes.
To remove every trace of the sterilant, the materials were then washed 4 -5 times with sterile
distilled water. Then the explants were cut into
pieces (0.5 - 1.0 cm) and grown on MS and B5
media supplemented with different concentrations and combinations plant growth regulators
(PGRs). For elongation, the induced shoot buds
were individually grown in aseptic condition on
a wide range of PGR supplemented MS media
and the vessels were maintained in the culture
room in the same light and temperature conditions. In order to promote root formation the
multiple shoot buds at a height of 2 - 3 cm were
separated and individually cultured on rooting
media. At first half and one fourth strength MS
medium without any PGR were used and then
half strength MS medium supplemented with
different concentrations and combinations of
auxins (IBA, IAA and NAA) were used for profuse rooting.

Shoot apex, leaf and nodal segments of field

grown plants were aseptically cultured on MS
medium supplemented with different concentrations of cytokinins (BAP and Kn) and auxins
(NAA and IAA) in different combinations.
Nodal segments underwent direct organogenesis
producing multiple shoot buds (MSBs) in different PGR containing media (Table 1).

In all cases the media were solidified with

0.8% (w/v) agar (Sigma) and pH was adjusted
to 5.8 before autoclaving for 30 minutes at
121C under a pressure of 1.1 kg/cm2. All culture vessels with inoculated explants were incubated in a culture room at 25 20C under a regular cycle of 14 hr light and 10 hr dark
After sufficient growth the well rooted plantlets were transferred to outside environment
and planted in pots containing a mixture of soil
and compost (1:1) through successive phases of
acclimatization. For ex situ conservation the in
vitro developed seedlings were cultured on full,
half and one fourth strength MS medium supplemented with different concentrations of abscisic acid (ABA).
Rahman and Bhadra

Table 1: Effect of different concentrations and

combination of PGRs in MS medium on induction of MSBs in nodal explants of V. negundo.
PGR supplements
in the media (mg/l)
BAP

Kn

BAP+NAA

BAP+IAA

Kn+NAA

Kn+IAA

1.0
2.0
3.0
1.0
2.0
3.0
2.0+0.2
2.0+0.5
2.0+1.0
3.0+0.2
3.0+0.5
3.0+1.0
2.0+0.2
2.0+0.5
2.0+1.0
3.0+0.2
3.0+0.5
3.0+1.0
2.0+0.2
2.0+0.5
2.0+1.0
3.0+0.2
3.0+0.5
3.0+1.0
2.0+0.2
2.0+0.5
2.0+1.0
3.0+0.2
3.0+0.5
3.0+1.0

Days % of explants No. of shoot

to
giving
buds* /explant
response
response
( x SE)
10-12
82
1.80.20
10-12
85
2.00.32
9-12
85
2.20.20
10-14
78
1.670.21
10-14
80
1.800.20
10-13
80
2.200.20
8-10
86
2.20.20
8-10
93
3.60.51
8-11
90
3.40.51
8-12
88
2.40.24
8-12
90
2.80.37
8-10
92
3.00.32
7-10
88
1.80.20
8-10
90
2.20.37
8-12
92
2.20.20
8-10
86
2.20.20
8-12
91
2.60.24
8-10
91
2.60.24
10-14
82
2.330.21
9-14
85
2.600.24
8-14
87
2.500.22
10-13
82
2.400.24
8-14
88
2.660.21
8-14
87
2.600.24
10-14
81
2.000.26
8-13
87
2.200.20
8-12
86
2.170.17
10-12
80
2.170.30
8-12
85
2.400.24
8-12
86
2.200.20

* Values are the mean of three replicates with 15

explants.

