Agrobacterium rhizogenes-mediated transformation of ... - CIMAP Staff

Plant Biotechnol Rep (2007) 1:169–174
DOI 10.1007/s11816-007-0029-0
ORIGINAL ARTICLE
Agrobacterium rhizogenes-mediated transformation
of Picrorhiza kurroa Royle ex Benth.: establishment
and selection of superior hairy root clone
Praveen Chandra Verma Æ Laiq ur Rahman Æ
Arvind Singh Negi Æ Dharm Chand Jain Æ
S. P. S. Khanuja Æ Suchitra Banerjee
Received: 16 April 2007 / Accepted: 15 May 2007 / Published online: 3 August 2007
Ó Korean Society for Plant Biotechnology and Springer 2007
Abstract A protocol for induction and establishment of
Agrobacterium rhizogenes-mediated hairy root cultures of
Picrorhiza kurroa was developed through optimization of
the explant type and the most suitable bacterial strain. The
infection of leaf explants with the LBA9402 strain resulted
in the emergence of hairy roots at 66.7% relative
transformation frequency. Nine independent, opine and
TL-positive hairy root clones were studied for their
growth and specific glycoside (i.e., kutkoside and picroside
I) productivities at different growth phases. Biosynthetic
potentials for the commercially desirable active
constituents have been expressed by all the tested hairy
root clones, although distinct inter-clonal variations could
be noted in terms of their quantity. The yield potentials of
the 14-P clone, both in terms of biomass as well as
individual glycoside contents (i.e., kutkoside and picroside
I), superseded that of all other hairy root clones along
with the non-transformed, in vitro-grown control roots of
P. kurroa. The present communication reports the first
successful establishment, maintenance, growth and selection
of superior hairy root clone of Picrorhiza kurroa with
desired phyto-molecule production potential, which can
serve as an effective substitute to its roots and thereby
prevent the indiscriminate up-rooting and exploitation of
this commercially important, endangered medicinal plant
species.
Keywords Agrobacterium rhizogenes Hairy roots
Glycosides Picrorhiza kurroa Growth index
Abbreviations
BAP benzyl amino purine
IBA indole-3-butyric acid
NAA a-naphthalene acetic acid
TL Left-terminus DNA
Introduction
CIMAP Publication No.: 2007-28J
P. C. Verma
National Botanical Research Institute,
Lucknow, UP 226001, India
L. ur Rahman A. S. Negi D. C. Jain
S. P. S. Khanuja
Central Institute of Medicinal and Aromatic Plants,
P.O. CIMAP, Lucknow, UP 26015, India
S. Banerjee (&)
Plant Tissue Culture Division,
Central Institute of Medicinal and Aromatic Plants,
P.O. CIMAP, Lucknow, UP 26015, India
e-mail: sbanerjee_cimapin@indiatimes.com
Plant-based molecules are continuously gaining widespread
acceptance due to their effective therapeutic
properties (Dubey et al. 2004). A wide range of such
plant-derived molecules of pharmaceutical interest accumulates
in plant roots as secondary metabolites, which
necessitates uprooting and killing of the whole plant for
harnessing the compound. Indiscriminate exploitation of
the natural resources through unregulated uprooting coupled
with the lack of attention towards strategic cultivation
practices have led to the endangered or threatened
conditions of several high-altitude important medicinal
plant species (Kumar et al. 1997). Realizing the threat of
extinction of such endangered medicinal plant species,
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170 Plant Biotechnol Rep (2007) 1:169–174
attention has already been focused towards developing
production alternatives of root-derived phytomolecules in
order to meet the growing demand of pharmaceutical
industries. The role of Agrobacterium rhizogenes-mediated
‘‘hairy root’’ cultures as an efficient production
alternative has undeniably proved its effectiveness in the
worldwide arena (Guillon et al. 2006a, b; Hu and Du
2006). The stride of hairy root technology from the
boundaries of research laboratories to industrial-scale
production strategies has magnificently been manifested
through the advent of the German company ROOTec
(http://www.rootec.com), devoted fully towards up-scaling
the hairy root technology as a production alternative at the
industrial level for two important phytomolecules of
endangered plant origin.
In the backdrop of these developments and in continuance
to our earlier research effort (Verma et al. 2002), it
was felt essential to focus our analogous research attention
towards another very important and critically endangered
medicinal plant species, i.e., Picrorhiza kurroa, which
yields clinically proven hepato-protective and immunomodulating
glycosides in its underground parts (Anonymous
2001; Gupta et al. 2006).
