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

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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) 123
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. References Anonymous (2001) Picrorhiza kurroa Monograph. Altern Med Rev 6:319–321 Banerjee S, Zehra M, Kukreja AK, Kumar S (1995) Hairy roots in medicinal plants. Curr Res Med Arom Plants 17:348–378 Byrne M, Mc Donnell R, Wright M, Carnes M (1987) Strain and genotype specificity in Agrobacterium soybean interaction. Plant Cell Tissue Organ Cult 8:3–5 Christey MC, Braun RH (2005) Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation. Methods Mol Biol 286:47–60 Constabel CP, Towers GHN (1988) Thiarubine accumulation in hairy root cultures of Chaenactis douglasii. J Plant Physiol 133:67–72 Dubey NK, Kumar R, Tripathi P (2004) Global promotion of herbal medicine: India’s opportunity. Curr Sci 86:37–41 Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151– 158 Giri A, Ravindra ST, Dhingra V, Narasu ML (2001) Influence of different strains of Agrobacterium rhizogenes on induction of hairy roots and artemisinin production in Artemisia annua. Curr Sci 81:378–382 Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006a) Harnessing the potential of hairy roots: dawn of a new era. Trends Biotechnol 24:403–409 Guillon S, Tremouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006b) Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol 9:341–346 Gupta PP (2001) Picroliv. Drugs Future 26:25–31 Gupta A, Khajuria A, Singh J, Bedi KL, Satti NK, Dutt P, Suri KA, Suri OP, Qazi GN (2006) Immunomodulatory activity of biopolymeric fraction RLJ-NE-205 from Picrorhiza kurroa. Int Immunopharmacol 6:1543–1549 Hamil JD, Parr AJ, Robins RJ, Rhodes MJC (1986) Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes. 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