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

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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