It has been clearly demonstrated that Ti plasmid present in Agrobacterium tumefaciens and Ri plasmid from A. rhizogenes cause the transformation of plant cells by introducing their T-DNA into genomic DNA of plant cells.
The Ri plasmid is the causative agent of so-called hairy root disease. Hairy root cultures are able to synthesize a variety of plant secondary products. This is of special interest in medicinal plants that synthesize and accumulate pharmaceutically important metabolites in their roots.
The first report of the induction of hairy roots in P. ginseng and the establishment of the culture followed by the infection with A. rhizogenes was demonstrated by Yoshikawa and Furuya (1987). The ginseng hairy root cultures grew more rapidly and produced saponins more effectively than the ordinary cultured roots obtained by hormonal control. Production of ginseng saponins, such as ginsenosides Rb and Rg in the hairy roots and ordinary cultured roots, as determined by TLC, was comparable. The total saponin contents per dry mass were 0.35-0.95 per cent for the transformed hairy roots and 0.38-0.91 per cent for the ordinary cultured roots. The highest contents were obtained when both hairy and ordinary roots were grown on a medium supplemented with growth regulators such as IBA and kinetin. Inomata et al. (1995) achieved the highest growth rate of ginseng hairy roots in batch culture under the effect of BA which also increased the ginsenoside production. Ginsenoside production by hairy root cultures, analyzed by HPLC, was reported by Ko et al. (1989); the results were comparable with those of Yoshikawa and Furuya (1987) (0.470.82 per cent). These authors found that the production of ginsenoside Rb1 reached a maximum level in an early stage of culture in contrast to the yields of Rg1 and Re which reached their maxima at a later stage of culture. Recently these authors reported on the cultivation of hairy root clone HRB-15 in a 3 l bubble type bioreactor (Ko et al., 1996). They established a unique two-step process of hairy root culture to maximize biomass and secondary metabolites. The ginsenoside synthesis was enhanced by yeast elicitation.
Hairy root cultures were further used for biotransformation studies. Kawaguchi et al. (1990) reported on the biotransformation of digitoxigenin by P. ginseng hairy roots and found that biotransformation involved esterification and high glycosylation ability. As a result, five new components and seven previously reported components were isolated as biotransformation products of digitoxigenin. Among the new compounds three esters (digitoxigenin stearate, digitoxigenin palmitate and digitoxigenin myristate) and two glucosides (3-epidigitoxigenin ^-D-gentiobioside and digitoxigenin ^-D-sophoroside) were determined.
Further attempts were aimed at the continuous production of glycosides by hairy root cultures cultivated in a bioreactor (Yoshikawa et al., 1993). As a result, a continuous glycosylation of (^Sj-2-phenylpropionic acid (PPA) was determined during two months. PPA was converted to (RS)-2-phenylpropionyl ^-D-glucopyranoside at a 71 per cent conversion ratio, and to (2RS)-2-O-(2-phenylpropionyl)-D-glucose (8 per cent), (2S)-2-phenylpropionyl 6-O-^-D-xylopyranosyl- ^-D-glucopyranoside (10 per cent) and a myo-inositol ester of (R)-2-phenylpropionic acid (5 per cent). Moreover, about half of the conversion products were excreted.
Lee et al. (1995) have established an efficient transformation system using P. ginseng cotyledonary explants and A. tumefaciens strain LBA 4404 harbouring the binary vector pBI121 carrying the CaMV 35S promoter-GUS gene fusion and the neomycin phosphotransferase gene (NPTII) as a selectable marker. All embryos derived from kanamycin-resistant calli exhibited a GUS-positive response which appeared in petiole, stem, root and flower in more than 50 per cent of regenerants, which was proved with X-gluc treatment and by Southern blot analysis using a DIG-labeled GUS NOS poly(A) probe. The mitotic stability of the GUS expression in protoplast-derived regenerants was determined by PCR and X-gluc treatment. The PCR method revealed the presence of the GUS gene in 92 per cent of regenerants and the X-gluc treatment in 78 per cent of them.
This Agrobacterium-mediated transformation system may be of practical use for introducing single-gene mediated traits, such as herbicide, insect, and disease resistance into this species.
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