Picrorhiza kurroa Royle ex Benth belongs to the
family Scrophulariaceae and is an endemic plant of the
alpine Himalayan range of India. The roots and rhizomes
of 3–4-year-old P.kurroa plants yield a crystalline
product called ‘‘kutkin,’’ which is usually a mixture of
two major C9-iridoid glycosides, i.e., picroside-I (6-O
-trans cinnamoylcatalpol) and kutkoside (10-O-vaniloylcatalpol)
(Kumar et al. 2004). Significant hepatoprotective,
anticholestatic, antiulcerogenic, antiasthematic,
antidiabetic, anti-inflammatory and immuno-regulatory
functions have already been ascribed to these glycosides
for which the extracts of the underground parts of this
plant finds applications as the major component in several
Indian herbal preparations (Ram 2001; Thyagarajan
et al. 2002).
In order to address the problem of unregulated trade of
the underground parts of P. kurroa and to impede the
adulteration of the raw materials to be used for herbal
preparations, it seems highly desirable to explore the immense
potential of the hairy root system of this presently
unexplored medicinal plant species. This communication
highlights the significant progress made in the afore-mentioned
directions that have helped to address the concern
involving this particular endangered medicinal plant species
P. kurroa through the establishment and selection of
fast-growing, high-yield hairy root clone(s). The current
research findings will help in bringing the prospect of
achievable, root-derived phytomolecules from hairy root
cultures of P. kurroa another step closer to industrial
exploitation.
Materials and methods
Induction and establishment of hairy roots
Picrorhiza kurroa plants (8–10 weeks), maintained under
in-vitro conditions on semisolid MS (Murashige and Skoog
1962) medium supplemented with 2.0 mg L –1 BAP and
0.1 mg L –1 NAA, were used as the explant source. The
young leaves and stem segments were inoculated through
pricking with a 48-h-old suspension culture of A. rhizogenes
strains, namely LBA 9402 and A 4 (kind gift from
Prof. D. Tepfer, INRA, Versailles Cedex, France), grown
in liquid YMB (Hooykass et al. 1977) medium (O.D 600 =
0.9–1.0). After 48 h of co-cultivation with the individual
bacterial strain, the explants were transferred onto the same
respective medium containing 1.0 g L –1 of cephalaxin
(Ranbaxy, India) under dark conditions. Similar types of
explants, pricked with a sterile needle devoid of the bacterial
suspension, were cultured under uniform conditions
as controls. The emerging hairy roots were subsequently
transferred to the half and full strengths of the B 5 medium
(Gamborg et al. 1968) containing 3% (w/v) sucrose for
their further proliferation. Once established, the individual
hairy root clones were transferred to liquid B 5 medium
with the same concentration of antibiotic and incubated on
a rotary shaker in the dark at 25 ± 1°C under constant
agitation (80 rpm). The antibiotic concentration was progressively
lowered and finally completely omitted after
4 months. The crushed hairy root extracts were streaked on
semisolid YMB medium to check for the presence of
A. rhizogenes at this stage. Roots excised from in vitrogrown
complete plantlets of P. kurroa were cultured under
identical conditions in liquid B 5 medium supplemented
with 1.0 mg L –1 IBA to serve as control roots.
Growth kinetic studies
The growth characteristics of 25 independently generated
hairy root clones were evaluated on the basis of total root
elongation (cm), lateral branching per centimeter of primary
roots and fresh weight (FW) increment after 15 days
of incubation in full- and half-strength liquid B 5 medium
containing 3% sucrose. On the basis of the apparent growth
behaviors with respect to these specific parameters, nine
individual root clones were selected for further studies.
All nine hairy root clones and the control, non-transformed
roots were subjected to growth kinetic analysis for
growth kinetic studies, 100 mg of actively growing hairy
roots from 15 days old cultures were transferred to 250-ml
Erlenmeyer flasks containing 50 ml of half-strength B 5
medium with 3% sucrose, and their growth performances
were determined following the method described earlier
(Verma et al. 2002). The selected superior hairy root clone
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Plant Biotechnol Rep (2007) 1:169–174 171
was cultured in 1 l half-strength B 5 medium for scale up
studies.
Characterization of hairy roots
To prove the transformed nature of these nine hairy root
clones, opines were extracted and detected by paper electrophoresis
according to the procedure of Morgan et al.
(1987) in parallel to extracts from non-transformed in vitro-grown
control roots. Their transformed nature was
further ascertained through PCR analysis according to the
procedure described by Rahman et al. (2004) using TLspecific
primer.
