Hairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, onemonth-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L −1 ) (as elicitor) and sucrose (20, 30, 40 and 50 g L −1 ) on the growth of hairy roots were evaluated. The results showed that, 30 g L −1 sucrose and 100 mg L −1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.
Agrobacterium rhizogenes is known as a natural tool of genetic engineering in many plant species. For the first time, hairy root induction in Agastache foeniculum using A. rhizogenes, rosmarinic acid content and the effect of different culture media and inoculation methods on hairy root growth rate were investigated. Hairy root culture of A. foeniculum was established by inoculation of the 1-month-old leaf explant with A4 strain of A. rhizogenes and the effectiveness of lightdark conditions and two inoculation methods (immersion and injection) were tested. Furthermore, in immersion method, the effects of inoculation time (3, 5 and 7 min) on root induction were investigated. In the second part of the study, the hairy root culture of A. foeniculum was studied using different basal culture media (MS, 1/2 MS and B5). Rosmarinic acid content in hairy roots and non-transformed roots was analyzed using high-performance liquid chromatography (HPLC). There was no significant difference between various inoculation methods in the ability of hairy roots induction. Observations showed that percentage of hairy root induction was higher when the explants were immersed for 5 min in bacterial suspension. Light conditions displayed the highest hairy root induction rates compared with dark condition. Various culture media are different in terms of types and amounts of nutrients and have influence on growth rate. The maximum growth rate (1.61 g fr wt/50 ml) of hairy roots were obtained in 1/2 MS medium. Rosmarinic acid content in transformed roots (213.42 µg/g dry wt) was significantly higher than non-transformed roots (52.28 µg/ g dry wt).
Key words: Agastache foeniculum, Agrobacteriumrhizogenes, culture medium, hairy roots, immersion, injection, rosmarinic acid
IZVLEČEK VPLIV RAZLIČNIH DEJAVNIKOV NA INDUKCIJO LASASTIH KORENIN PRI JANEŽNEM OŽEPU (Agastache foeniculum (Pursh) Kuntze)Bakterija Agrobacterium rhizogenes je znana kot naravno orodje genskega inženiringa pri mnogih rastlinskih vrstah. V tej raziskavi so prvič preučevali indukcijo lasastih korenin pri janežnem ožepu (Agastache foeniculum (Pursh.) Kuntze) z uporabo bakterije A. rhizogenes, vsebnost rožmarinske kisline in vpliv različnih gojišč ter inokulacijskih metod na rast lasastih korenin. Kulturo lasastih korenin janežnega ožepa so vzgojili iz izsečkov enomesečnih listov inokuliranih z bakterijo A. rhizogenes sev A4 z uporabo dveh inokulacijskih metod -potapljanje in vbrizgavanje, v razmerah na svetlem in v temi. Pri metodi potapljanja izsečkov v bakterijsko suspenzijo so preučevali tudi vpliv časa (3, 5 in 7 min) na indukcijo korenin. V drugem delu raziskave so preučevali vpliv različnih osnovnih gojišč (MS, 1/2 MS in B5) na kulturo lasastih korenin. Vsebnost rožmarinske kisline v transformiranih lasastih koreninah in netransformiranih koreninah so analizirali s HPLC metodo. Značilna razlika v indukciji lasastih korenin v odvisnosti od metod inokulacije ni bila ugotovljena. Opazovanja so pokazala, da je bil odstotek indukcije lasastih korenin večji, če so izs...
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