Genetic Transformation of Plant Mitochondria with mTP-DNA Nano-Complexes

Summary

Here, we present a protocol for gene delivery into plant cell mitochondria. This new method utilizes cell penetrating peptides with mitochondria targeting properties and a mitochondria-optimized aadA:gfp reporter cassette. It is suitable for transient and stable, organelle-specific gene transfer and expression in somatic and germ cells.

Abstract

We report a peptide based in vivo plant cell mitochondrial genetic engineering method. Peptide vectors were created by selecting mitochondrial protein sorting signal sequences that contain similar physical and chemical properties as a cell-penetrating peptide (CPP). In the presence of exogenous double-stranded DNA (dsDNA) the peptide vectors called mitochondrial targeting peptides (mTPs) form peptide-nucleic acid nanoparticles. The nanoparticles when incubated with plant cells first cross the cellular membrane and then cross the outer and inner mitochondrial membranes to deliver exogenous DNA into the mitochondria of plant cells. The exogenous DNA integrates into the mitochondrial genome and the transfected plant cells can be cultured into plantlets. A linear double stranded gene construct was delivered into protoplasts and microspores of AC Ultima spring triticale (X. Triticosecale Wittmack) plants using the mTP peptide vector system to study in vivo mitochondrial transfection of plant cells and transient mitochondrial gene expression.

Introduction

Mitochondria are organelles that supply energy for metabolic activities in eukaryotic cells. In plants, the mitochondria are involved not only with energy conversion during aerobic respiration and carbon metabolism in the Krebs cycle, but also to chloroplast function and stress responses¹. The ability to manipulate the mitochondrial genome will facilitate the study of plant primary and secondary metabolism. In addition, plant mitochondrial genetic engineering has many important applications in agricultural crop breeding and hybrid maintenance due to the mitochondria being linked to cytoplasmic male sterility².

In vivo mitochondrial genetic research in eukaryotic cells has been restricted due to the lack of ability to genetically manipulate the mitochondria in whole cells. Biolistic transformation of Chlamydomonas reinhardtii and two yeast species, Saccharomyces cerevisiae and Candida glabrata, has been successfully applied to mitochondrial transformation^3,4,5. A mitochondrial targeted adenoassociated virus has also recently proved to be effective as a mitochondrial DNA delivery method in a mouse model system⁶. In a recent report, a fusion peptide combining a mitochondrial-targeting peptide and cell-penetrating peptide was shown to deliver foreign DNA into the mitochondria of Arabidopsis thaliana leaf cells through infiltration⁷.

Isolated mitochondria served as the earliest in vitro model for mitochondrial genetic transformation research. An example of this is a study that examined mitochondrial transcription and RNA maturation processes using electroporated wheat isolated mitochondria⁸. Isolated mitochondria have also been transformed utilizing their natural competence to import double stranded linear DNA. The exogenous DNA was internalized via direct DNA uptake. The mechanism of active DNA import was independent of DNA sequence and involved the voltage-dependant anion channel (VDAC) translocase of the outer mitochondrial membrane (Tom20 and Tom40), specific for import of proteins and tRNA^9,10. The imported DNA was integrated into the mitochondrial genome through homologous recombination^11,12,13. If the exogenous DNA contains a gene controlled by a mitochondrial promoter, the DNA can be transcribed and processed into mature RNA molecules to form functional proteins in the mitochondria^11,13,14.

An alternative approach to genetic engineering of plant mitochondria that can be applied to intact plant cells is based on mitochondrial targeting peptides (mTPs). The experimental criteria applied to identify mTPs emulate the existence of a protein sorting signal with similar physicochemical properties as a cell penetrating peptide (CPP) called Transactivator of Transcription (Tat). Cell penetrating peptides are defined as short cationic peptides which are capable of transducing cargo varying in size, chemical properties, or function across cell membranes in a receptor independent manner¹⁵. Tat’s cell penetrating properties are derived from the peptides’ positive charge and the guanidinium head group of the arginine residues which facilitate electrostatic membrane interaction followed by translocation of the peptide and its cargo through the plasma membrane¹⁶. Tat and its attached cargo accumulate in the nucleus of transfected cells due to the presence of a protein sorting signal called a nuclear localization signal (NLS) within its peptide sequence¹⁷.

