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MB Sample ID: SA194045

Local Sample ID:	73072_1
Subject ID:	SU002144
Subject Type:	Plant
Subject Species:	Lithospermum officinale
Taxonomy ID:	475907

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Treatment:

Treatment ID:	TR002156
Treatment Summary:	A set of 50 bacteria previously isolated from another A/Sd-producing plant, Alkanna tinctoria L. (Tausch), was selected based on diversity and on phenotypic traits tested in vitro (Rat et al., 2021). The 50 strains were first grown in 35 ml Reasoner’s 2A broth (R2B, Acumedia) at 28°C, 100 rpm for 72 h. Before preparing the inocula, 5 ml of each bacterial culture was sampled and used to estimate bacterial concentrations via counting of colony-forming units (CFU), while the remaining culture was used as inoculum. The inoculum was centrifuged at 4°C, 14000 rpm for 10 min, the supernatant was discarded, and the pellet was preserved in 2 ml of R2B medium supplemented with 10% glycerol. Bacterial pellets were then stored at -20°C until inoculation. To prepare the inoculum, a pellet was suspended in 28 ml of sterile phosphate-buffered saline (PBS) at pH 7.4. The resuspended inoculum was then adjusted to a concentration of 104-106 CFU/ml, and ten µl were used to inoculate each plant. The bacterial suspension was first injected in the MSRmod medium with a micropipette. Then, a shoot tip of L. officinale of 3.5 cm length was selected and the top two-three leaves were removed by cutting. The plant was finally transferred in sterile conditions and inserted at the positions where the bacteria had been injected in the medium. Plants treated with only PBS were used as non-inoculated controls. To avoid light in the root compartment, which can inhibit the production of shikonin (Yazaki et al., 1999), the surface of the medium was covered with sterilized (in an oven at 145°C during 10 h) quartz sand and the lower half of the jar was wrapped with aluminium foil (Figure 1). The jars were then incubated in a growth chamber at 20ºC, 16:8 h light:dark, with a light intensity of 50 µmol m-2s-1. Plants were harvested for analysis after seven weeks of incubation

Treatment ID:

TR002156

Treatment Summary:

A set of 50 bacteria previously isolated from another A/Sd-producing plant, Alkanna tinctoria L. (Tausch), was selected based on diversity and on phenotypic traits tested in vitro (Rat et al., 2021). The 50 strains were first grown in 35 ml Reasoner’s 2A broth (R2B, Acumedia) at 28°C, 100 rpm for 72 h. Before preparing the inocula, 5 ml of each bacterial culture was sampled and used to estimate bacterial concentrations via counting of colony-forming units (CFU), while the remaining culture was used as inoculum. The inoculum was centrifuged at 4°C, 14000 rpm for 10 min, the supernatant was discarded, and the pellet was preserved in 2 ml of R2B medium supplemented with 10% glycerol. Bacterial pellets were then stored at -20°C until inoculation. To prepare the inoculum, a pellet was suspended in 28 ml of sterile phosphate-buffered saline (PBS) at pH 7.4. The resuspended inoculum was then adjusted to a concentration of 104-106 CFU/ml, and ten µl were used to inoculate each plant. The bacterial suspension was first injected in the MSRmod medium with a micropipette. Then, a shoot tip of L. officinale of 3.5 cm length was selected and the top two-three leaves were removed by cutting. The plant was finally transferred in sterile conditions and inserted at the positions where the bacteria had been injected in the medium. Plants treated with only PBS were used as non-inoculated controls. To avoid light in the root compartment, which can inhibit the production of shikonin (Yazaki et al., 1999), the surface of the medium was covered with sterilized (in an oven at 145°C during 10 h) quartz sand and the lower half of the jar was wrapped with aluminium foil (Figure 1). The jars were then incubated in a growth chamber at 20ºC, 16:8 h light:dark, with a light intensity of 50 µmol m-2s-1. Plants were harvested for analysis after seven weeks of incubation