Establishment of authentication systems for seven Panax species and a hairy root transformation system for P. ginseng

Abstract

Panax species belong to the Araliaceae family and regarded as the king of herbal medicinal plants owing to the abundance of pharmacologically valuable ginsenosides. Among 14 reported Panax species, five major species including P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. vietnamensis have been well known, and are broadly utilized as herbaceous medicinal plants in Korea, the USA, Canada, China, Japan, and Vietnam. However, their genetic diversity, evolutionary relationship, and origin remain largely unresolved. Each Panax species is pharmacologically and economically important, albeit with differences in efficacy and price. These lead to the fraudulent, intentional substitution or addition of a substance in a product for financial advantage. Panax species have been harvested mostly from the wild except four cultivated species including P. ginseng, P. quinquefolius, P. notoginseng, and P. vietnamensis. P. ginseng is one of the most known Panax species because it contains a relatively more diverse types of ginsenosides. However, cultivation of P. ginseng takes a long time (about 4 - 6 years) with extensive efforts to control quality from biotic and abiotic stresses. All these problems and limitations have affected the development of ginseng industry. To overcome these limitations and understand their genetic diversity and evolutionary relationship, I conducted a comparative analysis of whole chloroplast genomes from seven Panax species and established the authentication systems based on chloroplast genome sequences. Furthermore, I developed a hairy root transformation system for biomass and ginsenoside production in P. ginseng. In the first chapter, I conducted a comparative analysis of whole chloroplast genome sequences of five Panax species including P. ginseng, P. quinquefolius, P. notoginseng, P. japonicus, and P. vietnamensis. I identified the number of short and large repetitive sequences, and screened large numbers of InDels and SNPs among these five species. Based on the large InDel regions, I developed fourteen practical InDel markers for authentication among these five Panax species, and eight of those markers were species-specific markers that successfully discriminated one unique species from the others. These markers are reliable, easily detectable, and valuable for applications in the ginseng industry as well as in related research. In the second chapter, I completed the chloroplast genome sequences of two more basal Panax species (P. stipuleanatus and P. trifolius). Comparative analysis of the chloroplast genome sequences of the seven Panax species revealed the numbers of SNPs in protein-coding genes and whole chloroplast genome level. Phylogenetic analysis based on whole chloroplast genomes clearly showed evolutionary relationship between the seven Panax and their relative species in Araliaceae family. By comparing chloroplast and mitochondrial genomes, I discovered a large number of fragments from the chloroplast genome, which is approximately 38.6%, transferred into the mitochondrial genome. I developed 18 species-specific SNP markers from the chloroplast coding sequences after eliminating intraspecies polymorphic sites, and chloroplast gene transfer regions. All these markers successfully distinguished one species from another, and can be used to authenticate all the seven Panax species from each other, thereby furthering efforts to protect the ginseng industry from economically motivated adulteration. In the third or final chapter, I developed an efficient system for ginsenoside production through hairy root biomass mediated by Agrobacterium rhizogenes transformation. Among five transformed lines, I selected two lines which showed highest efficiency of transformation including Yunpoong cultivar and Ganghwa local landrace. PCR and RT-PCR analysis for rol genes in these two transformed root lines indicated that these are transgenic hairy roots induced by A. rhizogenes transformation. Transgenic hairy roots can grow faster than adventitious roots, and transgenic hairy root growth were not affected by auxin (IBA) application. Transgenic hairy roots produced the identical ginsenoside in comparison with those of adventitious roots. Even though total ginsenoside contents tended to be lower than those of adventitious roots, some major ginsenosides were biosynthesized in similar amounts or higher than those in adventitious roots. Bioreactor was indicated as an ideal system for large scale production of transgenic hairy roots. This study provided valuable genetic information which can be used for future studies. The evolutionary relationship for seven Panax and their relative species were elucidated by whole chloroplast genomes. Two kinds of molecular markers developed in this studies can be used to authenticate all the seven Panax species from the others, thereby furthering efforts to protect the ginseng industry from economically motivated adulteration. Transformation system established in this study can be applied for future related researches, and transgenic hairy roots produced here will be a valuable material resources for biomass and ginsenoside productions as well as other studies.