Multiple shoot buds were initiated within 8

12 days of culture. The response of shoot apex
was very poor. In a few combinations of media
one shoot apex produced only 1 - 2 MSBs. So
the results of induced MSBs from shoot apices
are not presented here. Leaf segments failed to
give response in these media combinations. This
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Int. J. Med. Arom. Plants

finding indicates the specificity of explant and

PGR interaction. Maximum number of nodal
explants (93%) produced MSBs on 2.0 mg/l
BAP + 0.5mg/l NAA. The average number of
MSBs (3.60.51) per explant was also highest
in this media combination (Figure 1A). The
lowest number of MSBs (1.670.21) per explant
was recorded in 1.0 mg/l Kn and in this media
composition the minimum number of explant
(78%) produced MSBs. Such positive response

308
In vitro Propagation and Conservation of Vitex negundo

of BAP in combination with NAA on induction

of MSBs has been noted in other medicinal
plant species such as Gloriosa superba (Hassan
and Roy 2005), Rauvolfia serpentina (Baksha et
al. 2007) and Boerhaavia diffusa (Roy 2008;
Biswas et al. 2009). On the other hand, Sahoo
and Chand (1998) and Islam et al. (2009) reported that MS medium supplemented with BA
individually enhanced the induction of MSBs in
V. negundo.

Figure 1: Different steps of in vitro propagation and conservation of V. negundo.

(A) Initiation and proliferation of MSBs on MS medium containing 2.0 mg/l BAP + 0.5 mg/l NAA.
(B) Elongation of MSBs on MS medium containing 2.0 mg/l BAP + 1.0 mg/l IAA.
(C) Induction and proliferation of roots in MS medium containing 1.0 mg/l NAA
(D) In vitro developed complete seedling after transplantation in small plastic pot.
(E) Growth of shoot buds in 0.4 mg/l ABA supplemented half strength MS medium.
(F) Growth of shoot buds in MS medium (as control).

Rahman and Bhadra

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In vitro Propagation and Conservation of Vitex negundo

Int. J. Med. Arom. Plants

% of explants giving response

In order to examine the efficiency of the

PGR combinations in the background of different nutrient compositions, both MS and B5 media were used separately as background of some
PGR combinations and the results are depicted
in Figure 2. Comparative study revealed that in
maximum cases MS was better than B5 in terms
of in vitro culture in V. negundo. Baskaran and
Jayabalan (2005) in their experiment on Eclipta
alba recorded the higher efficiency of MS medium compared to that of B5 for in vitro culture.

MS medium

95

B5 medium

90
85
80
75
70
BN-1 BN-2 BN-3 BN-4 BN-5 BN-6 BI-1 BI-2 BI-3 BI-4 BI-5 BI-6
Media composition

No. of MSBs/explant

MS medium

B5 medium

4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
BN-1 BN-2 BN-3 BN-4 BN-5 BN-6 BI-1 BI-2 BI-3 BI-4 BI-5 BI-6
Media composition

Figure 2: Comparison between MS and B5 medium in terms of induction of MSBs in nodal

explants of V. negundo.
BI-1=2.0 mg/l BAP+0.2 mg/l IAA, BI-2=2.0
mg/l BAP+0.5 mg/l IAA,
BI-3=2.0 mg/l BAP+1.0 mg/l IAA, BI-4=3.0
mg/l BAP+0.2 mg/l IAA,
BI-5=3.0 mg/l BAP+0.5 mg/l IAA, BI-6=3.0
mg/l BAP+1.0 mg/l IAA,
BN-1=2.0 mg/l BAP+0.2 mg/l NAA, BN-2=2.0
mg/l BAP+0.5 mg/l NAA,
BN-3=2.0 mg/l BAP+1.0 mg/l NAA, BN-4=3.0
mg/l BAP+0.2 mg/l NAA,
BN-5=3.0 mg/l BAP+0.5 mg/l NAA, BN-6=3.0
mg/l BAP+1.0 mg/l NAA.

