Objectives Here, we report the effect of overexpression of ginseng farnesyl diphosphate synthase on the transcription of three key regulatory enzymes involved in triterpene metabolism in hairy root of ginseng and Centella asiatica (L.) Urban. Materials and Methods A four-year-old root of Panax ginseng C.A. Meyer and Centella asiatica (L.) Urban whole plants were obtained from National Institute of Crop Science (Suwon, Korea) and Chonnam National University (Gwangju, Korea), respectively. Agrobacterium rhizogenes R1000 strain was kindly provided by Dr. In (Nongwoo Bio, Yeju, Korea). Results and Discussion The role of farnesyl diphosphate synthase (FPS) in triterpene biosynthesis (Fig. 1) was investigated. A pCAMBIA3101 vector was used to insert a exogenous gene into target plant genome (Fig. 2). After the transformation, we produced Panax ginseng and Centella asiatica hairy roots by introducing the coding region of the gene from Panax ginseng. In these hairy roots, integration of the transgenes into the C. asiatica nuclear genome was confirmed by PCR analysis using PgFPS (P. ginseng FPS) primers and by Southern hybridization using PgFPS-specific probe. FPS specific activity is increased 4-fold compared to controls. In RT-PCR analysis, overexpression of PgFPS in hairy roots was observed (Fig. 3) and two genes, cycloartenol and beta-amyrin synthase, related to triterpene biosynthesis were up-regulated. These results suggest that FPS overexpression might lead to an enhanced biosynthesis of triterpene saponins and phytosterols. However, we did not demonstrate whether or not the introduction of PgFPS gene in Centella asiatica genome directly enhances triterpene saponin production, although our results showed that gene expression related to triterpene saponin biosynthesis were obviously up-regulated. Therefore, additional experiments such as overexpression of FPS gene in triterpene saponin-deficient mutant plants will be required.

This study was carried out to compare chromosomal characteristics between Atractylodes japonica and A macrocephala. Cytogenetic analysis was conducted based on karyotype analysis and physical mapping using fluorescence in situ hybridization. As a result of karyotype analysis by feulgen staining, somatic chromosome numbers of A. japonica and A. macrocephala were 2n=24. The length. of the mitotic metaphase chromosomes of A. japonica ranged from 0.70 to 1.60μm with a total length. of 12.11μm and the homologous chromosome complement comprised six metacentrics, five submetacentrics and one subtelocentrics. On the other hand, the length of the mitotic metaphase chromosomes of A. macrocephala ranged from 0.90 to 2.35μm with a total length of 16.58μm and the homologous chromosome complement comprised seven metacentrics and five submetacentrics. The total length of A. japonica chromosomes was shorter than that of A. macrocephala, but A. japonica had one subtelocentrics (chromosomes 4) different from A. macrocepha1a. chromosomes. The F1SH technique using 17S and 5S rDNA was applied to metaphase chromosomes. The signals for 17S rDNA were detected on the telomeric regions of chromosomes 4 and 5 in both A japonica and A. macrocephala. The 5S rDNA signal was found in the short arm of chromosome 1.

To identify the variation of the RAPD patterns between two Atractylodes species, 52 kinds of random primers were applied to each eight of A japonica and A. macrocephala genomic DNA. Ten primers of 52 primers could be used to discriminate between the species and 18 polymorphisms among 67 scored DNA fragments (18 fragments are specific for A. japonica and A. macrocephala) were generated using these primers, 26.9% of which were polymorphic. RAPD data from the 10 primers was used for cluster analysis. The cluster analysis of RAPD markers showed that the two groups are genetically distinct. On the other hand, to identify the variation of the AFLP patterns and select the species specific AFLP markers, eight combinations of EcoRI/MseI primers were applied to the bulked A. japonica and A. macrocephala genomic DNA. Consequently, three combinations of EcoRI/MseI primers (EcoRI /Mse I ; AAC/CTA, AAC/CAA, AAG/CTA) used in this study revealed 176 reliable AFLP markers, 42.0% of which were polymorphic. 74 polymorphisms out of 176 scored DNA fragments were enough to clearly discriminate between two Atractylodes species.