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. 2019 Mar;5(3):282-289.
doi: 10.1038/s41477-019-0359-2. Epub 2019 Feb 18.

High-efficiency Generation of Fertile Transplastomic Arabidopsis Plants

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Free PMC article

High-efficiency Generation of Fertile Transplastomic Arabidopsis Plants

Stephanie Ruf et al. Nat Plants. .
Free PMC article

Abstract

The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.

Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Biolistic nuclear and plastid transformation of Arabidopsis thaliana. (a) Workflow of transformation experiments. Microcalli are induced from root tissue (6 days after germination) harvested from seedlings raised on synthetic medium. Biolistic bombardment is conducted after 5 days of incubation on callus-inducing medium and followed by selection for kanamycin resistance (nuclear transformation) or spectinomycin resistance (chloroplast transformation). Resistant shoots are rooted on agar slants and plants are grown to maturity in sterile containers. A timeline indicating the approximate duration of the individual steps in the protocol is given at the right. See text and Supplementary Figs. 1-4 and 6-8 for details. (b) Fertility of regenerated Arabidopsis plants. The plants are fertile and produce large amounts of seeds. Two ripe siliques in which the seeds can be seen are indicated by white arrowheads. These experiments were repeated independently for 22 transplastomic lines with similar results.
Fig. 2
Fig. 2
Construction of plastid transformation vectors and selection of transplastomic Arabidopsis plants. (a) Physical map of the targeting region in the plastid genome (ptDNA) of Arabidopsis and plastid transformation vectors. Genes above the line are transcribed from left to right, genes below the line are transcribed from the opposite strand of the ptDNA. The transgenes are inserted into the intergenic spacer between the trnfM and trnG genes within a cloned ptDNA fragment (Transformation vector). The location of two BglII restriction sites that were used for Southern blot analysis and the binding site of the hybridization probe is also indicated. The sizes of the transgene cassettes in the three vectors are given in kb. ZmPclpP: promoter of the plastid clpP gene from Zea mays (with the clpP 5’ UTR and the G10-derived Shine-Dalgarno sequence form phage T7; ref. 39); aadA: aadA gene from E. coli; syn-aadA: synthetic codon-optimized aadA gene; NtTrps16: 3’ UTR from the tobacco plastid rps16 gene; EcTrrnB: rRNA operon terminator from E. coli; CrPpsaA: promoter of the plastid psaA gene from Chlamydomonas reinhardtii; CrTatpB: 3’ UTR of the plastid atpB gene from C. reinhardtii. (b) Selection of a transplastomic line (white arrowhead) following bombardment of wild-type tissue with vector pCH8. These transformation experiments were repeated independently 507 times (cf. Table 1), and resulted in similar background growth of the bombarded calli. (c) Selection of a transplastomic line (white arrowhead) after bombardment of acc2 knock-out tissue (At-Δacc2) with vector pCH8. Note the much more efficient suppression of background callus growth from the acc2 knock-out tissue. For additional images, see Supplementary Figs. 4 and 6. The transformation experiments with the At-Δacc2 recipient line and vector pCH8 were repeated independently 98 times with similar results. (d) Southern blot analysis of transplastomic Arabidopsis lines. Total DNA extracted from regenerated plants growing under aseptic conditions was digested with BglII, separated by agarose gel electrophoresis and hybridized to a radiolabelled probe (cf. panel a). The sizes of hybridizing fragments are indicated in kb at the right. These experiments were repeated independently three times with similar results.
Fig. 3
Fig. 3
Homoplasmy of transplastomic lines obtained with vectors pCH8, pJF1153 and pJF1151, and demonstration of maternal transgene inheritance. The left plates show the comparison between the progenies of the selfed transplastomic line (Tp) and the selfed At-Δacc2 recipient line used for transformation. The right plates show the progenies of reciprocal crosses between the wild type (Wt) and the transplastomic line. Seeds were germinated in the presence of 100 mg/L spectinomycin. These assays were repeated independently two times with similar results.
Fig. 4
Fig. 4
Expression of the YFP reporter in transplastomic At-Δa-JF1151 plants. (a) Chlorophyll and YFP fluorescence of wild-type (Wt) and transplastomic (Tp) seedlings grown in the absence (Spec0) or presence (Spec100) of spectinomycin. The plates were scanned with a Typhoon imager that produces a red image of the chlorophyll fluorescence and a green image for the (yellow) YFP fluorescence. The images were taken 20 days after sowing. (b) Confirmation of chloroplast YFP expression in leaf mesophyll cells by confocal laser-scanning microscopy. BF: bright-field image; Chl: chlorophyll fluorescence; YFP: YFP fluorescence (colored in green, to match the color of the Typhoon image); Chl+YFP: overlay of the chlorophyll and YFP fluorescences. These experiments were repeated independently three times with similar results.
Fig. 5
Fig. 5
Immunoblot analysis of YFP accumulation in transplastomic Arabidopsis plants. Two independently generated transplastomic At-Δa-JF1151 lines were analyzed. The wild type (At-Wt) and a transplastomic At-Δa-CH8 line were included as negative controls. Samples of total soluble protein extracted from leaves (with the amounts given in µg) were separated by polyacrylamide gel electrophoresis and blotted. A dilution series of YFP purified from Escherichia coli (with the amounts given in ng) was loaded for semiquantitative assessment of protein accumulation in transplastomic plants. Note that the YFP recombinantly expressed in bacteria migrates slightly slower than the YFP in the transplastomic samples. This is due to the presence of a purification tag (His-tag) in the YFP isolated from E. coli. See Methods for further details. These experiments were repeated independently two times with similar results.

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References

    1. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL, Robertson D, Klein TM, Shark KB, Sanford JC. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science. 1988;240:1534–1538. - PubMed
    1. Svab Z, Hajdukiewicz P, Maliga P. Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA. 1990;87:8526–8530. - PMC - PubMed
    1. Maliga P. Plastid transformation in higher plants. Annu Rev Plant Biol. 2004;55:289–313. - PubMed
    1. Maliga P, Bock R. Plastid biotechnology: food, fuel, and medicine for the 21st century. Plant Physiol. 2011;155:1501–1510. - PMC - PubMed
    1. Bock R. Engineering plastid genomes: Methods, tools, and applications in basic research and biotechnology. Annu Rev Plant Biol. 2015;66:211–241. - PubMed

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