Rahman and Bhadra

Although nodal segments gave rise to

MSBs, they did not grow rapidly. In order to
induce rapid elongation, these MSBs were isolated and individually grown on different spectrum of same PGR combinations. The results
obtained from this experiment are summarized
in Table 2. Here also the role of PGR combination in growth and development of plant organ
was
proved.
The
highest
elongation
(2.170.17cm) of MSBs was developed on 2.0
mg/l BAP + 1.0 mg/l IAA (Figure 1B) and the
lowest elongation (1.130.08cm) was on 1.5
mg/l Kn + 0.5 mg/l NAA.
In order to promote root formation the multiple shoot buds at a height of 2 - 3 cm were separated and individually cultured on rooting
media. Response of rooting was very much dependent on type and concentration of auxins
supplements in the media. Among the three
types of auxins, NAA was superior to that of
IBA and IAA in terms of induction of number
of root per shoot (Table 3). The maximum number of roots (35.601.63) per shoot was formed
in half strength MS medium supplemented with
1.0 mg/l NAA (Figure 1C) and the minimum
number (2.600.40) was in half strength MS +
2.0 mg/l IAA. The highest and lowest elongation of root (2.620.11 and 1.330.12cm) was
recorded in half strength MS medium containing
1.0 mg/l IBA and 0.5 mg/l IBA respectively.
The efficiency of NAA in in vitro rooting has
been reported in many other medicinal plant
species including Lavandula vera (Andrade et
al. 1999), Curculigo orchioides (Wala and Jasrai
2003), Vitex trifolia (Hiregoudar et al. 2006)
and Rauvolfia serpentina (Baksha et al. 2007).
On the other hand, Jawahar et al. (2008) and
Islam et al. (2009) reported that IBA (0.5 mg/l)
supplemented half strength MS medium was
best for induction of root system in V. negundo.
After rooting the complete plantlets were transferred to outside environment and planted in
pots (Figure 1D) containing garden soil and
compost (1:1) through successive phases of acclimatization. Survival percentage of the regenerated plantlets under natural conditions was
about 82% and the plantlets were morphologically uniform having normal growth.

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Int. J. Med. Arom. Plants

Table 2: Data on elongation of MSBs of V. negundo when cultured on 0.8% (w/v) agar solidified
MS medium supplemented with different PGR combinations.
PGR supplements in the medium
(mg/l)

BAP+NAA

BAP+IAA

Kn+NAA

Kn+IAA

1.5+0.5
1.5+1.0
2.0+0.5
2.0+1.0
2.5+0.5
2.5+1.0
1.5+0.5
1.5+1.0
2.0+0.5
2.0+1.0
2.5+0.5
2.5+1.0
1.5+0.5
1.5+1.0
2.0+0.5
2.0+1.0
2.5+0.5
2.5+1.0
1.5+0.5
1.5+1.0
2.0+0.5
2.0+1.0
2.5+0.5
2.5+1.0

Initial length* (cm) of

individual shoot buds

Length* (cm) of individual

Increase in length* (cm) of
shoot buds after 30d of cul- shoot buds after 30d of culture

( x SE)
1.330.11
1.420.15
1.330.11
1.250.11
1.420.15
1.420.15
0.920.05
1.330.11
1.330.11
1.500.11
1.420.08
1.420.15
1.250.11
1.300.12
1.400.12
1.300.10
1.300.12
1.200.12
1.400.10
1.300.12
1.200.12
1.300.12
1.100.10
1.600.10

ture ( x SE)
2.580.08
2.840.11
2.750.17
2.830.17
2.840.21
2.750.17
2.170.15
3.160.21
2.580.08
3.670.20
2.830.21
3.250.34
2.380.08
2.550.17
2.600,19
3.100.19
2.650.10
2.600.18
2.600.10
2.750.16
2.800.20
3.000.16
2.350.19
3.050.17

( x SE)
1.250.11
1.420.15
1.420.15
1.580.15
1.420.15
1.330.11
1.250.11
1.830.21
1.250.11
2.170.17
1.410.15
1.830.21
1.130.08
1.250.11
1.200.12
1.800.12
1.350.10
1.400.19
1.200.12
1.450.12
1.600.19
1.700.12
1.250.11
1.450.12

* Values are the mean of three replicates with 15 explants.

Table 3: Effect of auxin, low nutrient conditions and their combinations on induction and growth of
roots in in vitro developed shoot buds of V. negundo.
PGR supplements in the media (mg/l) Days to root induction

% of rooting No. of root* /shoot bud

(

x SE)

Length* of individual
root (cm)
(

th MS without PGRs
MS without PGRs
MS+IBA

MS+IAA

MS+NAA

MS+
IBA+IAA
MS+
IBA+NAA

0.5
1.0
2.0
0.5
1.0
2.0
0.5
1.0
2.0
2.0+0.5
2.0+1.0
2.0+2.0
2.0+0.5
2.0+1.0
2.0+2.0

