Phymchendsny, Vol. 26, No. 12. pp. 3241-3245. 1987. 0031~9422/87 f3.00+ 0.00 Printed in Great Britain. 0 1987 Pergamon Journals Ltd. zyxwvutsrq RELATIONSHIP BETW EEN zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC AGROBACTERWM RHIZOGENES TRANSFORMED HAIRY ROOTS AND INTACT, UNINFECTED NICOTIANA PLANTS ADRIAN J. PARR and JOHN D. HAMILL Plant Cell Biotechnology Group, AFRC Institute of Food Research, Norwich, Colney Lane, Norwich NR4 7UA. U.K. (Receid 30 March 1987) Key Work Index-Mcotiunu; Solanaaae; tobacco; Agrobacrerium rhizogenes; hairy roots; biosynthetic capacity; transport; pyridine alkaloids. Abstract-Hairy root cultures were obtained following infection of a range of Nicotiana species with Agrobacterium rhizogenes. Such cultures synthesized alkaloids in amounts which closely reflected, in both qualitative and quantitative terms, the biosynthetic capacity of roots from the uninfected parent species or variety. Cultures also released alkaloids from the roots into the growth medium. Such release was not however correlated with the ability of intact plants to mobilizealkaloids from the roots to aerial parts. The predictable nature of many aspects of secondary product synthesis in hairy roots should be advantageous to the development of biotechnological processes. INTRODUCTION Infection of dicotyledonous plants with the bacterium Agrobacterium rhizogenes leads to the production of characteristic outgrowths of ‘hairy roots’ at the points of infection [ 11. Such roots arise from cells which have been genetically transformed by the acquisition of DNA (the transferred DNA, or T-DNA) from a plasmid (the R, plasmid) in the infecting bacterium [2,3]. This DNA codes for auxin synthesis, and other rhizogenic functions [4,5], such that the normal differentiated state of cells is altered in favour of root formation. Recent results [6] have shown that, when excised from the parent plant, the transformed hairy roots can be successfully cultured on a large scale in fermenter systems, with a growth rate similar to that for conventional cell suspensions. It has also been shown [7-IO] that hairy roots are able to synthesize secondary products characteristic of the roots of the original plant. Transformed hairy root cultures thus offer a novel approach to the biotechnological production of valuable plant secondary metabolites. Preliminary indications [7,8] suggested that one fea- ture of transformed root systems is that their biosynthetic capacity might closely reflect that of normal roots from the original plants. These system would thus offer ad- vantages over conventional cell culture systems, where secondary product synthesis is often at much lower levels than in the parent plant [l 11, and where the spectrum of products produced is also often different to that seen in plants [12]. In the present paper we report on investiga- tions to determine to what extent the analysis of whole plants can be used to predict the biosynthetic potential of transformed hairy root cultures. In plants of the genus Nicotiana, pyridine alkaloids are known to be synthesized in the roots, and then to some extent transported to the aerial parts [13]. Tansformed hairy roots can similarly synthesize alkaloids, and release a proportion into the growth medium [7]. We chose to examine a range of Nicotiana species of differing alkaloid content, com- position and distribution (e.g. 1143) in order to test whether plant root alkaloid content might be a useful indicator of alkaloid levels in transformed hairy roots, and whether shoot alkaloid content might indicate to what extent alkaloids are secreted by hairy root cultures. RESULTS Alkaloid synthesis As anticipated, substantial differences were observed between different Nicotiana species with regard to the alkaloid spectrum and content of intact plants (Table 1). Results broadly resembled those of ref. [14]. In some species however, plantlets grown aseptically in agar showed a lower alkaloid content and/or an increased proportion of the nicotine demethylation product, norni- cotine [IS]. This may indicate that conditions in sterile culture are sub-optimal for alkaloid synthesis in certain species of the genus Nicotiana, as has also been found for tropane alkaloid synthesis in Atropa belladonna [9]. Hairy roots cultures were easily obtained following infection of aseptic plantlets with Agrobacterium rhizo- genes. Southern blotting showed all lines to possess T, T- DNA (Table 2), at levels ranging from one integration event in N. cavicola and N. ajiiicana to five or six integra- tion events in N. hesperis and N. rustica. The majority of lines also possessed T, T-DNA (Table 2). although N. afiicana apparently lacked sequences homologous to the T, T-DNA probe used (covering ca SO”/,+ of the normal T, T-DNA region 1173). This is in line with observations that in some species T, T-DNA is apparently more important in determining the hairy root mor- phology [4,5]. 3241