Industrial Crops and Products 25 (2007) 169–177

Mycorrhizal fungi and growth and development of

micropropagated Scutellaria integrifolia plants
N. Joshee a , S.R. Mentreddy b,∗ , Anand K. Yadav a
a Agricultural Research Station, Fort Valley State University, 1005 State University Drive, Fort Valley, GA 31030, USA
b Department of Plant and Soil Science, Alabama A&M University, P.O. Box 1208, Normal, AL 35762, USA

Received 3 November 2005; received in revised form 15 August 2006; accepted 15 August 2006

Abstract
Scutellaria species have been used in many traditional medical systems and is well known among the Native American tribes as
a strong emmenagogue and as a female medicinal herb. The inoculation of arbuscular mycorrhiza fungi (AMF) into the roots of
micropropagated plantlets could help not only mass propagate these species, but also help grow in marginal, phosphorus deficient
soils. Leaves, shoot apices, and nodal segments from wild as well as from greenhouse-grown plants were used to initiate cultures in
Murashige and Skoog (MS) medium supplemented with cytokinins benzyladenine (BA), kinetin, thidiazuron (TDZ), naphthalene
acetic acid (NAA), and indole butyric acid (IBA) Among all the explants tested for shoot bud induction, only shoot tips and nodal
explants were responsive. Explants swelled and became rough on the surface at the end of 3-week incubation with many green
shoot buds. Two to 3 weeks after transfering to rooting media with or without IBA, all shoots developed roots. In vitro raised plants
were acclimatized in a mist chamber and transferred to 6-l containers in the greenhouse to study the role of AMF on plant growth
and development. Inoculation with AMF, showed positive effects on plant growth, particularly root development compared with
the control plants. Among the five AMF strains tested, S3004 increased plant height and fresh weights of shoot, root, and seed.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Bioactive compounds; Medicinal plant; Root:shoot ratio; Shoot bud induction

1. Introduction S. amoena, S. hypericifolia, S. rehderiana, S. tenax, and

Plant-based medicines are responsible for tremen-
dous economic impact worldwide (De Smet, 1997).
Scutellaria species have been extensively used in the tra-
ditional medical systems of China, Korea, India, Japan,
many European countries, and North America. The most
extensively used species is Scutellaria baicalensis or
Baical skullcap. Other medicinal Scutellaria species are
∗ Corresponding author. Tel.: +1 256 372 4250;
fax: +1 256 372 5429.
E-mail address: smentreddy@email.aamu.edu (S.R. Mentreddy).
S. viscidula (Song, 1981). The skullcaps (Scutellaria
spp., family Lamiaceae), are herbaceous, slender plants
scattered over temperate regions and tropical mountains
in different parts of the world. Of the 300 species world-
wide, Scutellaria is represented by about 90 species in
North America. These plants do well under full sun, lim-
ited feeding, and well-drained soil. In southeastern USA,
Scutellaria blooms from May to August. Skullcaps are
now beginning to be used in southern gardens for their
sun and heat tolerance as well as their bright and showy
blooms.
Skullcap is well known among the Cherokee and
other Native American tribes as a strong emmenagogue

