Optimization of Hairy Root Induction,Biomass Production of Chicory (Cichorium intybus L.)

Transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes strains are used as a tissue culture tool for secondary metabolite production since hairy roots are genetically and biologically stable and they are able to produce metabolite within a short time. Chicory (Cichorium intybus L.) is a medicinal plant from Asteraceae. This plant contains many important metabolites include chicoric asid, inulin, scoline, coumarin and flavonoids.

The seeds were surface-sterilized by rinsing with 2% benomil for 30 min and later sterilized with 5% sodium hypochlorite for 20 min and subsequently with 70% ethanol for 90 s. the wounded leaf of 20 and 28-day-old seedlings inoculated with Agrobacterium rhizogenes 11325. Bacterial colonies were cultured on liquid LB medium for 1 day. The explants were submerged in the solution for 15 min and were shaken gently. After that, the explants were drained on sterile filter paper and then transferred to MS solid medium. The explants were incubated at (25 ± 2) ◦C under dark condition for three different co-cultivation period (24, 48 and 72 hours). In the next step, the explants were transferred to MS solid medium supplemented with 500 mg/L cefotaxime. The explants were incubated at (25 ± 2) ◦C under a 16-h photoperiod condition for four weeks. At the end of incubation time, the percentage of hairy root induction, root number and root length were investigated. Genomic DNA of Cichorium intybus L. hairy root was extracted following the method of CTAB and subjected to PCR analysis. Non-transformed root DNA was used as a negative control and pRi11325 plasmid DNA was used as positive control. The pair of primers specific to the rolB fragment sequences was: 5-ATGGATCCCAAATTGCTATTCCCCACGA -3 and 5-TAGGCTTCTTTCATTCGGTTTACTGCAGC-3. The amplification protocol for the rolB was a 5 min melting at 94 ◦C followed by 35 cycles of a 5 min melting at 94 ◦C, a 45 s annealing at 55 ◦C and a 1 min elongation at 72 ◦C, and final elongation at 72 ◦C for 7 min. PCR products were electrophoretically separated on 0.8 % agarose gels (w/v) and stained with gel red. To determine the best media composition for growth of hairy roots, approximately 100 mg FW roots were cultured in basal liquid MS, solid MS and liquid 1/2MS medium supplemented with 500 mg/L cefotaxime on a rotary shaker (90 rpm) at 25 ◦C in dark for 5 weeks, and increase in fresh weight and dry weight was recorded at 5 week later. Three replicates were made for each experimental set. Non-transformed roots were cultured on same mediums.

Each of the two different explants – leaf and petiole showed different levels of hairy root induction in different co-culture time. Hairy root induction was observed 7 days after co-cultivation. There were low adventitious roots formed from control explants. 72 hours co-cultivation period was found to be more efficient in inducing hairy root phenotype in both explant types. The infection of explants by A. rhizogenes is a process that implies a succession of events, including the transfer and integration of T-DNA, into the host plant genome. Therefore, the time of contact between A. rhizogenes and the explants determines the transformation efficiency. A maximum of transformation frequency (53.33%) was observed in leaf explants in 72 hours co-cultivation followed by 33.33% in petiole explants in 72 hours co-cultivation. In leaf explants maximum root number (8.5 roots per explant) and maximum length of root (9.16 cm) induced from 20-day-old seedling in 72 hours co-culture. In petiole explants maximum root number (3.66 roots per explant) and maximum root length (11.46 cm) induced from 28-day-old seedling in 72 hours co-culture. The “hairy root” phenotype characterized by high branching and fast growth was previously reported in “reactive species” like Datura innoxia but not for “difficult species”. The high phenolic content of woody plant species could be responsible for the reduced hairy root phenotype as reported for gingko or pine. Three different culture media (solid MS, liquid MS and 1/2 MS media) were tested to determine the best suitable media for the maximum Biomass of hairy roots line.

Genetically transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes are suitable source for production of bioactive molecules due to their genetic stability and generally show fast growth in culture media free of growth hormones. This study describes the protocol for hairy root induction which could further be useful for the production of secondary metabolites and biomass.