The Agrobacterium strain GV3101 containing TRV-VIGS vectors was grown at 28 °C in Luria–Bertani medium supplemented with 10mM MES, 20mM acetosyringone, 50mg l–1 gentamicin, and 50mg l–1 kanamycin for ~24h. Agrobacterium cells were harvested and suspended in the infiltration buffer (10mM MgCl2, 200mM acetosyringone, and 10mM MES, pH 5.6). A mixture of Agrobacterium cultures containing pTRV1 and pTRV2 and its derivatives, in a ratio of 1:1 (v/v), were placed in the dark at room temperature for 4h before inoculation. For vacuum infiltration, Silwet L-77 was added to a final concentration of 0.01% (v/v).
Nicotiana benthamiana infiltration was performed as described byLiu et al. (2002a ). Agrobacterium cultures containing pTRV1 and pTRV2 or its derivatives (1:1, OD600=1.0) were injected into the lower leaf of four-leaf stage plants by using a 1ml needleless syringe. For axil injection, 20 μl of bacterial suspension (1:1, OD600=4.0) were injected per plant into the leaf axil using a 1ml syringe with a needle at the four-leaf stage period.
Since rose leaves and axils are hard to inject, the vacuum infiltration method was used for rose cuttings, and seedlings were infiltrated as described previously (Ma et al., 2008 (link); Yan et al., 2012 (link)). Four-week-old rose cuttings were removed from the soil, the roots were rinsed with distilled water, and whole plants were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD600=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. This was repeated once. Treated plants were briefly washed with distilled water and then planted in pots. For seedlings, the seeds generated were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD600=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. The infiltrated seeds were briefly washed with distilled water and placed on wet filter paper for several days, and then the seedlings were planted in pots.
Strawberry was vacuum infiltrated in the same way as rose cuttings and seedlings. After the release of the vacuum, the plants were briefly washed with distilled and planted in pots.
Arabidopsis and chrysanthemum infiltration was performed as described by Burch-Smith et al. (2006) (link). A mixture of Agrobacterium culture containing pTRV1 and pTRV2-GFP (OD600=1.5) was infiltrated into the two leaves of 2-week-old Arabidopsis plants and the lower leaves of six- to eight-leaf-stage chrysanthemum plants using a needleless syringe. The infected chrysanthemum plants were transferred into pots.
Nicotiana benthamiana infiltration was performed as described by
Since rose leaves and axils are hard to inject, the vacuum infiltration method was used for rose cuttings, and seedlings were infiltrated as described previously (Ma et al., 2008 (link); Yan et al., 2012 (link)). Four-week-old rose cuttings were removed from the soil, the roots were rinsed with distilled water, and whole plants were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD600=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. This was repeated once. Treated plants were briefly washed with distilled water and then planted in pots. For seedlings, the seeds generated were submerged in infiltration mixture containing pTRV1 and pTRV2 or its derivatives (OD600=1.0) and subjected to a vacuum at –25 kPa twice, each for 60 s. The infiltrated seeds were briefly washed with distilled water and placed on wet filter paper for several days, and then the seedlings were planted in pots.
Strawberry was vacuum infiltrated in the same way as rose cuttings and seedlings. After the release of the vacuum, the plants were briefly washed with distilled and planted in pots.
Arabidopsis and chrysanthemum infiltration was performed as described by Burch-Smith et al. (2006) (link). A mixture of Agrobacterium culture containing pTRV1 and pTRV2-GFP (OD600=1.5) was infiltrated into the two leaves of 2-week-old Arabidopsis plants and the lower leaves of six- to eight-leaf-stage chrysanthemum plants using a needleless syringe. The infected chrysanthemum plants were transferred into pots.
Free full text: Click here
