Two cultivars of industrial chicory were assessed is this study. VL-70 seeds and sterile in vitro plants of K1793 were obtained from ILVO Flanders Institute for Agriculture, Fisheries and Food Research, Melle, Belgium. Seeds of VL-70 lines were surface sterilized with 70% ethanol (2 min), after which the seeds were transferred to 5% sodium hypochlorite solution (15 min). After this, the seeds were rinsed three times with UHP H2O and then sown on solid Murashige and Skoog medium [20 (link)]. Hairy roots of C. intybus VL-70 line were initiated by Agrobacterium rhizogenes infection using altogether three bacterial strains: LBA9402, A4, and 15834. The agropine-type strains were A4 (kindly provided by Dr. David A. Tepfer, Versailles, France), LBA9402, and 15834 (kindly provided by Prof. Ulf Nyman, Copenhagen, Denmark). The strains LBA9402 and 15834 were cultivated in YMB (yeast mannitol broth) culture medium added with 100 ppm rifampicin. The strain A4 was cultivated in APM culture medium [21 (link)] added with 0.1 ppm biotin. Bacteria were cultivated on solid medium at +28 °C, 48 h prior to infection. Infections were performed by sterile syringe needle dipped in the bacterial mass and infecting a sterile explant.
For establishment of K1793 hairy roots, sterile in vitro plant leaves were infected with A. rhizogenes LBA9402 as described above. Explants were then placed on modified Gamborg B5-media [22 (link),23 (link)] and were incubated in the dark for a 48-h co-cultivation period.
After the co-cultivation, all the explants were transferred onto bacteriological media plates with cefotaxime sodium 500mg/L to kill the excess bacteria. After 10–14 days, hairy roots started to appear on the wound site, and they were excised from the explant and cultivated on solid modified B5 medium. The presence of rolB and the absence of virD genes were confirmed by PCR as earlier described [12 (link)]. For biomass determination, 100 mg FW of hairy roots from both cultivars were weighed in 100 mL Erlenmeyer flasks and 20 mL of modified B5 medium was added. Roots were cultivated in dark as described in [23 (link)]. Hairy roots were harvested by filtering after 7, 14, 21, or 28 days of cultivation and freeze-dried before analyses. Three in vitro plants of lines VL-70 and K1793 were transferred to soil and cultivated for a period of 3 months to form a taproot. Taproots were collected and freeze-dried before analysis.
For establishment of K1793 hairy roots, sterile in vitro plant leaves were infected with A. rhizogenes LBA9402 as described above. Explants were then placed on modified Gamborg B5-media [22 (link),23 (link)] and were incubated in the dark for a 48-h co-cultivation period.
After the co-cultivation, all the explants were transferred onto bacteriological media plates with cefotaxime sodium 500mg/L to kill the excess bacteria. After 10–14 days, hairy roots started to appear on the wound site, and they were excised from the explant and cultivated on solid modified B5 medium. The presence of rolB and the absence of virD genes were confirmed by PCR as earlier described [12 (link)]. For biomass determination, 100 mg FW of hairy roots from both cultivars were weighed in 100 mL Erlenmeyer flasks and 20 mL of modified B5 medium was added. Roots were cultivated in dark as described in [23 (link)]. Hairy roots were harvested by filtering after 7, 14, 21, or 28 days of cultivation and freeze-dried before analyses. Three in vitro plants of lines VL-70 and K1793 were transferred to soil and cultivated for a period of 3 months to form a taproot. Taproots were collected and freeze-dried before analysis.
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