0926-6690/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2006.08.009
170 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177
and female medicinal herb (Moerman, 1998). Cherokee
women use skullcap herb to maintain healthy menstrual
cycles. A decoction of the root is taken after childbirth
to stimulate the reproductive system. The dry root of S.
baicalensis is a well known Chinese medicine (Tang and
Eisenbrand, 1992).
1.1. Annual consumption and market demand
The amount of skullcap harvested and sold in world
markets increased 2.5 times from approximately 6364 kg
in 1997 to 15,910 kg in 2001 (Greenfeild and Davis,
2004), The annual consumption of skullcap increased
by 23% between 2000 and 2001. The dollar value
of the 2001 harvest was between US$ 185,000 and
195,000, which was 3.5 times higher than 1997. Skull-
cap is currently recommended as an alternative seda-
tive treatment to kava which could increase the demand
for this herb. The price of skullcap has steadily risen
during the past 5 years and is currently around US$
8.80–17.60 kg−1 . Organically grown skullcap fetch pre-
mium prices, for example US$ 17.60–33.00 kg−1 in
Canada (Porter, 2006). The demand for skullcap in the
world markets is predicted to grow at an annual rate
of 20–30% (Greenfeild and Davis, 2004). During 2001,
about 70% of the world market demand for skullcap was
supplied by sources outside of North America.
1.2. Bioactive compound and micropropagation of
skullcaps
About 50 different types of flavonoids have been
isolated from Scutellaria (Tang and Eisenbrand, 1992;
Zhang et al., 1994; Miyaichi and Tomimori, 1994,
1995; Ishimaru et al., 1995; Zhou et al., 1997). Chem-
ical compounds extracted from Scutellaria spp. are
flavones, flavonoids, chrysin, baicalin, iridoids, neo-
clerodane derivatives, scutapins, isoscutellarein, wogo-
nin, etc. (Chou, 1978; Chou and Lee, 1986; Miyaichi et
al., 1988a, 1988b, 1989; Tomimori et al., 1984, 1985,
1986a, 1986b, 1990).
Few reports are available indicating the successful
micropropagation of Scutellaria (Li et al., 2000; Sinha
et al., 1999; Stojakowska et al., 1999). In vitro propaga-
tion will be helpful in rapidly providing large number
of uniform, insect- and disease-free plants to investi-
gate efficacy of medicinal components. By using the
hairy-root culture system, Hirotani (1999) isolated a new
flavone glucoside, 15 known flavonoids, and five known
phenylethanoids. Baicalin was produced in the callus
cultures of S. baicalensis (Shin and Lee, 1995). Potential
problems with medicinal plant preparations are contam-
ination with insects, bacteria, fungi, and environmental
pollutants, seasonal variability in the bioactive com-
pounds, degradation of active ingredients in processing
and storage of plant materials, and the lack of under-
standing of the unique physiology of medicinal plants
(Li et al., 2000).
1.3. Recent medical/clinical studies
Crude extract from S. barbata were found to be selec-
tively toxic to Staphylococcus aureus (Sato et al., 2000).
Flavonoids from S. baicalensis have been reported to
have inhibitory effect on human immunodeficiency virus
(Li et al., 1993), human T cell leukemia virus type I
(Baylor et al., 1992), and mouse skin tumor promotion
(Konoshima et al., 1992). It has been suggested that
baicalin may be significant in the lipid metabolism of
adipose cells (Eun et al., 1994; Chung et al., 1995).
Scutalpin C, a diterpenoid, has been shown to pos-
sess strong insect antifeedant activity against the larvae
of Spodoptera littoralis (Munoz et al., 1997). Anti-
inflammatory activity of skullcap formulations has been
associated with cyclooxygenase-2 (COX-2) enzyme
inhibition (Chi et al., 2001).
1.4. Arbuscular mycorrhizal fungi association
Arbuscular mycorrhizal fungi are ubiquitous in nature
and have been reported to form obligate symbiotic asso-
ciation with most angiospermic plants (Kendrick and
Birch, 1985) including several medicinal plant species
(Srivastava and Basu, 1995; Venkateshwar Rao et al.,
2000). The term vesicular–arbuscular mycorrhiza was
originally applied to the root endophytes that form sym-
biotic association with more than 90% of the vascular
plant families. However, because a major suborder lacks
the ability to form vesicles in roots of host plants, AMF
is currently the preferred acronym (Jarstfer and Sylvia,
2001). Arbuscular mycorrhizal fungi have been reported
to improve plant water relations and drought tolerance
of host plants through increased P-uptake in P-deficient
soils and/or through enhanced soil moisture extraction
(Rao and Lee, 1991). Scutellaria species are known to
thrive well in drought prone environments and soils poor
in fertility (Knight, 1999), suggesting possible associa-
tion with mycorrhiza.
About 90 species of Scutellaria are known to grow
wild in North America (Joshee et al., 2002). They have
not been brought into cultivation and their association
with AMF is not known. A survey of current literature
reveals that inoculation of AMF into the roots of micro-
propagated plantlets plays a beneficial role (Lovato et al.,
 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177
1996; Gange and Ayres, 1999; Rai, 2001). The purpose
of this study was to determine the effects of AMF on plant
height and dry matter production, and the most compat-
ible strain(s) of AMF with micropropagated Scutellaria
integrifolia plants. Thus, a greenhouse study on micro-
propagated plants of S. integrifolia was conducted using
four known and a commercially available, but unknown
mixture of AMF strains (BioVam, commercial mixture
of strains, T & J Enterprises, Spokane, WA).
2. Materials and methods
2.1. Plant materials
Fresh seeds of S. integrifolia were collected from
wild population growing at the Center for Georgia Nat-
ural Heritage program, Georgia Department of Natu-
ral Resources, Social Circles, GA (33.663◦ N latitude,
83.720◦ W longitude). Seedlings were raised in the
greenhouse and about 2-month-old plants were used to
obtain leaf, internode, node, and shoot tips to prepare
explants and initiate cultures. Apical parts up to the
fourth node from the shoot tip of a vegetative shoot were
used for making explants. Shoot tips with two leaves
and node and internodes approximately 1.0–1.5 cm long
were selected as explants to initiate cultures. Petio-
lated leaves with incisions on petiole, midrib, and lam-
ina varied in size from 0.5 to 1.0 cm at the time of
culture.
2.2. Sterilization and shoot induction
Plant material collected from greenhouse was kept
under running tap water for 1 h and then sterilized
with 10% Clorox solution for 15 min. After the Clorox-
treated plant material was washed three times with auto-
claved distilled water, it was soaked for 30–60 min in
a 1% plant preservative mixture (PPM) procured from
Phytotechnology Lab, Shawnee Mission, KS. The MS
(Murashige and Skoog, 1962) medium containing 3%
sucrose, 0.25% phytagel, and various concentrations
of growth regulators with necessary modifications was
used. Because we wanted to identify the best explant for
adventitious shoot bud induction, 30 explants each of
leaf, internode, nodal segments, and shoot tips were inoc-
ulated on MS basal medium supplemented with 2.2 ␮M
BA and 0.54 ␮M NAA for 4 weeks. This treatment was
