Assessment of Genetic Diversity in Podophyllum peltatum by Molecular Markers*

The quality and quantity of medicinal plants are serious issues for the pharmaceutical and dietary supplement industries. Traditionally, this material has been harvested from the wild, and while cultivation procedures have been developed for supplements such as St. John’s Wort, this is still the general method employed for the majority of medicinal plants. Increasing public demand for these products is creating a serious environmental problem as demand is out pacing the supply and endangering the survival of many of these species in the wild. There are two basic uses for medicinal plants; direct use as dietary supplements or as “chemical factories” for the production of plant-derived drugs such as podophyllotoxin, a lignan produced by the phenylpropanoid pathway. Thus, the parameters used to evaluate the source differ slightly depending on the application. As chemical factories, yield is a combination of biomass and drug content, which defines the quality of the plant material. In cultivation, yield is measured by the amount of compound produced per hectare and not on a single plant basis. Growing genetically defined plant material improves biomass quality and helps to protect the world’s germplasm from extinction. Unfortunately, there are few breeding programs for medicinal plants. We are presently trying to achieve these goals for several medicinal plants using a variety of quantitative methods. One of the medicinal plant species we are adapting to cultivation is Podophyllum peltatum L., Berberidaceae, also known as North American mayapple. The genus Podophyllum is a rich source of podophyllotoxin, an aryltetralin lignan that has important biological activities (Leander and Rosen 1988) and is the precursor of semisynthetic chemotherapeutic drugs such as etoposide, teniposide, and etopophos (Stähelin and Wartburg 1991; Imbert 1998). Currently, the patent for etoposide has expired and new clinical trials on various cancer therapies are under way (Ekstrom et al. 1998; Holm et al. 1998; Ajani et al. 1999). These factors have caused the sales of podophyllotoxin in the US to double from 1994 to 1999. In addition, new improved etoposide derivatives such as NK 611, GL 331, and TOP 53 are in pre-clinical development and are showing good results (Huang et al. 1996; Pagani et al. 1996; Utsugi et al. 1996; Raßmann et al. 1999). Podophyllum emodi is presently the commercial source of podophyllotoxin for the pharmaceutical industry. It has been so intensively harvested from the wild that it is now endangered in its natural habitat, an area of the Himalayas (Foster 1993; Bhadula et al. 1996). In P. emodi, only rhizomes and roots are rich in podophyllotoxin (Jackson and Dewick 1984), and it takes seven or more years to establish a well-developed underground root/rhizome system. The majority of the roots and rhizomes are harvested resulting in the total destruction of the plants. In contrast, P. peltatum stores podophyllotoxin 4-O-β-D-glucopyranoside in leaf blades, and recent developments have shown that this compound can be easily converted to podophyllotoxin (Canel et al. 2000, 2001). The amount of podophyllotoxin 4-O-β-D-glucopyranoside varies from plant to plant, but some contain as much as 3% on a dry weight basis (Canel et al. 2001). These findings suggest that P. peltatum is a better candidate for development as a sustainable crop than P. emodi because as a perennial, it generates leaves annually from the rhizome and the leaves can be harvested late in the season without damaging the plant. Therefore, it would be desirable to identify superior genetic material that could be adapted for mayapple cultivation. Podophyllum peltatum is found throughout the wooded landscape of the eastern half of North America (Meijer 1974). It has an extensive rhizogenous system that allows it to spread and survive as established colonies (de Kroon et al. 1991; Landa et al. 1992; Gerber et al. 1997). Studies have compared phenotypic characteristics between and within colonies, and while there can be variation between colonies, there appears to be little variation within smaller colonies (Parker 1989). Fertility studies between and within colonies indicate that seed set is lower when crosses are made within a colony (Laverty and Plowright 1988). It has