Mitochondrial proteins transcribed from nuclear DNA are translated in the cytosol and targeted to the mitochondria within the cell by short peptide sequences on the N-terminus, C-terminus and/or contained within the protein sequence¹⁸. These short peptide sequences are referred to as pre-sequences and mitochondrial or matrix targeting peptide (mTPs) sequences for mitochondrial proteins^19,20. Mitochondrial translocases of the outer/inner membrane (TOM/TIM) bind to protein mTPs and facilitate the transport of the nascent cytosolic proteins across the double membrane of mitochondria²¹. In addition, mTPs were found to be able to transport other cargoes into cell mitochondria, including cancer drugs^22,23 and RNAs²⁴.

Organelle (chloroplast and mitochondria) genome transformation is associated with a challenge of high number of organelles per cell and high number of genomes per organelle. In chloroplast transformation, generation of transplastomic plants with genetically uniform, homogeneous chloroplast genomes (homoplastomic plants) can be achieved by means of discriminatory selection that favors transgenic copies of ptDNA over the unmodified genomes, and maintains them during replication and sorting²⁵. Application of a selection agent during post-transfection tissue culture enables enrichment of chloroplasts with genetically engineered genomes. Therefore, efficient selection and regeneration procedures are needed for successful generation of transformed plants. Similar rules are expected to apply also for generation of homoplasmic plants with stably transformed mitochondrial genomes. Cells that survive selection will contain only a subset of transfected mitochondria out of the entire population of mitochondria in the cell. Therefore, a selectable marker is required to select for mitochondria expressing the selectable marker gene while eliminating mitochondria that have not taken up the DNA construct.

The most common selectable marker used for organelle transformation is the bacterial aadA gene, which encodes aminoglycoside 3` adenyl transferase and confers high levels of resistance to spectinomycin and streptomycin²⁶. However, the selection was for the contrast between green tissue and chlorotic tissue rather than for survival and growth²⁷. The aadA gene was used initially as a marker for transformation of nuclear genomes²⁷. Soon after, aadA was successfully employed as a marker for transformation of plastids²⁸. In plastid transformation, the dominant aadA selectable marker gene replaced the hitherto used recessive marker which also conferred resistance to spectinomycin but originated from the plastid 16S rRNA gene²⁸.

In addition to selectable markers, reporter genes are useful tools to monitor transformation events by imaging expression of the delivered genes. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria was reported to function as a sensitive and nondestructive reporter of in vivo gene expression^29,30. The wild-type GFP has been used for expression studies in plants in various transformation systems³¹. Mutants of GFP with increased stability and enhanced fluorescence have been isolated to improve detection^31,32,33. Moreover, the use of fusion proteins where the coding region of a reporter gene is joined in-frame with a second gene of interest has been particularly useful. Fusion of the gfp reporter gene and the aadA selectable marker gene (aadA:gfp) allows for visual deletion of gene expression through green fluorescence of GFP as well as for selection of transfected cells through resistance to antibiotics (dual selection)³⁴.

Our protocol presents a new method for gene delivery into the mitochondria of intact plant cells. The method is based on mTP peptide nano-carriers, which combine cell penetrating and mitochondria targeting properties. To demonstrate the versatility of the mTP-DNA protocol, we tested it in both somatic and germ cell systems and showed binding and delivery of a linear double-stranded DNA reporter cassette (aadA:gfp) into the mitochondria of triticale protoplasts and microspores, followed by documented expression in organello. Furthermore, we demonstrated that plantlets with exogenous DNA integrated into the mitochondrial genome can be regenerated from microspores transfected with aadA:gfp DNA.

