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

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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 123
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) 123
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