8-10
6-7
15-20
4-5
4-5
12-15
15-20
20-25
8-12
8-12
7-12
--12-15
8-13
7-12
8-12

70
72
75
82
88
72
78
77
85
85
90
--75
92
95
95

2.800.22
3.600.40
3.600.40
4.200.37
11.200.73
3.600.40
3.800.37
2.600.40
33.002.24
35.601.63
32.802.44
--8.601.08
6.000.32
13.200.58
6.800.37

x SE)

1.550.12
1.540.16
1.330.12
2.620.11
2.600.14
1.620.19
1.720.13
1.690.10
1.480.13
1.960.17
2.290.22
--2.440.15
2.260.11
2.530.14
2.370.19

* Values are the mean of three replicates with 15 explants; --= No response
In vitro regeneration of plants ultimately results in development of complete plantlets and
because of their rapid growth it becomes very
difficult to keep the culture for long period for
conservation. There are several methods of ex
situ conservation such as cryopreservation, algiRahman and Bhadra

nate encapsulation, use of growth retardants and

low temperature storage for in vitro conservation of genetic resources. But in the present
study only ex situ conservation based on induced growth retardant was followed. And for
this purpose abscisic acid (ABA) at different
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Int. J. Med. Arom. Plants

concentrations and low nutrient conditions were

used in MS medium to keep the growth of in
vitro seedlings stunted so that they can be kept
for longer period. Full strength MS medium was
used here as control and the other media compositions were used to examine the poor growth of
seedlings. The data on elongation of seedlings
were recorded after 30 days of culture and the
results are summarized in Table 4. It was found
that the growth of seedlings was lowest
(0.710.10cm) in half strength MS medium containing 0.4mg/l ABA (Figure 1E) followed by
that in one fourth MS + 0.4 mg/l ABA and one
fourth MS + 0.2 mg/l ABA. In control medium

311
In vitro Propagation and Conservation of Vitex negundo

the growth was 1.920.15cm (Figure 1F). In

comparison, growth in half strength MS +
0.4mg/l ABA was about three times less than
that of full strength MS medium. It was found
that the seedlings could survive normally under
this stunted growth condition for more than 8
months without nay subculture. Agarwal et al.
(1992) succeeded in slow growth storage of in
vitro developed shoots of Vanilla walkeriae an
endangered orchid using low nutrient media.
Charoensub and Phansiri (2004) found that
mannitol added to the media could reduce the
growth of in vitro derived plantlets of Plumbago
indica.

Table 4: Data on elongation of in vitro developed shoots of V. negundo grown on different low nutrients and ABA containing media.
Media with or without ABA
Initial length*
Length* (cm) of shoot
(mg/l)
(cm) of shoot buds after 30d of culture
( x SE)
( x SE)
MS (Control)
1.250.11
3.270.17
MS
1.580.15
3.330.17
MS
1.660.11
3.080.08
MS+0.1mg/l ABA
1.750.11
3.330.11
MS+0.2mg/l ABA
1.420.15
2.710.10
MS+0.4mg/l ABA
1.330.17
2.410.14
MS+0.1mg/l ABA
1.580.15
2.750.16
MS+0.2mg/l ABA
1.580.08
2.410.08
MS+0.4mg/l ABA
1.670.17
2.380.13
MS+0.1mg/l ABA
1.170.11
2.210.14
MS+0.2mg/l ABA
1.420.08
2.210.14
MS+0.4mg/l ABA
1.500.18
2.250.09

Increase in length* (cm) of shoot after

30d of culture
( x SE)
1.920.15
1.750.21
1.420.15
1.580.15
1.290.19
1.080.14
1.170.12
0.830.11
0.710.10
1.040.10
0.790.10
0.750.09

* Values are the mean of three replicates with 15 explants.

Conclusion

Acknowledgement

From the overall results of this research

work it can be concluded that the protocol for
production of complete seedlings under in vitro
condition could be developed in V. negundo and
this protocol could be used for mass propagation
by our herbal industries. It was proved that induction of MSBs through direct organogenesis
were dependent on PGR supplements in the medium. The results of in vitro aided ex situ conservation indicated that it is possible to go for
this kind of conservation strategy on short term
basis until cryopreservation facilities can be developed. Here the selection of growth retardant,
its concentrations as well as low nutrient condition of the medium need to be identified properly.

The first author gratefully acknowledges the

financial support of the University Grants
Commission of Bangladesh in the form of Ph.D.
scholarship.

Rahman and Bhadra

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