Chemical analysis
The time course production of the desired secondary
metabolites, i.e., kutkoside and picroside I, was determined
by subjecting these dried root samples to a chemical
extraction process. The extraction of glycosides and HPLC
analysis for kutkoside and picroside I was carried out
according to the procedure reported by Gupta (2001) with
minor modifications. The HPLC analysis was performed on
reverse phase HPLC (Agilent HP 1100) with C 18 column
(Waters Co., USA). The mobile phase (acetonitrile: water
0–15 min at 15–20% and 16–35 min at 20–80%) was
pumped at a flow rate of 1 ml min –1 . (Verma 2003). The
cycle time of analysis was about 35 min. The compounds
were identified on the basis of their retention time and
comparison of UV spectra with the authentic standards.
The quantification was repeated thrice for each sample, and
the data were subjected to statistical analysis.
strain compatibility has received substantial research
attention over the years (Banerjee et al. 1995; Giri et al.
2001; Hu and Du 2006). The clonal nature of individual
hairy root lines has made it mandatory to screen and select
the best performer among a wider, independently generated,
heterogeneous background as reported in earlier
analogous studies (Christey and Braun 2005).
Characterization of hairy roots
The transformed nature of the nine selected hairy root
clones was confirmed through PCR analysis by the presence
of rol B (670 bp) sequences from TL DNA of Ri
plasmid (Fig. 1). The transformed nature was further confirmed
by the presence of mannopine (Fig. 2).
Growth behavior of hairy roots
Nine potentially superior clones were selected on the basis
of their growth performances among the 25 independently
generated hairy root lines (data not presented). The
Table 1 Frequency of hairy root induction from different explants
following co-cultivation with two different strains of A. rhizogenes
Bacterial strain
B5 medium
Leaf
Stem segment
LBA 9402 66.7 ± 0.33 8.76 ± 1.56
A4 NR NR
NR no response
Results and discussion
Induction and establishment of hairy roots
Visible roots were formed on the leaf explants after
3 weeks of inoculation with the A. rhizogenes strain LBA
9402 at 66.7% relative transformation frequency. On the
other hand, the A 4 strain of A. rhizogenes appeared ineffective
in inducing hairy roots in P. kurroa even after repeated
trials. This is in agreement with earlier reports
where bacterial strain specificity was found to play a
determining role in establishing hairy roots (Byrne et al.
1987; Porter 1991; Zehra et al. 1999; Torregrosa et al.
2002).
Among the two explants tested, leaf explants proved to
be relatively better than the the stem explant (Table 1) both
in terms of transformation frequencies and further growth
potentials of the resulting hairy root clones. The imperative
role of explants in determining the plant genotype-bacterial
Fig. 1 PCR amplification of TL genes (670 bp) in hairy roots of
P.kurroa transformed with A. rhizogenes LBA 9402. Lane M 100 bp
molecular marker (larger band is 500 bp); lane 1-8 randomly selected
hairy root clones; nt non-transformed control in vitro grown roots of
P. kurroa
Fig. 2 Opine assay of transformed hairy root tissues of P. kurroa;
lane 1 standard mannopine; lane 2 untransformed control root; lane
3–5 randomly selected transformed hairy root clones (n neutral
sugars; m mannopine)
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172 Plant Biotechnol Rep (2007) 1:169–174
productivity potentials of the selected clones, both in terms
of biomass and secondary metabolites of interest at three
different growth phases, are summarized in (Figs. 3, 4, 5).
In terms of biomass productivity, a gradual increase in GI
up to 40 days of culture could be noted with all these root
clones, among which three clones (i.e., 7C, 3E and 4E)
continued their growth till 60 days of culture, while the
remaining six exhibited a decline in growth beyond
the 40th day of culture. Statistical analysis revealed that the
biomass production of four hairy root clones, i.e., lA, 2B,
2D and 14P, differ significantly from that of the rest at their
respective optimum growth phases (Fig. 3). The highest
growth increment was exhibited by the 14P clone (Fig. 6),
which was noted to be 29.6-fold after 40 days of culture.
On the contrary, the control, non-transformed roots could
attain a maximum of only 6.24 growth increments after
60 days of culture in 1.0 mg L –1 IBA supplemented B 5
medium, which was 4.1 times less than that of the 14P line.
35
30
7C 4E 1A 2B 2D 3E 1D 2F P14 Nr
Chemical profiling
Biosynthetic potentials for the production of the root
specific commercially desirable active constituents (i.e.,
(% DW)
content
Picroside I
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
7C 4E 1A 2B 2D 3E 1D 2F P14 Nr
20 40 60
Culture Period (No. of day)
Fig. 5 Comparative analysis of picroside I productivities of nine
independent hairy root clones and that of the non-transformed
in vitro-grown control roots during three different growth phases.