Protocol

1. Preparation of culture media and cells

1. Stock solutions
   1. NPB-99 Macro-salts (10x). Prepare 1 L of 10x Macro-salts stock solution by dissolving 14.15 g of KNO₃, 2.32 g of (NH₄)₂SO₄, 2.0 g of KH₂PO₄, 0.83 g of CaCl₂·2H₂O and 0.93 g of MgSO₄·7H₂O in 500 mL of ddH₂O. Stir the solution at room temperature and bring the volume to 1 L with ddH₂O.
   2. NPB-99 Micro-salts (100x). Prepare 1 L of 100x Micro-salts stock solution by dissolving 40 mg of KI, 0.5 g of MnSO₄·7H₂O, 0.5 g of H₃BO₃, 0.5 g of ZnSO₄·7H₂O, 1.25 mL of 1 mg/mL CoCl₂·6H2O, 1.25 mL of 1 mg/mL CuSO₄·5H₂O and 1.25 mL of 1 mg/mL Na₂MoO₄·2H₂O in 500 mL of ddH₂O. Stir the solution at room temperature and bring the volume to 1 L with ddH₂O.
   3. Fe-EDTA (100x). Prepare 100 mL of 100x Fe-EDTA stock solution by dissolving 278 mg of FeSO₄.7H₂O and 372 mg of Na₂EDTA in 50 mL of ddH₂O. Stir the solution at room temperature and bring the volume to 100 mL with ddH₂O.
   4. NPB-99 vitamins (100x). Prepare 500 mL of 100x NPB-99 vitamins stock solution by dissolving 250 mg of thiamine-HCl, 25 mg of pyridoxine-HCl and 25 mg of nicotinic acid in 450 mL of ddH₂O. Stir the solution at room temperature and bring the volume to 500 mL with ddH₂O.
   5. GEM Macro-salts (10x). Prepare 1 L of 10x Macro-salts stock solution by dissolving 19 g of KNO₃, 1.65 g of (NH₄)NO₃, 1.7 g of KH₂PO₄, 4.4 g of CaCl₂·2H₂O and 3.7 g of MgSO₄·7H₂O in 500 mL of ddH₂O. Stir the solution at room temperature and bring the volume to 1 L with ddH₂O.
   6. GEM Micro-salts (100x). Prepare 1 L of 100x Micro-salts stock solution by dissolving 2.2 g of MnSO₄·7H₂O, 0.62 g of H₃BO₃, 0.86 g of ZnSO₄·7H₂O, 20 mL of 1 mg/mL CoCl₂·6H₂O, 15 mL of 1 mg/mL CuSO₄·5H₂O and 25 mL of 1 mg/mL Na₂MoO₄·2H₂O in 500 mL of ddH₂O. Stir the solution at room temperature and bring the volume to 1 L with ddH₂O.
   7. GEM vitamins (100x). Prepare 1 L of 100x GEM vitamins stock solution by dissolving 100 mg of thiamine-HCl, 100 mg of pyridoxine-HCl, 100 mg of nicotinic acid, 40 mg of ascorbic acid, 50 mg of pantothenate, 20 mg of riboflavin, 20 mg of folic acid, 20 mg of biotin, 790 mg of betaine chloride and 1000 mg of choline-HCl in 500 mL of ddH₂O. Bring the volume to 1000 mL with ddH₂O.
      NOTE: Filter sterilize the stocks solutions 100x NPB-99Macro-salts (step 1.1.1), 100x NPB-99 Micro-salts (step 1.1.2), 100x Fe-EDTA (step 1.1.3), 10x GEM Macro-salts (step 1.1.5) and 100x GEM Micro-salts (step 1.1.6) using a 0.22 μm filter and store at 4 °C. Filter sterilize the 100x vitamin stock solutions (steps 1.1.4 and 1.1.7) using a 0.22 μm filter, aliquot 10 mL into 15 mL plastic tubes and store at -20 °C.
2. Solutions and media
   1. NPB-99 extraction and wash solution
      1. Prepare 1 L of NPB-99 extraction and wash solution by starting with 500 mL of ddH₂O in a 2 L beaker on a stir plate and adding 100 mL of the 100x NPB-99Macro-salts stock solution, 10 mL of the 100x NPB-99 Micro-salts stock solution, 10 mL of the 100x Fe-EDTA stock solution, 10 mL of the 100x NPB-99 vitamins stock solution.
      2. Supplement the solution with 50 mg of myo-inositol, 500 mg of L-glutamine, 90 g of maltose monohydrate, 200 μL of 1 mg/mL 2,4-D stock solution, 200 μL of 1 mg/mL kinetin and 1 mL of 1 mg/mL PAA.
      3. Bring volume to 1 L with ddH₂O and adjust pH to 7.0.
   2. NPB-99 culture medium
      1. Prepare 200 mL of NPB-99 culture medium by starting with 100 mL of ddH₂O in a 400 mL beaker on a stir plate and adding 20 mL of the 100x NPB-99Macro- salts stock solution, 2 mL of the 100x NPB-99 Micro-salts stock solution, 2 mL of the 100x Fe-EDTA stock solution, 2 mL of the 100x NPB-99 vitamins stock solution.
