Transformed root culture

Mukundan, U. et al., pH-mediated release of betalains from transformed root cultures of Beta vulgaris L., Appl. Microbiol. BiotechnoL, 50, 241, 1998. 

Robins, R. J., Parr, A. J. and Walton, N. J. 1991. Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures. 2. on the relative contributions of L-arginine and L-ornithine to the formation of the tropane ring. Planta, 183 196-201. 

N. J. 1989. Regulation of secondary metabolism in transformed root cultures. In Primary and Secondary Metabolism of Plant Cell Cultures II (Kurz, W. G. W ed.), pp. 58-72. Berlin Springer-Verlag. 

Porsteffen, A., Drager, B. and Nahrstedt, A. 1992. Two tropine reducing enzymes from Datura stramonium transformed root cultures. Phytochemistry, 31 1135-1138. 

McLauchan, W. R., McKee, R. A. and Evans, D. M. 1993. The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv.SC58. Planta, 191 440 45. 

The usefulness of GC-MS analysis for biosynthetic studies was demonstrated by Patterson and O Hagan [74] in their investigation of the conversion of littorine to hyoscyamine after feeding transformed root cultures of Datura stramonium with deuterium-labeled phenyllactic adds. This study complements previous investigations on the biosynthesis of the tropate ester moiety of hyoscyamine and scopolamine [75], where GC-MS played a key role. It also has general relevance in the biosynthetic pathway of tropane alkaloids in the entire plant kingdom [76]. 

Two enz)mies of pyrrolidine alkaloid formation responsible for the conversion of putrescine to the N-methylpyrrolinium ion have been investigated in some detail. PMT, partially purified from cultures of Hyoscyamus niger and fully characterized from Datura stramonium, has been cloned by differential screening of complementary deoxyribonucleic acid (cDNA) libraries from high- and low-nicotine-yielding N. tabacum plants (Hibi et ah, 1994). The enzyme shows considerable sequence homology to spermidine synthase but is distinct from this enz)mie as it only shows PMT activity when expressed in Escherichia coli. MPO has been isolated in pure form from N. tabacum transformed root cultures (McLauchlan et ah, 1993). It is quite widely spread in... 

Both untransformed (Hashimofo and Yamada, 1994) and transformed root cultures of Datura, Hyoscyamus, Atropa and Duboisia species (Robins and Walton, 1993) accumulate high levels of fhe fropane alkaloids, hyoscyamine and scopolamine (Fig. 2.3). These medically imporfanf fropane alkaloids present not only an interesting biochemical problem but also a realistic... 

A detailed study of H6H has allowed the genetic manipulation of scopolamine formation. The alkaloid spectrum of transformed root cultures of A. belladonna contains hyoscyamine and scopolamine in a ratio between 10 1... 

Drager, B., Portsteffen, A., Schaal, A., McCabe, P.H., Peerless, A.C.J. and Robins, R.J. (1992) Levels of tropinone reductase activities influence the spectrum of tropane esters found in transformed root cultures of Datura stramonium. Planta, 188, 581-6. 

Rhodes, M.J.C., Robins, R.J., Parr, A.J. and Walton, N.J. (1990) Secondary metabolism in transformed root cultures, in Secondary Products from Plant Tissue Culture (eds B.V. Charlwood and M.J.C. Rhodes). Oxford University Press, Oxford, pp. 201-25. 

Robins, R.J., Woolley, J.G., Ansarin, M., Eagles, J. and Goodfellow, B.J. (1994a) Phenyl-lactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures. Planta, 194, 86-94. 

Teuber M, Azemi ME, Namjoyan F, Meier A-C, Wodak A, Brandt W, Drager B. Putrescine N-methyltransferases—a structure-function analysis. Plant Mol. Biol. 2007 63 787-801. McLauchlan WR, McKee RA, Evans DM. The purification and immunocharacterization of N-methylputrescine oxidase from transformed root cultures of Nicotinia tabacum. Planta 1993 191 440-445. 

O Hagan D, Robins RJ, Wilson M, Wong CW, Berry M. Hu-orinated tropane alkaloids generated by directed biosynthesis in transformed root cultures of Datura stramonium. J. Chem Soc. Perkins Trans. 1. 1999 2117-2120. 