selected on the basis of our preliminary studies on other
Scutellaria species. After induction, explants were trans-
ferred to growth regulator-free MS basal medium for
an additional 4 weeks for elongation. As shoot tip was
identified as the best explant (Table 1), additional exper-
171
Table 1
Effect of explant type on regeneration of shoots in Scutellaria inte-
grifolia after 8 weeks of culture on MS medium supplemented with
2.2 ␮M BA and 0.54 ␮M NAA
Explant No. of explants showing Response Average no.
regeneration of shoots/ (%) of shoot
no. of explants cultured buds/explant
Leaf 0/30 0.0 0.0
Stem internode 0/30 0.0 0.0
Stem node 30/30 100 13.06
Shoot tip 30/30 100 17.63
iments were done using shoot tip explants. Cultures
were initiated in 100 mm × 15 mm Petri dishes contain-
ing 25 ml culture medium. Explants were incubated for
1 week in dark and then for additional 3 weeks in light at
23–25 ◦ C. Shoot induction was determined as a response
to various cytokinins at different concentrations and BA
(0.44, 2.2, 4.4, and 8.8 ␮M), kinetin (0.46, 2.3, 4.6, and
9.2 ␮M), and TDZ (0.45, 2.25, 4.5, and 9.0 ␮M) were
tested during the initial studies. In the same experiment,
BA (2.2 ␮M), KIN (2.3 ␮M), and TDZ (2.25 ␮M) with
0.54 ␮M NAA were also included as additional treat-
ments. The number of elongated adventitious buds mea-
suring 5 mm or more was evaluated after 8 weeks in
culture.
2.3. Multiplication studies using liquid culture
system
The Liquid Lab® Rocker system developed by South-
ern Sun, Hodges, SC and Caisson Laboratories, North
Logan, UT was used for the multiplication studies. Two,
white fluorescent lights measuring 91.5 cm long were
used to illuminate cultures from the bottom. The Liq-
uid LabTM Vessel is a lightweight polycarbonate plastic
container (27 cm × 10 cm × 10 cm). The vessel is auto-
clavable and can work with both liquid and semi-solid
media. The vessel includes a microporous patch for gas
exchange along with a septaport for the addition and
testing of fluids. Depending on plant size, the vessel can
hold plant material equivalent to as much as 8–20, 118 ml
autoclavable glass jars (dimensions: 42 mm × 72 mm).
This shoot multiplication study using liquid culture
medium was carried out with the explants cultured for 6
weeks on a shoot induction medium containing 2.2 ␮M
BA + 0.54 ␮M NAA, because this treatment showed the
best induction. Shoot tip explants after 6 weeks culture in
the induction medium were transferred to culture vessels
with 75 ml of MS basal medium with 0.1% PPM. After
6 weeks of culture at 14 h of photoperiod, the number of
shoots was counted.
172 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177
2.4. Root induction
Elongated shoots on basal MS medium measuring
3–5 cm long were excised and transferred to the MS
basal medium and two rooting media (MS + 4.9 ␮M
IBA, MS + 9.8 ␮M IBA) and incubated the first week
in dark and then in the diffused light at 14 h photope-
riod for another 3 weeks. All media, chemicals, and
growth regulators were procured from Caisson Labora-
tories, Rexburg, ID, and Phytotechnology Laboratories,
Shawnee Mission, KS. All media were adjusted to pH
5.8–6.0 before autoclaving for 15 min at 121 ◦ C.
2.5. Plant material and arbuscular micorrhiza
fungi (AMF) treatments
To determine the role of mycorrhizal fungi in plant
growth and subsequent development of micropropa-
gated plants, acclimated plants were established in 6-
l pots containing sterilized soil-less mixture with low
P. At the time of potting, 5 mg of inoculum of the
different AMF strain: S3000 (Gigaspora margarita),
S3004 (Glomus etunicatum), S3029 (G. etunicatum),
S3064 (Glomus intradicas), and CS (Commercial mix-
ture of strains) was added to each treatment pot. A con-
trol treatment without any AMF was also included for
comparison.
Each treatment including control comprised 20 pots
with five replications of four pots per each treatment.
The pots were arranged in a randomized complete block
design in the greenhouse and were watered regularly to
avoid moisture deficit (Fig. 1H). Two pots per replication
were destructively sampled once at 75 days after planting
(DAP) and again at final harvest at 98 DAP to assess the
effect of AMF on plant height, total plant fresh weight,
and the root:shoot ratio on fresh weight basis.
2.6. Statistics
Shoot bud induction experiments were carried out
with 5 replicates and each replicate consisted of 6–10
explants. All data were subjected to statistical analysis
using SAS (1996). Means separation was by the Dun-
can’s Multiple Range Test.
3. Results and discussion
3.1. Plant material
All the four types of explants in the initial experiment
survived, but no adventitious buds were induced in the
leaf and internode explants. Shoot tips and nodes both
exhibited high rate of shoot bud induction (Table 1), and
thus, shoot tips were used for all the future experiments.
Some of the leaves at the cut end of the petiole and midrib
and internodes at wounds exhibited root induction.
3.2. Shoot induction
Explants on MS basal medium elongated and grew
normally, but did not show any shoot bud induction
(Fig. 1A). Shoot tips slightly elongated and then swelled,
and became rough on the surface at the end of 3-week
incubation in the medium supplemented with cytokinin
alone and cytokinin plus 0.54 ␮M NAA. Explants devel-
oped little callus with many green shoot buds that elon-
gated into shoots upon being transferred to the MS basal
medium for 3–4 weeks. Both shoot tips and nodal seg-
ments were responsive to all cytokinins. However, at the
end of the experiment, maximum number of shoots was
obtained with 2.2 ␮M of BA. Good response was also
obtained with TDZ (Fig. 1F) and kinetin showed the
poorest results (Table 2).
An interesting result was that BA concentrations
higher than 2.2 ␮M inhibited shoot bud formation
(Fig. 1B–D). Leaf explants showed no response to any
cytokinin, but they developed callus and roots at the peti-
ole ends in the presence of auxins, especially IAA. The
BA treatment was the most effective cytokinin and its
shoot induction response was further improved by sup-
plementing with 0.54 ␮M NAA (Fig. 1E and F). The
exposure to cytokinin for three to 4 weeks was sufficient
for shoot bud induction on explants. These were then
transferred to basal medium for further shoot elonga-
tion. For an additional 4 weeks, shoot buds elongated to
form 3–5 cm long shoots. The microshoots after elonga-
tion to 3–5 cm made a dense clump of 30–50 shoots per
explant.
The role of BA and TDZ on shoot bud induction has
been documented for S. discolor (Sinha et al., 1999) and
S. baicalensis (Li et al., 2000). Depending on the explant,
TDZ induced 9–20 shoots/explant (Li et al., 2000),
whereas for S. discolor the 4.44 ␮M BA + 0.54 ␮M NAA
induced the highest number of shoots. Furthermore, for
S. discolor, the addition of 0.54 ␮M NAA to the BA treat-
ments improved the total number of shoots produced.
Elongated shoots were cut and processed for rooting and
acclimatization (Fig. 1F and G).
3.3. Shoot multiplication using liquid rocker system
Liquid Lab® Rocker system considerably improved
the rate of multiplication over the semi-solid medium.
Five culture vessels with two explants each were placed
 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177 173