      2. Supplement the solution with 10 mg of myo-inositol, 100 mg of L-glutamine, add 200 μL of the 10 mg/mL arabinogalactan solution, 18 g of maltose monohydrate, 20 g of ficoll, 40 μL of 1 mg/mL 2,4-D solution, 40 μL of 1 mg/mL kinetin and 200 μL of 1 mg/mL PAA.
      3. Bring volume to 200 mL with ddH₂O and adjust pH to 7.0.
   3. GEM Medium
      1. To prepare 2 L of GEM medium, first make 1 L of 0.6% (w/v) solidifying agent 1 solution by adding 6.0 g of solidifying agent 1 and a large magnetic stir bar to 1 L of ddH₂O in a large (2 or 3 L) Erlenmeyer flask and stirring for 5 min to disperse solidifying agent 1, and then autoclave for 20 min and cool to 65 °C.
      2. Make 2x GEM medium solution by adding to 500 mL of ddH₂O in a 2000 mL beaker, 200 mL of the 10x GEM Macro-salts stock solution, 20 mL of the 100x GEM Micro-salts stock solution, and 20 mL of the 100x Fe-EDTA stock solution.
      3. Supplement the 2x GEM solution with 30 g of maltose monohydrate, 10 g of sucrose, 0.7 g of xylose, 0.7 g of ribose, 0.4 g of myo-inositol, 0.71 g of U2.5 Amino Acid Mixture, 1.5 g of L-glutamine, 2 mg of spermine, 8 mg of spermidine and 20 mL of 100x GEM vitamins stock solution.
      4. Add soluble organic acids: 2.0 g of malic acid, 0.4 g of fumeric acid, 0.04 g of succinic acid, 0.04 g of α-ketoglutaric acid, 0.01 g of pyruvic acid, 0.01 g of citric acid and 2 mL of plant preservative mixture. Bring volume to 1 L with ddH₂O and adjust pH to 5.8 with KOH. Filter sterilize the 2x GEM medium solution using a 0.22 μm filter.
      5. Mix the 2x GEM medium with 0.6% solidifying agent 1 solution by continuous stirring on a hot stir plate. Add 8 mL of streptomycin solution (50 mg/mL; final concentration: 200 mg/mL), mix and aliquot 25 mL medium into 9 cm Petri dishes; once solidified, store plates at 4 °C.
         NOTE: Avoid melting solidified agent 1 and GEM medium: solidified and then re-melted agent 1 will not re-solidify.
   4. Rooting Medium
      1. Prepare 1 L of rooting medium by starting with 500 mL of ddH₂O and adding 100 mL of the 10x GEM Macro-salts stock solution, 10 mL of the 100x GEM Micro-salts stock solution, 10 mL of the 100x Fe-EDTA stock solution, 1.48 g of NH₄NO₃, 0.83 mL of 1 mg /mL KI and 10 g of sucrose. Bring volume to 1 L with ddH₂O and adjust pH to 6.0 with 1 N KOH.
      2. Transfer the solution into a 2 L bottle and add 6 g of agar. Autoclave for 15 min and cool to 65 °C.
      3. Add 0.5 mL of filter sterilized PAA (1 mg/mL) solution and 8 mL of streptomycin solution (50 mg/mL; final concentration: 400 mg/mL), mix and aliquot the rooting medium into 250 mL magenta vessels (50 mL) or 15 mL glass tubes (5 mL). Once medium is solidified, store it at 4 °C.
3. Preparation of plant cells
   1. Isolate microspore cells at the mid-late uninucleate stage from the surface-sterilized anthers of triticale (X. Triticosecale Wittmack, cv. Ultima)³⁵ according to the previously published protocol³⁶.
   2. Isolate and purify protoplasts from Triticale leaves by following the protocol published previously³⁷.
   3. Adjust the final cell concentration to 2 x 10⁵ cells/mL (500 μL = 100,000 cells) with the NBP-99 medium (microspores) or CPW solution (protoplasts)³⁷.
      NOTE: Other triticale cultivars as well as other plant species (e.g., wheat can be used as a source of cells for transfection).

