*not tested for essential oil production 62 Potentialities of Hairy Root Cultures for In Vitro Essential Oil Production A. Cristina Figueiredo 1* • José G. Barroso 1 • Luis G. Pedro 1 • Johannes J.C. Scheffer 2 1 Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, Centro de Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisbon, Portugal 2 LACDR, Leiden University, Gorlaeus Labs, PO Box 9502, 2300 RA Leiden, The Netherlands Corresponding author: *acsf@fc.ul.pt Keywords: Achillea millefolium, Anethum graveolens, batch culture, bioreactor, Levisticum officinale, Mygliol, Pimpinella anisum ABSTRACT Roots are much more than an organ that provides transport of water and solutes from the soil to the above ground parts of a plant. The recognition that roots possess a unique physiology and that they can contribute with biologically active chemicals to the environment expanded the field of root research. In this context, hairy roots became a unique experimental system, due to their high biomass growth allied to their biosynthetic capacity. In addition, hairy roots constitute a good system for the study of metabolic pathways, of root growth and primary and secondary metabolism, of their behaviour under stress-inducing environments, of the interrelationships of roots with biotic and abiotic factors, as well as the production of regenerants. The production of essential oils by hairy roots demonstrates a biosynthetic ability that can attain higher yields than in undifferentiated cultures and is sometimes equal to or higher than the parent plant. This capacity is strictly correlated with the differentiated state of the cultures, the level of production being severely impaired or lost when the hairy root phenotype is lost. Other factors, e.g. the type and/or age of the inoculums, the gap between subcultures, the combinations of nutritional and environmental stressing factors, including different medium composition, photoperiod conditions, and cultivation in a two-phase system and biotransformation, can also affect biomass growth and product performance. The possibility of growing and maintaining in vitro hairy root cultures of different species widens the indispensable basic knowledge needed to manipulate, in a controlled way, their molecular, biochemical and ecological potential. 1. INTRODUCTION The renewed interest in natural products in Western countries has brought essential oils to the vanguard of international trade. These oils are important, not only in the perfume industry, but also as antimicrobial and antioxidant components in agro-food industries. However, the steady supply of the market of essential oils faces some problems, mainly due to environmental (climate, pests, seasonal and geographic variations), political and social conditions, to the large amounts of plant material needed, as well as space and manual labor needs (Figueiredo 1998). In vitro plant cultures, mainly of hairy roots, appear in this context, as a potential approach to explore the improvement of essential oil accumulation. The advantages of hairy root cultures, for a large production of secondary metabolites, are mainly due to 1) their high growth rate in the absence of phytohormones, with a high ratio of biomass production per unit time, 2) their cellular integrity and longevity, which determine a Table 1 Factors detrimental to the in vitro synthesis of secondary metabolites.  The lack of differentiated structures  Accumulation in the glycosidic form  Mutations and chromosomal aberrations  Cellular defence mechanism  Non-regulation of the terpene biosynthetic pathway in the same way as the in vivo plants  Lack of specific tonoplast carriers  Reduced amounts of oxygen present in culture  Catabolic activity Table 2 Methodologies tested for enhancement of secondary metabolites production by plant cell cultures.  Calli cultures  Biotransformation  Cytodifferentiation induction  Immobilization  Ploidy increase  Hairy roots  Culture medium (medium composition and growth regulators)  Culture in mists*  Environmental conditions (photoperiod, temperature, addition of carbon dioxide)  Culture in a two-stage system (growth and production medium)*  Elicitation  Co-culture system*  Culture in a two-phase system