Fig. 1. In vitro micropropagation of Scutellaria integrifolia. (A) Shoot tip explant on basal MS medium. (B) Multiple shoots on MS + 2.2 ␮M BA.
(C) Shoot primordia and callus induction on MS + 8.8 ␮M BA. (D) Multiple shoot induction on MS + 2.2 ␮M BA + 0.54 ␮M NAA. (E) Multiple
shoot induction MS + 2.25 ␮M TDZ + 0.54 ␮M NAA. (F) Rooting of elongated shoots on MS + 4.9 ␮M IBA, arrow indicates growing roots. (G)
Acclimatized micropropagated plants. (H) Experimental set up to study the role of AMF on the growth and development of micropropagated plants
in the greenhouse. (I) Plant response to the various strains of arbuscular micorrhiza fungi: (1) control; (2) S3000; (3) S3004; (4) S3029; (5) S3064;
(6) BioVam or commercial strain.
174 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177
Table 2
The effect of various cytokinins and NAA treatments on adventitious shoot induction in Scutellaria integrifolia
Sl. no. BAP (␮M) No. of shoots/explant KIN (␮M)
1 0.44 6.4 (±2.07)a,b aC 0.46
2 2.2 23.6 (±5.68) aB 2.3
3 4.4 20.4 (±4.77) aB 4.6
4 8.8 16.6 (±3.05) aB 9.2
5 2.2 + 0.54 NAA 48.0 (±10.17) aA 2.3 + 0.54 NAA
6 Control 2.00 (±0.0) D
Cytokinin concentrations (rows 1–4) in ␮M correspond to 0.1, 0.5, 1.0, and 2.0 mg l−1 whereas NAA concentration corresponds to 0.1 mg l−1 .
a Values in parentheses denote standard deviation from the mean.
b Mean separation within columns followed by the same upper case letters and within rows followed by the same lower case letters are not
significantly different (Duncan’s Multiple Range Test at P ≤ 0.05).
on the rocker system at 16 h photoperiod and final shoot
number for each explant was counted after 6 weeks.
An average of 233.40 (±61.13) shoots/explant were
obtained that is almost five times larger than the semi-
solid medium. Elongated shoots can be cut and easily
rooted in a liquid root induction medium that can be
transferred directly to a mist bed for acclimatization.
3.4. Root induction
At the end of 1-month incubation, roots were present
in all shoots planted on the MS basal and two other root-
ing media containing IBA. The root induction was almost
a week earlier in the media supplemented with IBA than
the basal medium. Although at the lower concentrations
of IBA (4.9 ␮M) the rooting was more favorable (Fig. 1F)
than at 9.8 ␮M IBA as higher concentration also induced
callusing and swelling in addition to rooting. Follow-
ing this finding, all of the rootings were made in the
MS + 4.9 ␮M IBA combination.
3.5. Acclimatization and planting in the greenhouse
Healthy plantlets were gently removed from the root-
ing medium and agar was washed off from the roots
under tap water. For acclimatization, plantlets were
transferred into a mist chamber in the greenhouse. After
2 weeks, the plants became sturdy and started growing.
At this stage, the plants were transferred to 6-l con-
tainers to determine the role of AMF on growth and
development (Fig. 1H). Additional plants were main-
tained in the greenhouse pots for further growth and seed
collection.
An efficient and reproducible protocol was devel-
oped for the micropropagation of the medicinal plant
S. integrifolia. Clean and uniform plants will be helpful
in future studies for phytochemical screening, develop-
ing hairy root cultures to study secondary metabolites,
No. of shoots/explant TDZ (␮M) No. of shoots/explant
5.6 (±1.0) aC 0.45 4.0 (±1.82) aC
17.6 (±3.85) aA 2.25 19.4 (±5.59) aB
15.2 (±3.70) aAB 4.5 14.8 (±4.15) aB
13.0 (±3.54) aB 9.0 13.6 (±2.70) aB
19.4 (±3.21) bA 2.25 + 0.54 NAA 28.0 (±7.0) bA
and raising planting material for greenhouse and field
studies.
3.6. Mycorrhizal studies
Comparisons of control and plants treated with dif-
ferent strains of AMF are presented in Fig. 1I. At 75
days after planting, the plant height ranged from 20.7 cm
for plants inoculated with S3029 (G. etunicatum) to
31.4 cm for plants inoculated with S3064 (G. intradi-
cus) (Table 3). Plants inoculated with the AMF strain
S3029 were significantly shorter than plants in the con-
trol treatments and those receiving AMF strains S3064
(G. intradicas) and S3004 (G. etunicatum), but were sim-
ilar to plants receiving S3000 (Gigaspora margarita) and
commercial mixture.
The fresh weight of shoots ranged from 7.0 to
16.9 g/plant (Table 3). Plants inoculated with the AMF
strain S3004 produced more shoot than plants inoculated
with any other strain, whereas those plants inoculated
with S3029 produced the lowest shoot weight. Plants
inoculated with the commercial mixture, S3064, and
S3029 had significantly lower shoot fresh weight than
the plants inoculated with S3004 and S3000, but simi-
lar to the control plants. Plants inoculated with S3004
also had higher root fresh weight than plants inoculated
with AMF strains S3029, commercial mixture, S3064,
and control plants. Plants inoculated with commercial
mixture had a higher root to shoot ratio than the plants
inoculated with S3000, but was similar to the rest of
the treatments. Plants inoculated with the AMF strain
S3004 produced a significantly greater amount of seed
than plants in the treatments S3029, commercial mix-
ture, and S3000. The plants treated with strain S3004
produced nearly 53% greater amount of seed than the
average yield of other treatments.
At harvest, however, the trends at 98 DAP changed
markedly from those observed at 75 DAP and summa-
 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177 175