2. Preparation of DNA and peptides

1. Construction of a vector carrying a reporter gene construct for mitochondrial expression
   NOTE: The wheat mitochondrial aadA:gfp reporter cassette (aadA:gfp, Figure 1A) is a 3424 base pair long plant mitochondrial transfection vector designed to target insertions in between the trnfM-1 and rrn18-1 genes of the triticale (X Triticosecale Wittmack) and wheat (Triticum aestivum) mitochondrial genome.
   1. Design the vector using a bioinformatics software.
   2. Start with the nucleotide sequence 300379-300878 (GenBank accession No. AP008982.1) as the left sequence flanking the cassette for its integration into the mitochondrial genome via homologous recombination (5’ HR) with the trnfM-1 target insertion sequence.
   3. Introduce a multiple cloning site following the trnfM-1 target insertion sequence.
   4. Add the selection marker gene cassette sequence: (i) the T. aestivum mitochondrial atpA gene promoter (P_atpA; GenBank accession No. X54387.1), (ii) aadA:gfp fusion gene³⁴ (GenBank accession No. ABX39486) and (iii) the T_cobA terminator (GenBank accession No. AP008982.1, nucleotides: 62871-62565).
      NOTE: Other selectable marker genes and mitochondrial gene expression elements (promoters and terminators) may be used.
   5. End the design with the nucleotide sequence 300880-301373 (GenBank accession No. AP008982.1) as the right sequence flanking the cassette for its integration into the mitochondrial genome via homologous recombination (3’ HR) with the rrn18-1 target insertion sequence.
   6. Order synthesis of the designed reporter gene construct by a DNA synthesis service. The construct will be delivered as a bacterial plasmid vector containing the reporter gene cassette (e.g., pMK_Wheat_Mito_aadA-gfp).
   7. Perform plasmid preparation following the manufacturer protocol and using the kits summarized in the Table of Materials.
2. DNA cargo preparation
   1. Linearize plasmid DNA by digestion with a single-cutter restriction enzyme (e.g., PstI) according to the supplier’s protocol.
   2. Purify the linearized plasmid DNA following the manufacturer protocol and using the kit summarized in the Table of Materials.
      NOTE: Good quality of DNA is crucial for transfection.
   3. Dilute DNA in nuclease-free/protease-free H₂O to obtain 150 ng/μL stock solution.
   4. Per each transfection, aliquot 10 μL of the 150 ng/μL DNA stock solution (total DNA amount: 1.5 μg) into sterile 1.5 mL tubes.
   5. Add 40 μL of nuclease-free/protease-free H₂O into each tube (final volume: 50 μL) and mix the tube content by gentle pipetting. Scale up for multiple transfections by preparing the DNA working stock solution in a volume proportional to the number of transfections.
3. mTP peptide carrier preparation
   1. Order synthesis of the mTPs by peptide synthesis service. The peptide will be delivered as white powder, which should be stored at -20 °C.
   2. Prepare 1 mg/mL peptide stock by dissolving 1 mg of the peptide powder in 1 mL of nuclease-free/protease-free H₂O. Aliquot 100 μL of the stock into sterile 0.6 mL tubes and store them at -20 °C until use.
   3. Thaw the peptide stock on ice prior to transfection. Do not exceed three thawing-freezing cycles.
   4. Prepare the working stock solution by aliquoting the nuclease-free/protease-free H₂O into sterile 1.5 mL tubes, adding the appropriate volume of the 1 mg/mL peptide stock (Table 1), and mix gently. Scale up for multiple transfections by preparing the peptide working stock solution in a volume proportional to the number of transfections.

3. Transfection of triticale microspores with mTP-DNA nano-complexes (Figure 1B)

1. Formation of mTP-DNA nano-complexes
   1. Aliquot 50 μL of the DNA working stock solution into a sterile 1.5 mL tube and add 50 μL of the peptide working stock solution, mix gently.
   2. Incubate the peptide-DNA mixture for 10 min at room temperature; mix gently by tapping the tube bottom every 5 min. Do not exceed 15 min, because prolonged incubation will result in aggregation of the peptide-DNA complexes.
2. Delivery of mTP-DNA nano-complexes into triticale microspores
   1. Aliquot 500 μL of the 2 x 10⁵ cells/mL microspore cell suspension (step 1.2; total 100,000 cells per transfection) into sterile 2 mL tubes.
   2. Add 100 μL of the peptide-DNA mixture (step 2.1) to microspores and mix gently. Include control treatments by omitting the peptide or DNA component, or both.
   3. Incubate the peptide-DNA-microspores transfection mixture for 1 h at room temperature; mix gently by tapping the tube bottom every 5 min.
   4. Add 400 μL of the NPB-99 medium (step 1.1.1) and continue incubation at room temperature for 24 h (confocal microscopy) or 48 h (qRT-PCR) or 1 h (microspore culture and selection).
      NOTE: Samples for confocal microscopy analysis should be incubated in the dark.

