Formation of Agrobacterium rhizogenes-mediated transformed hairy root cultures of Rubia tinctorum L. and production of secondary metabolites

Plant secondary metabolites have been widely used in various sectors; i.e, medicine, food,agriculture. For this reason researches especially on the production of secondary metabolites viatissue culture techniques have been accelerated.Hairy root cultures which have biochemical and genetic stabilities, fast growing abilities inmedia without plant growth hormones are the most suitable and preferable tissue culture techniques.Agrobacterium rhizogenes has been used regularly for gene transfer in many dicotyledonous plants.As a result of the infection of this bacteria, T-DNA of root inducing plasmid (Ri-plasmid) is transfered tothe plant cell and integrated to genomic DNA. The proliferative and multibranched hairy roots areformed in infection regions. These roots have very important potentials in the production of some plantsecondary metabolites.Rubia tinctorum L. `Madder? used by local people in dyeing of carpets with its roots for manyyears has diuretic, antispasmodic, antiscorbutic and antibacterial properties. In this thesis, hairy rootcultures of Rubia tinctorum L. and their secondary metabolites were investigated.After surface sterilization of seeds, micropropagation of plantlets was achieved in MS mediacontaining 0,5 mg/l IBA. In transformation studies, the leaf and nod explants of the plantlets wereinoculated by 4 different strains (15834, A4, R1000, 8196) of Agrobacterium rhizogenes. The highesthairy root occurrence was determined as 61,2% in the nod explants inoculated with 15834 in solid MSmedium. The obtained hairy roots were cultured in liquid MS, B5 and SH media. Especially the hairyroots obtained from MS medium were golden color; the roots from B5 media were red and also thecolor of liquid B5 media turned red. The roots obtained from SH media were yellowish and thin.The amounts of secondary metabolites in hairy roots formed by inoculation with differentstrains (A4, R1000 and 8196) in MS medium were analyzed with HPLC. The highest amounst ofanthraquinones in hairy roots obtained with R1000 strain were determined as 1.13 mg/g for lucidinprimeveroside; 8.4 mg/g for ruberythric acid, and 0.072 mg/g for alizarin.0,1µM methyl jasmonate as an elicitor and high amount of sucrose (%12) were added into B5media in order to increase the amount of secondary metabolite. In the dried hairy roots obtained fromB5 media containing sucrose without methyl jasmonate (B5S), the amounts of lucidin primeveroside,ruberythric acid and alizarin were determined as 13,45 mg/g, 93,2 mg/g and 0,78 mg/g, respectively.The hairy roots belonging to B5 media containing methyl jasmonate and 120 g sucrose (B5SMeJA)the amounts of lucidin primeveroside, ruberythric acid, and alizarin were found as 12,31 mg/g, 86,8mg/g 0,14 mg/g, respectively. In the collected roots from nature, the amounts of lucidin primeveroside,ruberythric acid, except alizarin metabolites were higher than the hairy roots.Key wordsRubia tinctorum, Agrobacterium rhizogenes, hairy root, secondary metabolite, alizarin