Fig 1.
Selection of optimal explant types for transformation.
Calli generated from: A: petiole segments; B: stem segments; C: leaf discs.
Fig 2.
The effect of kanamycin on callus formation from petiole segments.
A: Calli generated from petiole segments on medium without kanamycin for 30 days; B-E: Petiole segments on medium containing 5, 10, 15 and 20 mg/L kanamycin, respectively; F: The calculated differentiation proportion of petiole segments under different kanamycin concentrations. Data are the mean values ± SE of three biological replicates. * and ** indicate significant differences between control and treatments at P<0.05 or P<0.01 (Student’s t-test), respectively.
Fig 3.
Optimizing parameters for Agrobacterium-mediated petiole segment transformation in V. amurensis.
A: The transformation efficiency of petiole segments on different Agrobacterium strains (EHA105, GV3101 and LBA4404). B: The effect of bacterial concentration (OD600 = 0.5, 1.0, 1.5 and 2.0) during transformation. C: The effect of infection time (4, 8, 12 and 16 min) on transformation. D: The effect of co-cultivation times (1, 2, 3 and 4 days) on transformation. All of the data are the mean values ± SE of three biological replicates. Lower and upper case letters indicate significant differences between treatments at P<0.05 or P<0.01 (Student’s t-test), respectively.
Fig 4.
eGFP fluorescence observation and molecular analysis of transformants.
A: Transformed calli generated from the end of the petiole segments of V. amurensis after 30 days cultivation. B, C: Bright-field and eGFP fluorescence results of callus emphasized by a red mark in A. D: Transformed calli generated from the end of the petiole segments of V. amurensis after 60 days cultivation. E: Different transgenic callus lines were cultivated in new medium. F, G: Bright-field and eGFP fluorescence results of digested cells from one transgenic callus line. H: Amplification of NPTII gene fragments from the genomic DNAs of non-transformed calli (NT), pSAK277-eGFP plasmid (P) and 5 transgenic lines (T1-T5). I: Amplification of eGFP gene fragments from the cDNAs of non-transformed calli (NT) and 5 transgenic lines (T1-T5). The plasmid of pSAK277-eGFP (P) was used as positive control for PCR amplification. Fluorescence microscope (ECLIPSE 80i, Nikon). GFPuv was excited at 465–495 nm and emitted through a 515–555 nm bandpass filter.