4. Nucleic acids extraction from the transfected cells and plant tissues

1. Harvest microspore cells by centrifugation, freeze the cell samples in liquid nitrogen and store at -80 °C until use.
2. Disrupt frozen cells with ceramic or metal bead by aggressive shaking in a cell/tissue disrupting machine (2 times 2 pulses for 60 s at 4,000 x g).
   NOTE: Cells may be also disrupted by vortexing with metal beads in lysis buffer for 15 min at maximum speed (1,000 x g) at room temperature.
3. Isolate gDNA and RNA following the manufacturer protocol and using the kits summarized in the Table of Materials, with some modifications, including additional on-column DNase I digestion. Alternatively, the additional DNase I digestion can be done on the purified RNA samples, followed by clean up on column, according to the manufacturer protocol.
4. Asses the quantity and quality of DNA and RNA using agarose gel electrophoresis (to ensure DNA and RNA integrity) and spectrophotometrically (to quantify the DNA and RNA).

5. Analysis of transient expression of the delivered transgene

1. Real-time RT-PCR (qRT-PCR)
   1. Generate cDNA from 1 μg of total RNA isolated from transfected and control cells following the manufacturer protocol and using the kits summarized in the Table of Materials.
   2. Perform real-time RT-PCR according to protocol outlined in Table 2, using GFP1L/GFP1R (for aadA::gfp) and EF_F1/EF_R1 (for EF1α) primers (Table 3). Calculate relative expression of the aadA::gfp gene in relation to the expression of the endogenous EF1α gene, using the standard curve method.
2. Confocal microscopy
   1. Add fluorescent dye (3.0 μL of 1.0 mM mitochondria-specific dye; final concentration: 3 μM) to 1 mL of transfected cells 24-48 h after transfection to stain cell mitochondria.
   2. Observe cells using a confocal microscope to examine the subcellular localization the GFP protein (excitation/emission wavelength: 490 nm/520 nm) by collecting fluorescence emissions in z-confocal planes of 10–15 nm and analyzing the images using appropriate software. Compare it to localization of the cell mitochondria stained with mitochondria-specific dye (excitation/emission wavelength: 554 nm/576 nm).

6. Selection and regeneration of putative transformants

1. Microspore embryogenesis
   1. Incubate the transfected and control microspores for 1 h at room temperature and then transfer to 35 mm Petri dishes containing 2.5 mL of NBP-99 medium (total volume: 3.5 mL) and 4 ovaries. Add streptomycin (7 μL of 50 mg/mL stock; final concentration: 100 mg/L).
   2. Seal 35 mm plates with semi-transparent flexible film and place them in a 150 mm Petri dish with 60 mm dish containing distilled water. Seal the 150 mm dish with semi-transparent flexible film to keep moisture in.
   3. Culture the cells in the dark at 27 °C to induce embryogenesis. Carry microspore control cultures with or without streptomycin to determine the effect of antibiotics on microspore embryogenesis and green plant regeneration.
2. Embryo germination
   1. After 4 weeks of culture, transfer the developing embryos onto GEM plates supplemented with streptomycin (final concentration: 200 mg/L).
   2. Culture embryos at 16 °C beneath wide spectrum 40 watts bulbs delivering 80 µM m^-2 s^-1 (a 16-h light period) for embryo germination.
      NOTE: Only a few embryos will germinate into green plantlets. Some embryos will develop into albino plants or may form roots only, while the majority of embryos will be aborted.
3. Rooting of green plantlets
   1. After 3 to 4 weeks, transfer green plantlets into magenta vessels containing the rooting medium with streptomycin (final concentration: 400 mg/L) and continue plantlet cultivation as described above (step 6.2.2).
      NOTE: This selection procedure results in regeneration of haploid putative aadA:gfp plants. In order to obtain fertile, doubled haploid plants, haploid plants may be treated with colchicine to induce chromosome duplication^38,39 or cultivated in soil for spontaneous genome duplication.

7. PCR screening of putative transformants

1. gDNA isolation
   1. Collect about 100 mg of leaf samples from the regenerated plants, freeze the tissue samples in liquid nitrogen and store at -80 °C until use.
   2. Disrupt frozen cells with ceramic or metal beads by shaking aggressively in a cell/tissue disrupting machine (2 times 2 pulses for 60 s at 4,000 x g) or by crushing leaf tissue with a pestle in tubes frozen in liquid nitrogen.
   3. Isolate genomic DNA from young leaves of the primary transformants (T₀) at the end of in vitro culture steps using a kit summarized in the Table of Materials, following the manufacturer’s protocol.
2. PCR
   1. Perform PCR using GFP1L/GFP1R primers (Table 3) according to the protocol outlined in Table 4.
   2. Separate the PCR samples by electrophoresis in a 1.0% agarose gel containing ethidium bromide in 1x TAE buffer for 1 h.