Concomitant to the enhancement of the signal for C-3 in the C NMR spectrum, the signals for H2 and H4 decreased in the H-NMR spectrum of all the tropine moieties compared with those in authentic samples. This indicates the incorporation of deuterium at C2 and C4 derived from C Hj CXX)". As the diminution of the signals for H2 and H4 was not identical at both positions, we suppose a sequential incorporation of labeled acetate (example given in Fig. 15b). From these data we assume that the biosynthesis from N-methyl-pyrrolidinium ion to tropine is a two-step process which does not involve a four carbon unit (acetoacetyl coenzyme A) but two units of acetyl coenzyme A, which were added sequentially as has been suggested for the biosynthesis of cocaine [28, 37]. On the other hand it has been reported that l,2- C2-acetate was incorporated with an equal efficiency at C2 and C4 by non-transformed root cultures of H. albus [36]. Further investigations are required to clarify this matter. 

Tropinone is stereospecifically reduced to yield both tropine (3a-hydroxytropine), which led to the formation of tropane alkaloids, and pseudotropine (3p-hydroxytropine), the precursor of calystegines. These stereospecific reductions are catalyzed by two different tropinone reductases, tropinone reductase I and II (TRI and TRII). Both enzymes have been isolated from many Solanaceous species. Thus, TR I and TR II were isolated from D. innoxia roots and the crude extract favoured the production of pseudotropine over tropine [151]. Also, from transformed root cultures of D. stramonium two different tropinone reductases were obtained. In this species, TRI showed about 5-fold larger activity than TRII, and TRI displayed a pronounced pH-dependency, while TRII was more tolerant to different pH values [152]. Moreover, two tropinone reductases were also isolated from H. niger root cultures. TRI-reduction was reversible, whereas TRII-reduction was essentially irreversible [153]. In subsequent studies it was found that the accumulation of both TRs was the highest in the lateral roots of H. niger throughout development, with different cell-specific patterns [154]. 

In this section, transformation of some medicinal plants with A. rhizogenes and production of useful secondary metabolites by transformed root cultures are described. In addition, production of transgenic plants is discussed. 

Fig. (55). Alkaloid yields from transformed root cultures.

A Shoot regeneration on the transformed roots cultured on HF 1/2 MS solid medium at 25°C in the dark for 6 months. B Aberrant feature of leaves (three leaves) observed in the transformed shoot cultured on HF B5 solid medium at 25°C under 16 hr light. C Comparison between transformed (left) and non-transformed (right) plants cultured on HF B5 solid medium at 25°C under 16 hr light. 

Transformed cultures of C. ipecacuanha, one of the recalcitrant woody plant species for Agrobacterium-mQdaaiQd transformation, have been established by a co-culture method. This work is the first report on transformation of ipecac as far as we know and this proved a capability of genetic transformation of ipecac by use of Agrobacterium-mQdiaiQd transformation. Growth of the transformed roots with only Tl-DNA was inferior to the previously reported hairy roots. However, optimization of culture conditions, basal medium and auxin addition could improve fresh mass and ipecac alkaloid productivity. The transformed root cultures presented herein may be a promising tool for an alternative source of ipecac alkaloids and for biosynthetic and transport studies of ipecac alkaloids. 

Embryogenic callus was also obtained from the non-transformed roots cultured with 0.5 mg/1 NAA, Fig. (62). Mikimopine, however, was only detected in the MAFF clones. Fig. (63). 

One of the advantages of the ability to isolate calystegines from transformed root cultures is that this should enable experiments to be conducted much more easily than in whole plants and the biosynthetic pathway may therefore be elucidated in the near future. 

Monitoring nitrogen metabolism in a transformed root culture of Datura stramonium. Fliniaux and colleagues reported the application of long-range data in their... 

Major progress has been achieved using labelling and enzymatic methods applied to in vitro tissue cultures, in particular with genetically transformed root cultures. 

Considerable interest has been shown recently in genetically transformed root cultures for the production of secondary metabolites. In order to develop hairy root cultures in the laboratory, surface-sterilized plant tissue is inoculated with a suspension of A. rhizogenes and, after a period of incubation (generally between 1-6 weeks) at 24-28°C, transformed roots emerge at the infection sites. After elimination of the excess bacteria, the excised roots are incubated in liquid culture medium. The genetically transformed roots grow faster than do untransformed roots, they are highly branched and they can be cultivated in hormone-free... 

Table 7. Some Examples of Tropane Alkaloid Production by Agrobacterium rhizogenes-Transformed Root Cultures...

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