Values are the mean of triplicate results and error bars show standard
deviations
Growth index
25
20
15
10
5
0
20 40 60
Culture Period (No. of Day)
Fig. 3 Comparative analysis of biomass yield (GI) production
potential of nine independent hairy root clones and that of the nontransformed
in vitro-grown control roots during three different growth
phases. Values are the mean of triplicate results, and error bars show
standard deviations
(% DW)
Kutkoside Content
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
7C 4E 1A 2B 2D 3E 1D 2F P14 Nr
20 40 60
Culture Period (No. of day)
Fig. 4 Comparative analysis of Kutkoside productivities of nine
independent hairy root clones and that of the non-transformed in vitro
grown control roots during three different growth phases. Values are
the mean of triplicate results, and error bars show standard deviations
Fig. 6 The selected superior P14 hairy root clone of Picrorhiza
kurroa cultured on hormone-free, half-strength B5 medium after
10 days of culture (a) and after 40 days of culture (b)
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Plant Biotechnol Rep (2007) 1:169–174 173
picroside-I and kutkoside) have been expressed by all the
nine tested hairy root clones of P. kurroa, although distinct
inter-clonal quantitative variations could be noted (Figs. 4,
5). In seven of the nine hairy root lines studied (i.e., 1A,
1D, 2B, 2D, 4E, 2F and P14), the product synthesis was
found to be related to growth and maximum production of
the total glycoside, and as well that of the selected individual
ones (i.e., picroside I and kutkoside) coincided with
the exponential growth phase. Although the remaining two
root clones (7C and 3E) complied with the growth-related
total glycoside production pattern, their picroside I and
kutkoside production potentials deviated from the abovementioned
trend. Growth-related production behavior of
the active constituents has also been noted in a number of
other plant species by several investigators (Hamill et al.
1986; Constabel and Towers 1988; Toivonen et al. 1991;
Verma et al. 2002).
It is interesting to note that the biosynthetic potential of
the 14P line, in terms of picroside-I and kutkoside content,
superseded that of all other hairy root clones along with the
non-transformed control roots of P. kurroa. Additionally,
this selected 14P hairy root clone produced almost three
times higher amounts of the total as well as individual
glycosides compared to that of the control non-transformed
roots at their respective optimum production phases.
Moreover, the prescribed ratio between the two important
components of Picrorhiza glycosides (i.e., picroside-I and
kutkoside), as noted usually in the naturally grown roots
(Kumar et al. 2004), has excellently been manifested by
this selected 14P hairy root clone.
The clonal nature of the individually selected hairy root
lines resulting from site-specific insertion of Ri T-DNA and
the difference in the incorporated copy numbers might
have finally been responsible for such inter-clonal variation
in the biosynthetic potentials in terms of the targeted
compounds, as has already been reported in several other
medicinal plant species (Wysokinska and Chmiel 1987;
Mano et al. 1986, 1989; Yoshimatsu et al. 1990; Inomata
et al. 1993; Zehra et al. 1999) (Fig. 6).
Scale-up studies
The biomass yield of the hairy root line 14P, when cultured
in 1-l medium, was found to be 10.43 g (DW) in 40 days of
culture. In contrast, the 3-year-old field-grown P. kurroa
plant is reported to yield 0.75 g dry roots/plant (Nautiyal
et al. 2001), which is nearly 14 times less than that of the
selected 14P hairy root line. Consequently, the kutkoside
and picroside yield was noted to be nearly four-fold higher
in the 14P line than the reported 3-year-old field-grown
plant.
Conclusion
The outcome of the present investigation elucidates successful
induction, growth, maintenance and selection of the
rapidly growing hairy root line (14P) of P.kurroa with
in vitro desired phytomolecule production potential. The
superiority of the selected 14P hairy root clone in terms of
root biomass and kutkoside/picroside yield over the reported
3-year-old field-grown P. kurroa plant (Nautiyal et al. 2001)
undoubtedly highlights the significance of the present research
endeavor, which can endow us with a substitute for
the exploitation of this commercially important, endangered,
hitherto unexplored medicinal plant species.
Acknowledgments The authors wish to express their sincere thanks
to Dr. Sushil Kumar (Ex-Director, CIMAP, Lucknow) for providing
the necessary research facilities and constant encouragement. Heartfelt
thanks are also due to Dr. R Pal (Senior Scientist, CDRI, Lucknow)
for his constructive suggestions and requisite guidance.
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