Results: 4

1.
Fig. 3.

Fig. 3. From: Determining the transgene containment level provided by chloroplast transformation.

Phenotypes of seedlings selected for paternal transmission of transgenic chloroplasts as evidenced by the presence of green tissue in F1 seedlings. (A) Seedling with small green islands in one cotyledon (line F1-spec81). (B) Seedling with green sectors in a cotyledon and a green meristem (line F1-spec7). (C and D) Same seedling after 3 (C) and 5 (D) weeks of continued growth in the presence of spectinomycin. (Scale bars: 0.5 cm.)

Stephanie Ruf, et al. Proc Natl Acad Sci U S A. 2007 April 24;104(17):6998-7002.
2.
Fig. 2.

Fig. 2. From: Determining the transgene containment level provided by chloroplast transformation.

Selection for paternal transmission of transgenic plastids. (A) Identification of a seedling displaying green cotyledon sectors on spectinomycin-containing medium. (B) Close-up of the seedling (line F1-spec1) in comparison to a green wild-type seedling (left) grown in the absence of spectinomycin. The frame marks the window observed under the UV stereomicroscope in C and D. (Scale bar: 0.5 cm.) (C) Detection of GFP fluorescence by using long pass filter GFP2. Whereas the wild-type seedling shows the red autofluorescence of the chlorophyll, the F1-spec1 seedling shows both red chlorophyll and green GFP fluorescence resulting in yellow overall fluorescence. (D) Detection of GFP fluorescence by using bandpass filter GFP3 (which excludes chlorophyll autofluorescence from detection). Only the green sectors in the F1-spec1 seedling show GFP fluorescence indicating that they contain paternally inherited transgenic chloroplasts.

Stephanie Ruf, et al. Proc Natl Acad Sci U S A. 2007 April 24;104(17):6998-7002.
3.
Fig. 4.

Fig. 4. From: Determining the transgene containment level provided by chloroplast transformation.

Test for presence of transgenic plastids and their maternal inheritance after purification to homoplasmy. (A) RFLP analysis to confirm the presence of paternally inherited transgenic plastids in the selected lines. Two lines with paternal plastids (F1-spec1 and F1-spec7) are shown in comparison to a spontaneous spectinomycin-resistant mutant (F1-spec9), the paternal parent (Nt-pDK53), and a maternal parent (Nt-nms1) containing wild-type plastids. For the two lines with paternally inherited plastids, tissue samples from the first and third regeneration rounds were analyzed (indicated by the final number in the line designation). Weak bands at wild-type size correspond to promiscuous plastid DNA in the nuclear genome that is frequently seen in such RFLP analyses (e.g., see refs. 23 and 24). (B) Seed assays confirming homoplasmy of plants regenerated from green seedling tissue. The lower Petri dish shows the progeny from line F1-spec1 germinated on spectinomycin-containing medium. Antibiotic resistance of all seedlings confirms homoplasmy for the paternally inherited transgenic chloroplasts. The upper Petri dish displays the wild-type control, which is sensitive to the antibiotic.

Stephanie Ruf, et al. Proc Natl Acad Sci U S A. 2007 April 24;104(17):6998-7002.
4.
Fig. 1.

Fig. 1. From: Determining the transgene containment level provided by chloroplast transformation.

Generation of parental lines for the screen for occasional paternal transmission of transgenic chloroplasts. (A) Physical map of the transgenic plastid genome in the paternal tobacco line Nt-pDK53. Genes above the line are transcribed from left to right, and genes below the line are transcribed in the opposite direction. Two transgenes are integrated into the intergenic spacer upstream of the tRNA-Val gene (trnV): the selectable marker gene aadA conferring resistance to spectinomycin and streptomycin (12), and the gfp gene serving as a reporter gene. Location of the EcoRV and XhoI restriction sites used for RFLP analysis is indicated. Prrn, chimeric rRNA operon promoter; TpsbA, 3′ untranslated region from the psbA gene; Trps16, 3′ untranslated region from the rps16 gene. (B) GFP accumulation in transgenic chloroplasts. (Left) GFP. (Center) Chlorophyll. (Right) Overlay. Green GFP fluorescence and red autofluorescence of chlorophyll were detected by confocal laser scanning microscopy. The green GFP fluorescence is only visible in chloroplasts of the transplastomic paternal line Nt-pDK53, and not in the male sterile maternal line Nt-nms1. (Scale bars: 8 μm.)

Stephanie Ruf, et al. Proc Natl Acad Sci U S A. 2007 April 24;104(17):6998-7002.

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