Table 3
Plant growth parameters and dry seed weight of S. integrifolia in response to different arbuscular mycorrhiza fungi (AMF) strains, 75 days after
planting (DAP)
Strain AMF spp. Plant height (cm) Shoot (g/plant) Root (g/plant) Root:shoot (ratio) Seed dry wt. (g/plant)

S3064 G. intradicas 31.4 a 10.8 bcd 7.8 b 0.74 ab 1.63 abc

S3004 G. etunicatum 29.4 a 16.9 a 12.8 a 0.60 ab 2.22 a
Control No AMF 27.9 a 12.2 abc 7.6 b 0.60 ab 1.99 ab
S3000 G. margarita 26.1 ab 14.0 ab 8.6 ab 0.52 b 1.20 bcd
BioVama Unknown 24.8 ab 9.0 cd 6.1 b 0.94 a 0.97 cd
S3029 G. etunicatum 20.7 b 7.0 d 5.0 b 0.62 ab 0.74 d

Means followed by the same letters within columns are not significantly different, Duncan’s Multiple Range Test (DMRT), P < 0.05.
a Commercial mixture.

rized in Table 4. The control plants were significantly
taller than those in treatment S3064, but were similar to
those as the rest of the treatments. Plant height ranged
from 26.4 cm for S3064 to 34.2 cm for the control plants.
The commercial (Bio Vam) treatment produced more
total fresh plant biomass than plants inoculated with
S3029 and S3004. The shoot and root fresh weights were
similar in all treatments, although the plants inoculated
with S3064 tended to produce higher amounts than the
rest of the treatments. The root to shoot ratio was greater
than 1 in all treatments indicative of a lower shoot weight
relative to root weight (Fig. 1I). The root to shoot ratio of
plants receiving the AMF strain S3000 was greater than
the plants receiving the AMF strain S3029, but was sim-
ilar to that of plants in other treatments. The ratio ranged
from 1.12 for plants inoculated with S3029 to 2.71 for
plants receiving strain S3000. Control plants had a root
to shoot ratio of 1.36, which was lower than the over all
mean for all treatments including the control.
Even though the differences between AMF treated
and control plants were statistically not significant,
trends reflecting beneficial effects due to AMF were clear
at both the 75 and 98 DAP. Preliminary results (data not
shown) from root colonization assays indicated that the
roots of control plants were highly infected with sapro-
phytic fungi, whereas there was no such infection in the
AMF treated plants. This indicates that there was a bene-
ficial effect of AMF inoculation as the establishment and
growth of saprophytic fungi may have been inhibited by
the mycorrhizal fungi.
In general, plant height, shoot, root, and seed fresh
weights at 75 DAP were higher for the plants treated with
AMF S3004 than the control plants, but such differences
were not apparent at final harvest. A similar increase
in shoot and root weights due to VAM inoculation was
reported for a range medicinal plants (Srivastava and
Basu, 1995) and sorghum (Rao and Lee, 1991). Arbus-
cular mycorrhizal fungi have been shown to increase
biomass production in forage legumes (Medina et al.,
1990) and corn (Sylvia, 1999). Plants inoculated with
the commercial mixture tended to produce more root
biomass than shoot biomass (Fig. 1 I6). The AMF strain
S3004 tended to promote shoot and root growth and may
have enabled the plants to mature earlier as reflected by
a greater amount of seed than those in the control treat-
ment and all other strains. Strains S3064 and commercial
mixture enhanced greater root to shoot ratio than the con-
trol treatment. At final harvest, there were no significant
differences between the control plants and AMF treated
plants for all traits measured. The root to shoot ratio of

