Fig 1.
Paromomycin is a gentler selection agent than kanamycin for seedlings that weakly express the nptII transgene.
Seedlings of three nptII-positive mutant lines and corresponding nptII-negative wild type (Columbia ecotype) were grown for seven days on ½ x MS medium with 1% sucrose containing the indicated concentrations of (A) kanamycin and (B) paromomycin. The graphic below each image summarizes the consensus phenotype under each condition with a rubric that focuses on seedling pigmentation. The percentages represent the proportion of seedlings within the sample population exhibiting the pigmentation phenotype indicated in the box. Each sample has about 25 seedlings, and the experiment was replicated at least three times.
Fig 2.
Paromomycin acts as a less harsh selection agent than kanamycin for weakly resistant seedlings.
Representative close-up images of 7-day-old Arabidopsis seedlings grown on ½ strength MS medium with 1% sucrose. The chosen mutants are all from the SALK T-DNA collection and are compared to their wild-type (WT) Columbia. Optimal concentrations of kanamycin and paromomycin were added. All images are at the same magnification.
Fig 3.
Paromomycin resistant seedlings maintain the quantum yield of photosystem II photosynthesis.
The quantum yield of photosystem II is defined as the ratio of variable fluorescence over maximal fluorescence (Qymax = Fv/Fm) when dark-adapted seedlings are exposed to a saturating pulse of light using the Fluorocam. Panels (A), (B), and (C) display the same four genotypes under the indicated antibiotic conditions. Each sample has about 25 seedlings. Error bars indicate standard deviations. An ordinary one-way ANOVA with a Tukey’s multiple comparison test was done to compare the genotypes. Data series that share the same letter are not significantly different.
Fig 4.
Root length is useful in selecting for strongly resistant seedlings only.
Root lengths were measured daily for seedlings grown on vertical plates containing ½ strength MS media with 1% sucrose as the control (blue). Antibiotic treatments consisted of 30μM paromomycin (red) and 86μM kanamycin (black). (A) Wild type. (B) pab4. (C) gcn2-2. (D) gcn1-5. Error bars indicate standard error of the mean.
Fig 5.
Survival of seedlings on soil depends on the prior antibiotic treatment.
Seedlings of four genotypes were germinated on the indicated concentrations of antibiotics and transplanted to soil on day 7. No antibiotic was applied after transplantation. Survival was scored as the ability of the plant to complete all stages of its life cycle and produce seeds. (A) Wild type. (B) pab4. (C) gcn2-2. (D) gcn1-5. The difference between paromomycin and kanamycin survival rates was evaluated using a Fisher’s exact test. Data represent one experiment out of three biological replicates. Not significant (ns).
Fig 6.
Vegetative rosettes grow faster in plants with weak paromomycin resistance than in false positives.
Seedlings of four genotypes were germinated on the indicated concentrations of antibiotics and transplanted to soil on day 7. Rosette diameters were measured from n = 16 plants using a standard ruler starting on day 6 after transplantation until they were fully grown. Error bars indicate standard deviations. (A) Wild type. (B) pab4. (C) gcn2-2. (D) gcn1-5. Note that the growth rate of paromomycin-treated wild type (A) is strongly reduced compared to pab4 (B) and gcn2-2 (C) and gcn1-5 (D).
Fig 7.
Plants with weak paromomycin resistance flower earlier than non-transgenic false-positives.
Seedlings of four genotypes were germinated on the indicated concentrations of antibiotics and transplanted to soil on day 7. Plants were scored as flowering once the inflorescence began to emerge from the center of the rosette. Data are cumulative traces from n = 16 plants per line. (A) Wild type. (B) pab4. (C) gcn2−2. (D) gcn1−5. Note that the flowering of paromomycin-treated wild type (A) was delayed compared to pab4 (B), gcn2−2 (C), and gcn1−5 (D). Pretreatment of the moderately resistant gcn2−2 with kanamycin also strongly delayed flowering; kanamycin-treated pab4 did not reach the flowering stage in this experiment.