Functional genomics meets medicinal plants - innovative plant breeding to optimize plant secondary metabolites

Medicinal plants have been used for thousands of years - and the trend is growing

Medicinal plants have played a vital role in human health for thousands of years. In recent decades, the global use of these valuable plants has increased significantly. This is due to a number of factors: An ageing population, a growing interest in natural and environmentally friendly products, and an increasing desire for self-treatment. Market research predicts that the global market for herbal medicines will grow at an annual rate of around 11% until 2033. Notably, the European market is expected to become the second largest market after the Asia-Pacific region in the coming years. In Europe, Germany plays a key role as one of the leading processors of medicinal plants. The importance of the sector, which is dominated by small and medium-sized enterprises, is due not only to the globally significant production of phytopharmaceuticals, but also to a strong extraction and distribution industry.

Securing the supply of medicinal plants and protecting their populations is of great importance. Of the 50,000 to 80,000 species of plants used for medicinal purposes, around 15,000 are threatened by overexploitation and habitat destruction, while only around 900 species are cultivated. Sourcing plant raw materials is becoming increasingly challenging, partly due to crop failures caused by natural disasters, which are increasing as a result of climate change. However, high quality, reproducible plant material is essential for the supply of botanicals and botanical blends. Breeding new varieties enables adaptation to market needs and focuses on higher yields of active compounds, elimination of undesirable ingredients and tolerance to stress factors. Despite decades of cultivation of some medicinal plants, around 60 per cent of the species grown on a large scale have not been bred as breeding has often resulted in inferior varieties with low yields and highly variable levels of active ingredients. Unlike conventional crops such as maize or wheat, modern medicinal plant breeding is still in its early stages. Researchers and breeders lack basic data on the vast majority of medicinal plants, such as genetic information, physiological conditions, seed characteristics and the relationship between quality and efficacy and cultivation factors.

Fennel (Foeniculum vulgare Mill. subsp. vulgare var. vulgare) is one of the most important medicinal plants in Germany. It is a hardy, biennial or perennial herbaceous plant with yellow flowers and feathery leaves. Fennel is used to relieve digestive disorders and inflammation of the mucous membranes (catarrh) of the upper respiratory tract. The medicinal use of fennel is largely attributed to the antispasmodic, secretolytic, secretomotor, and anti-inflammatory effects of its essential oil, which is extracted from the fruits. The dried fruits are traditionally prepared as a tea. They are also used as dry extracts for dragees, alcoholic extracts for tinctures, and fennel oil for lozenges. Major constituents of the essential oil include trans-anethole (50-70 percent), fenchone (12-25 percent), and estragole (2-8 percent). The sweet anethole is considered an antispasmodic and digestive aid and is thought to work with the bitter fenchone to contribute to the expectorant effect of fennel seeds. The European Pharmacopoeia defines bitter fennel (Foeniculi amari fructus) as having a minimum content of 4 percent essential oil, of which at least 60 percent must be anethole and 15 percent fenchone. The estragole content in the essential oil must not exceed five percent.

Undesirable ingredient estragole: reduction as one important breeding goal

Because excessive use of estragole may pose a health risk, the European Medicines Agency (EMA) has established the following regulatory guidelines: For adults and adolescents, a maximum dose of 0.05 mg estragole per day, while for children under 12 years of age, a dose of 1.0 μg/kg body weight is recommended. Use is not recommended for children under 4 years of age. There are currently no legal regulations in the food sector. The European Food Safety Authority (EFSA) is currently assessing the safety of foods containing fennel and an opinion is expected in 2025. Estragole is considered potentially carcinogenic and probably mutagenic. Prolonged intake may lead to degradation products that can cause liver and genetic damage.
Reducing or even eliminating estragole in fennel is therefore an important breeding objective that we are working on at the Münster location. Despite the high agronomic and pharmaceutical relevance of fennel, we face several challenges as there is little breeding-related data available for fennel. Previous attempts to reduce the estragole content have often been accompanied by a simultaneous reduction in the desired ingredients. For example, a medium to low estragole content has been described for the Magnafena variety, but the trans-antheol content of the fruit's essential oil does not always reach the minimum level of 60 percent required by the European Pharmacopoeia.

A first step in the development of molecular markers and metabolic engineering to minimize estragole was the establishment of a tissue culture from grain fennel explants in our facility. Tissue culture allows for the cultivation of cell aggregates under sterile, controlled conditions. Another important milestone was the establishment of an efficient transformation protocol. For the first time, it is now possible to introduce genetic material into fennel cells and regenerate fennel plantlets. To test the efficiency of the transformation, we used a reporter gene whose expression is indicated by a characteristic red coloration of the fennel shoot. This red coloration serves as an indicator of successful integration and expression of the introduced gene. Furthermore, we were able to analyze the first molecular basis of the biosynthetic pathway for trans-anethole and estragole in grain fennel and identify the underlying genes. In future work, we will functionally characterize the identified genes to better understand their specific roles and functions in the biosynthesis of trans-anethole and estragole. To this end, we are using genome editing techniques such as CRISPR/Cas9 technology, which allows us to specifically knock out genes.
The technologies and know-how that we are generating through our research and development work on grain fennel offer promising prospects for transfer to other plants that also accumulate estragole as major constituents of the essential oil - such as tarragon (60-75 percent estragole), chervil (60 percent estragole) or basil (23-88 percent estragole).