Table 4
Plant growth parameters of S. integrifolia in response to different arbuscular mycorrhiza fungi (AMF) strains 98 days after planting (DAP)
Strain AMF spp. Plant Total plant Shoot dry Root dry Root:shoot AMF
height fresh wt. weight weight (ratio) infection
(cm) (g/plant) (g/plant) (g/plant) (%)

S3064 G. intradicas 26.4 b 20.8 ab 2.78 a 2.88 a 1.16 ab 19.6 a

S3004 G. etunicatum 28.3 ab 18.8 b 1.94 a 2.47 a 1.38 ab 0.0 b
Control No AMF 34.2 a 25.5 ab 2.41 a 2.87 a 1.36 ab 0.0 b
S3000 G. margarita 27.3 ab 22.7 ab 1.66 a 2.72 a 2.71 a 0.0 b
BioVama Unknown 31.1 ab 30.6 a 1.76 a 2.76 a 1.69 aba 0.0 b
S3029 G. etunicatum 27.2 ab 17.0 b 2.34 a 2.55 a 1.12 b 0.0 b

Means followed by the same letters within columns are not significantly different, DMRT, P < 0.05.
a Commercial mixture.
176 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177
plants treated with AMF strain S3000 was nearly twice
the control plants. Marked increase in root to shoot ratio
has been reported for VAM inoculated sorghum (Rao
and Lee, 1991).
4. Conclusion
An efficient plant regeneration system for S. inte-
grifolia using shoot tip explant was developed. The
selection of an appropriate explant is crucial to the suc-
cessful development of any in vitro propagation system.
Greenhouse-grown plants were used as the source mate-
rial for explants. The incorporation of a rocker system
utilizing liquid culture medium enhanced multiplication
many-fold and provided the ease of operation by elimi-
nating agar removal step. Furthermore, as liquid culture
is devoid of gelling agent, this process of shoot induc-
tion in the semi-solid and multiplication in liquid media
could greatly reduce the cost of production.
Inoculation with AMF, showed positive effects on
plant growth, particularly root development. The study
showed that there is preferential colonization of S. inte-
grifolia roots by a specific AMF strain. The present study
showed the need for further experiments to evaluate com-
patibility and methods for improving Scutellaria root
colonization by AMF and the presence of possible host
spp. × AMF strain interactions.
Acknowledgements
We thank Shizhou (Shirley) Wang, Fort Valley State
University Agricultural Research Station, for techni-
cal assistance, Tom Patrick, Georgia Natural Heritage
Program, Georgia Department of Natural Resources,
Wildlife Resources Division, for identifying wild pop-
ulations of S. integrifolia and collection of seeds. We
also thank Drs. D.M. Sylvia, University of Pennsylva-
nia, and A.K. Alagely, University of Florida, for pro-
viding the Arbuscular Mycorrhizae Fungi strains. This
research was supported by the USDA-CSREES 1890
Capacity Building Grants Research Project (GEOX-
2002-02989), 2002- Harnessing Agri-Biotechnology to
Establish Scutellaria as a medicinal crop.
References
Baylor, N.W., Fu, T., Yan, Y.-D., Ruscetti, F.W., 1992. Inhibition
of human T cell leukemia virus by the plant flavonoid baicalin
(7-glucuronic acid,5,6-dihydroxyflavone). J. Infect. Diseases 165,
433–437.
Chi, Y.S., Cheon, B.S., Kim, H.P., 2001. Effect of wogonin, a
plant flavone from Scutellaria radix, on the suppression of
cyclooxygenase-2 and the induction of inducible nitric oxide syn-
thase in lipopolysaccharide-treated RAW 264.7 cells. Biochem.
Pharmacol. 61, 1195–1203.
Chou, C.-J., 1978. Rivularin: a new flavone from Scutellaria rivularis.
J. Taiwan Pharm. Assoc. 30, 36–43.
Chou, C.-J., Lee, S.-Y., 1986. Studies on the constituents of Scutellaria
indica root (I). J. Taiwan Pharm. Assoc. 38, 107–118.
Chung, C.P., Park, J.B., Bae, K.H., 1995. Pharmacological effects of
methanolic extracts from the root of Scutellaria baicalensis and its
flavonoids on human gingival fibroblast. Planta Med. 61, 150–153.
De Smet, P.A.G.M., 1997. The role of plant derived drugs and herbal
medicines in healthcare. Drugs 54 (6), 801–840.
Eun, J.S., Suh, E.S., So, J.N., Oh, S.H., 1994. Effect of baicalin on the
differentiation of 3T3-Li cells. Yakahak Hoeji. 38, 48.