8. Analysis of transgene insertion into mitochondrial genome

1. Amplification and cloning of the left and right junctions
   1. Perform PCR according to protocol outlined in Table 4 by using 3 sets of primers for each junction. Left junction primer sets: trnfMF3/trnfMR2, trnfMF6/trnfMR4 and trnfMF5/trnfMR4; right junction primer sets: rrn18-1F3/rrn18-1R4, rrn18-1F3/rrn18-1R6 and rrn18-1F4/rrn18-1R5 (Table 3).
   2. Separate the PCR samples by electrophoresis in a 1.0% agarose gel containing ethidium bromide in 1x TAE buffer for 1 h.
   3. Purify the PCR products using a gel extraction kit (Table of Materials).
   4. Clone the PCR products into a PCR cloning vector (Table of Materials), according to the manufacturer’s protocol.
      NOTE: Other PCR cloning kits may be used.
2. Sequencing and sequence alignment
   1. Order sequencing of the cloned PCR products through a DNA sequencing service, using the universal M13 and T7 primers.
   2. Conduct sequence alignments using a bioinformatics software as described previously³⁷. Use pairwise or multiple alignment to generate the consensus sequence for each junction. Then, apply multiple genome alignment using aadA::gfp construct sequence with 500 bp extensions of the 5’ and 3’ HR (homology regions) as a target (reference) sequence to analyze nucleotide sequence similarity between the consensus junction sequences and the reference sequence.

Results

DNA binding and protection properties of mTPs

The ability of mTPs to non-covalently bind nucleic acids is an essential property for translocation of dsDNA cargoes into the plant mitochondria. The minimum peptide amount needed to bind to and completely saturate the linearized dsDNA in preparation for microspore transfection was determined in a gel mobility shift assay and a nuclease protection assay. Titration of the mTP peptide was performed by adding increasing amounts of the...

Discussion

Genetic engineering of mitochondria is a key tool in plant biology and biotechnology but has been very limited in its utility so far due to restrictions in DNA delivery into mitochondrial organelles. Biolistics and electroporation have proven to be popular methods of gene delivery into eukaryotic and prokaryotic cells⁴⁰, but there are limitations when applying these technologies to in vivo plant mitochondrial genetic transformation. Frequently mitochondrial transformation rates are low