Gange, A.C., Ayres, R.L., 1999. On the relation between arbuscular
mycorrhizal colonization and plant ‘benefit’. Oikos 87, 615–621.
Greenfeild, J., Davis, J.M., 2004, Skullcap (Scutellaria later-
ifolia L.). Medicinal Herb Production Guide. http://www.
naturalmedicinesofnc.org, July 12, 2006.
Hirotani, M., 1999. Genetic transformation of Scutellaria baicalensis.
In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry,
vol. 45, Transgenic Medicinal Plants, Springer, Heidelberg, pp.
271–283.
Ishimaru, K., Nishikawa, K., Omoto, T., Asai, I., Yoshihira, K., Shi-
momura, K., 1995. Two flavone 2 -glucosides from Scutellaria
baicalensis. Phytochemistry 40, 279–281.
Jarstfer, A.G., Sylvia, D.M., 2001. Isolation, culture and detection of
arbuscular mycorrhizal fungi. In: Hurst, C.J., Knudsen, G.R., McK-
iernan, M.J., Stetzenbach, L.D., Walter, M.V. (Eds.), Manual of
Environmental Microbiology, 2nd ed., Am. Soc. Microbiol. Wash-
ington, D.C., pp. 406–412.
Joshee, N., Patrick, T.S., Mentreddy, S.R., Yadav, A.K., 2002. Skull-
cap (Scutellaria species): a potential medicinal crop. In: Janick, J.,
Whipkey, A. (Eds.), Trends in New Crops and New Uses. ASHS
Press, Alexandria, VA, pp. 580–586.
Kendrick, W.B., Birch, S.M., 1985. Mycorrhizae: applications in agri-
culture and forestry. In: Moo-Young, M. (Ed.), Comprehensive
Biotechnology, 3. Pergamon Press, Oxford, pp. 109–152.
Knight, J., 1999. Grow Sheet Scullcap (Scutellaria baicalensis).
Herbs for Health. http://www.herbsforhealth.com/library/weekly/
aa073099.htm.
Konoshima, T.M., Kokumai, M., Kozuka, M., Iinuma, M., Mizuno, T.,
Tanaka, H., Tokuda, H., Nishino, A., Iwashima, A., 1992. Stud-
ies on inhibitors of skin tumor promotion. XI. Inhibitory effects
of flavonoids from Scutellaria baicalensis on Epstein-Barr virus
activation and their anti-tumor-promoting activities. Chem. Pharm.
Bull. 40, 531–533.
Li, B.-Q., Fu, T., Yan, Y.-D., Baylor, N.W., Ruscetti, F.W., Kung, H.F.,
1993. Inhibition of HIV infection by baicalin- a flavonoid com-
pound purified from Chinese herbal medicine. Cell. Mol. Biol.
Res. 39, 119–124.
Li, H., Murch, S.J., Saxena, P.K., 2000. Thidiazuron-induced de
novo shoot organogenesis on seedlings, etiolated hypocotyls and
stem segments of Huang-qin. Plant Cell Tissue Organ Cult. 62,
169–173.
Lovato, P.E., Gianinazzi-Pearson, V., Trouvelot, A., Gianinazzi, S.,
1996. The state of mycorrhizas and micropropagation. Adv. Hort.
Sci. 10, 46–52.
Medina, O.A., Kretschmer, A.E., Sylvia, D.M., 1990. Growth
response of field-grown Siratro (Macroptillium atropurpureum
Urb.) and Aeschynomene americana L. to inoculation with
selected vesicular–arbuscular mycorrhizal fungi. Biol. Fert. Soils 9,
54–60.
 N. Joshee et al. / Industrial Crops and Products 25 (2007) 169–177
Miyaichi, Y., Tomimori, T., 1994. Studies on the constituents of Scutel-
laria species XVI. On the phenol glycosides of the root of Scutel-
laria baicalensis Georgi. Nat. Med. 48, 215–218.
Miyaichi, Y., Tomimori, T., 1995. Studies on the constituents of Scutel-
laria species. XVII. On the phenol glycosides of the root of Scutel-
laria baicalensis Georgi (2). Nat. Med. 49, 350–353.
Miyaichi, Y., Imoto, Y., Kizu, H., Tomimori, T., 1988a. Studies on the
Nepalese crude drugs. IX. On the flavonoid constituents of the root
of Scutellaria scadens. Buch. -Ham. D. Don. Chem. Pharm. Bull.
36, 2371–2376.
Miyaichi, Y., Imoto, Y., Kizu, H., Tomimori, T., 1988b. Studies on
the Nepalese crude drugs. X. On the flavonoid and stilbene con-
stituents of the leaves of Scutellaria scadens. Buch. -Ham. D. Don.
Shoyakugaku Zasshi 42, 204–207.
Miyaichi, Y., Imoto, Y., Tomimori, T., Lin, C.-C., 1989. Studies on
the constituents of Scutellaria species. XI. On the flavonoid con-
stituents of the root Scutellaria indica. L. Chem. Pharm. Bull. 37,
794–797.
Moerman, D.E., 1998. Native American Ethnobotany. Timber Press,
Portland, OR, p. 927.
Munoz, D.M., De La Torres, M.C., Rodriguez, B., Simmonds,
M.S.J., Blaney, W.M., 1997. Neo-clerodane insect antifeedant
from Scutellaria alpina ssp. ja valambarensis. Phytochemistry 44,
593–597.
Murashige, T., Skoog, F., 1962. Revised medium for the rapid growth
and bioassay with tobacco tissue culture. Physiol. Plant. 15,
473–497.
Porter, B., 2006. Skullcap Production in Saskatchewan. http://www.
agr.gov.sk.ca/docs/crops/horticulture/skullcap.asp. July 12, 2006.
Rai, M.K., 2001. Current advances in mycorrhization in micropropa-
gation. In Vitro Cell. Dev. Biol. Plant 37, 158–167.
Rao, M.S.S., Lee, K.K., 1991. The effects of VAM (Glomus clarum)
and phosphorus on the growth, development, and dry matter parti-
tioning of sorghum (Sorghum bicolor L. Moench) with and without
drought stress cycles. Sorghum Newsletter 32, 60.
SAS, 1996. SAS System for Windows. SAS Inst., Cary, NC.
Sato, Y., Suzuki, S., Nishikawa, T., Kihara, M., Shibata, H., Higuti, T.,
2000. Phytochemical flavones isolated from S. barbata and antibac-
terial activity against methicillin-resistant Staphylococcus aureus.
J. Ethnopharmacol. 72 (3), 483–488.
Shin, S.W., Lee, H.K., 1995. Production of baicalin by cell cultures
of Scutellaria baicalensis. Korean J. Pharmacognosy 26, 159–
163.
Sinha, S., Pokhrel, S., Vaidya, B.N., Joshee, N., 1999. In vitro micro-
propagation and callus induction in Scutellaria discolor COLEBR.
A medicinally important plant of Nepal. Indian J. Plant Genet.
Resour. 12 (2), 219–223.
177
Song, W.Z., 1981. Studies on the resource of the Chinese herb Scutel-
laria baicalensis Georgi. Acta Pharm. Sin. 16, 139–145.
Srivastava, N.K., Basu, M., 1995. Occurrence of vesicular arbuscu-
lar mycorrhizal fungi in some medicinal plants. In: Adholeya,
A., Singh, S. (Eds.), Mycorrhizae: Biofertilizers for the Future.
Third National Conference on Mycorrhiza, TERI, Delhi, India, pp.
58–61.
Stojakowska, A., Malarz, J., Kohlmuenzer, S., 1999. Micropropagation
of Scutellaria baicalensis Georgi. Acta Soc. Bot. Pol. 68, 103–107.
Sylvia, D.M., 1999. Fundamentals and applications of arbuscular myc-
orrhizae: A “biofertilizer” perspective. In: Siqueira, J.O., Moreira,
F.M.S., Lopes, A.S., Guilherme, L.R.G., Faquin, V., Furtini Neto,
A.E., Carvalho, J.G. (Eds.), Soil Fertility, Biology, and Plant Nutri-
tion Interrelationships. Viçosa: SBCS, Lavras: UFLA/DCS, pp.
705–723.
Tang, W., Eisenbrand, G., 1992. Chinese Drugs of Plant Origin.
Springer, Berlin, pp. 919–929.
Tomimori, T., Miyaichi, Y., Imoto, Y., Kizu, H., Namba, T., 1984.
Studies on the constituents of Scutellaria species V. On the fla-
vanoid constituents of Ban Zhi Lian, the whole herb of Scutellaria
rivularis Wall (1). Shoyakugaku Zasshi 38, 249–252.
Tomimori, T., Miyaichi, Y., Imoto, Y., Kizu, H., Namba, T., 1985.
Studies on Nepalese crude drugs. V. On the flavonoid constituents
of the root of Scutellaria discolor Colebr. (1). Chem. Pharm. Bull.
33, 4457–4463.
Tomimori, T., Miyaichi, Y., Imoto, Y., Kizu, H., 1986a. Studies on
the constituents of Scutellaria species. VIII. On the flavonoid con-
stituents of Ban Zhi Lian, the whole herb of Scutellaria rivularis
Wall (2). Shoyakugaku Zasshi 40, 432–433.
Tomimori, T., Miyaichi, Y., Imoto, Y., Kizu, H., Namba, T., 1986b.
Studies on Nepalese crude drugs. VI. On the flavonoid constituents
of the root of Scutellaria discolor Colebr. (2). Chem. Pharm. Bull.
33, 406–408.
Tomimori, T., Imoto, Y., Miyaichi, Y., 1990. Studies on the constituents
of Scutellaria species. XIII. On the flavonoid constituents of the
root of Scutellaria rivularis Wall (1). Chem. Pharm. Bull. 38,
3488–3490.
Venkateshwar Rao, G.C., Manoharachary, C., Kunwari, I.K., Rajesh-
war Rao, B.R., 2000. Arbuscular mycorrhizal fungi associated with
some economically important spices and aromatic plants. Philip-
pine J. Sci. 129 (1), 1–5.
Zhang, Y.-Y., Guo, Y.-Z., Onda, M., Hashimoto, K., Ikeya, Y., Okada,
M., Maruno, M., 1994. Four flavonoids from Scutellaria baicalen-
sis. Phytochemistry 35, 511–514.
Zhou, Y., Hirotani, M., Yoshikawa, T., Furuya, T., 1997. Flavanoids and
phenylethanoids from hairy root cultures of Scutellaria baicalen-
sis. Phytochemistry 44, 83–87.