Materials

Name	Company	Catalog Number	Comments
Chemicals
(NH4)2SO4	Sigma	A3920	Component of 10x Macrosalts solution
2,4-Dichlorophenoxyacetic acid (2,4-D)	Sigma	D8407	Component of NPB-99 medium
Agar	Sigma	A7921	Solidifying agent 2; Component of rooting medium
a-ketoglutaric acid	Sigma	K1750	Component of GEM medium
Arabinogalactan (Larcoll)	Sigma	L0650	Component of NPB-99 and GEM media
Ascorbic acid	Sigma	A4544	Component of 100x GEM vitamins solution
Betaine chloride	Sigma	B7045	Component of 100x GEM vitamins solution
Biotin	Sigma	B3399	Component of 100x GEM vitamins solution
CaCl2, 2H2O	Sigma	C7902	Component of 10x Macrosalts solution
Choline-HCL	Sigma	C-1879	Component of 100x GEM vitamins solution
Citric acid	Sigma	C4540	Component of GEM medium
CoCl, 6H2O	Fisher	C371	Component of 100x Microsalts solution
CuSO4, 5H2O	Fisher	C489	Component of 100x Microsalts solution
FeSO4, 7H2O	Sigma	F8263	Component of 100x Fe-EDTA solution
Ficoll	Sigma	F4375	Component of NPB-99 culture medium
Folic Acid	Sigma	F8509	Component of 100x GEM vitamins solution
Fumaric acid	Sigma	F8509	Component of GEM medium
Gelrite	Sigma	G1910	Solidifying agent 1; Component of GEM medium
Glutamine	Sigma	G8540	Component of NPB-99 and GEM media
H3BO3	Sigma	B9545	Component of 100x Microsalts solution
KH2PO4	Sigma	P5655	Component of 10x Macrosalts solution
KH2PO4	Sigma	P9791	Component of 10x Macrosalts solution
KI	Sigma	P8166	Component of 100x Microsalts solution
Kinetine	Sigma	K0753	Component of NPB-99 medium
KNO3	Sigma	P8291	Component of 10x Macrosalts solution
Malic acid	Sigma	M1000	Component of GEM medium
Maltose	Sigma	M5895	Component of NPB-99 and GEM media
MgSO4, 7H2O	Sigma	M5921	Component of 10x Macrosalts solution
MitoTracker Orange CM-H2TMRos	Invitrogen	M7511	Mitochondria-specific dye for confocal microscopy analysis
MnSO4, H2O	Sigma	M7899	Component of 100x Microsalts solution
Myo-Inositol	Sigma	I3011	Component of NPB-99 and GEM media
Na2EDTA	Sigma	E5134	Component of 100x Fe-EDTA solution
NH4NO3	Sigma	A3795	Component of rooting medium
Nicotinic acid	Sigma	N0761	Component of 100x NPB-99 and GEM vitamins solutions
Pantothenate	Sigma	A7219	Component of 100x GEM vitamins solution
Phenylacetic acid (PAA)	Sigma	P6061	Component of NPB-99 and GEM media
Plant preservative mixture (PPM)	Plant Cell Technologies		Component of GEM medium
Pyridoxine- HCl	Sigma	P8666	Component of 100x NPB-99 and GEM vitamins solutions
Pyruvic acid	Sigma	P2256	Component of GEM medium
Riboflavin	Sigma	C4540	Component of 100x GEM vitamins solution
Ribose	Sigma	R7500	Component of GEM medium
Spermidine	Sigma	5292	Component of GEM medium
Spermine	Sigma	S4264	Component of GEM medium
Streptomysin sulphate	Sigma	S9137	Selection agent
Succinic acid	Sigma	S7501	Component of GEM medium
Sucrose	Sigma	S7903	Component of GEM medium and rooting medium
Thiamine-HCl	Sigma	T1270	Component of 100x NPB-99 and GEM vitamins solutions
U2.5 Amino Acid Mixture	sigma	U-7756	Component of GEM medium
Xylose	Sigma	X1500	Component of GEM medium
ZnSO4, 7H2O	Sigma	Z1001	Component of 100x Microsalts solution
Kits & Enzymes
All Prep DNA/RNA mini kit	Qiagen	80204	gDNA and RNA extraction
DNeasy Plant Mini Kit	Qiagen	69106	gDNA isolation
GeneRuler 1 kb DNA Lader	Thermo Scientific	11823963	DNA size marker for agarose elecrtophoresis
GeneRuler Low Range DNA Lader	Thermo Scientific	10212840	DNA size marker for agarose elecrtophoresis
pCR 2.1 Vector TA Cloning Kit	Invitrogen	K204001	Cloning of PCR amplicons
Plasmid Maxi kit	Qiagen	12163	Plasmid isolation
QIAquick Gel extraction kit	Qiagen	28706	Extraction of PCR products from agarose gels
QIAquick PCR purification kit	Qiagen	28104	Purification of linearized plazmid
QuantiTect SYBR Green PCR Master Mix	Qiagen	204143	Real time RT-PCR
RNase-free DNase set	Qiagen	79256	Dnase I treatment
SuperScript III First-Strand Synthesis Kit	Invitrogen	18080051	cDNA synthesis
Taq DNA polymerase kit	Qiagen	201205	Standard PCR
Other materials
150 mm Petri dishes	ThermoFisher	150350	Microspore culture, multi 35 mm Petri dish container
35 mm Petri dishes	Fisher Scientific	0875100A	Microspore culture
60 mm Petri dishes	Fisher Scientific	0875113A	Microspore culture, water reservoir
90 mm Petri dishes	ThermoFisher	101IRR20	Embryo germination
Cell Lyzer	Bertin Technologies		Disruption and homogenization of plant cells and tissues
Magenta vessels	Sigma	V 8505	Plantlet rooting
Semi-transparent flexible film (Parafilm)	ThermoFisher	5833-0001	Plate sealing
Sylvania Gro-lux wide spectrum bulbs (40 watts)	Sylvana		Growth chamber lights
Vacucap 60PF 0.2 UM	VWR	CA28139-704	Filter sterilization of media and solutions