Methods

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Plant materials

Seeds of Nicotiana tabacum cv. ‘SR1’ were obtained from the Leaf Tobacco Research Center. Nicotiana benthamiana, N. tabacum ‘SR1’, and Torenia fournieri cv. ‘Blue and White’ plants were grown in soil in a green room at 25–30°C under long-day conditions (16 h light/8 h dark), and Solanum lycopersicum cv. ‘Micro-Tom’ plants were grown in soil in a greenhouse at 20–30°C. Young leaves and mature pollen from newly opened flowers were used for bombardment.

Plasmid construction

The plasmid vectors used for particle bombardment in this study are listed in Table S1. The constructs LAT52p::mApple (YMv32), LAT52p::Venus (YMv35), LAT52p::mTFP1 (YMv37), and AtRPS5Ap::H2B-tdTomato (DKv277) were obtained from previous studies (Adachi et al. 2011; Mizuta et al. 2015). 35Sp::H2B-mClover (DKv700) and 35Sp::H2B-tdTomato (DKv744) were provided by Dr. Daisuke Kurihara, and 35Sp::mTFP1 (DKv327) was provided by Dr. Noriko Inada. The AtRPS5Ap::sGFP vector (sSNv10), in which the sGFP gene is driven by the Arabidopsis thaliana RPS5A (RIBOSOMAL PROTEIN SUBUNIT 5A; At3g11940) promoter, was produced by inverse PCR of the AtRPS5Ap::H2B-sGFP vector (Maruyama et al. 2013) and self-ligation of the PCR product to remove the H2B (HISTONE 2 B; At1g07790) sequence. The sequences of AtUBQ10p::sGFP (sSNv25), AtUBQ10p::tdTomato (sSNv26), and AtUBQ10p::H2B-mClover (sSNv28) followed by the Nos-terminator sequence were isolated from the DKv909, DKv922, and DKv916 vectors, respectively (Kurihara et al. 2015), by Hin_dIII/_Eco_RI digestion. Each fragment was cloned into the pGreen0029 vector (Hellens et al. 2000) using _Hin_dIII and _Eco_RI sites. The CRISPR/Cas9 vector targeting the _NbPDS3 gene (sSNv21) was constructed based on a previous study (Tsutsui and Higashiyama 2017). The AtRPS5A promoter was replaced with the AtUBQ10 (UBIQUITIN 10; At4g05320) promoter to drive Cas9 gene expression, and the target sequence for the NbPDS3 gene was then introduced via _Aar_I digestion (sSNv21). The primers used for plasmid construction are listed in Table S2.

Plant transformation

The sSNv28 vectors were introduced into Agrobacterium tumefaciens strain LBA4404 harboring the pSoup plasmid (Hellens et al. 2000) by electroporation, and N. benthamiana leaf discs were infected with Agrobacterium. The infected leaf discs were cultured on callus induction medium (1× Murashige and Skoog Basal Medium, 3% (w/v) sucrose, 0.8% (w/v) Bacto agar, adjusted to pH 5.8 with KOH] containing 0.05 mg/L 1-naphthaleneacetic acid, 0.5 mg/L 6-benzylaminopurine, 100 mg/L kanamycin sulfate, and 300 mg/L cefotaxime sodium. The plants that regenerated from calli were transferred to medium lacking hormones to induce root germination and were eventually transferred to soil.

Calculating the percentage area of pollen nuclei to cytoplasm

To calculate the percentage of area of pollen nuclei to the cytoplasm, we analyzed fixed raw and raw pollen grains. Wild-type pollen grains were fixed with a 9:1 mixture of ethanol and acetic acid (v/v) for 10 min, and the samples were directly stained with DAPI solution and incubated for more than 10 min. After staining, the pollen grains were washed twice with water, mounted on glass slides, and observed under an inverted fluorescence microscope (Eclipse Ti2; Nikon, Tokyo, Japan). The pollen from UBQ10p::H2B-mClover was collected in a liquid pollen germination medium 0.01% (w/v) boric acid, 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4, and 10% (w/v) sucrose adjusted to pH 6.5 with KOH (Wang and Jiang 2011), and the sample was immediately observed under a fluorescence microscope. ImageJ software (https://imagej.nih.gov/ij/index.html) was used to calculate generative nucleus to cytoplasm (GN/C) ratio of 20 pollen grains.

Biolistic delivery of plasmid DNA

The gold particles (0.6-μm diameter) used for biolistic delivery were washed with absolute ethanol, rinsed twice with sterilized water, and suspended in sterilized water to prepare a 30 mg/mL gold solution. The gold solution was dispensed (10 μL per shot) and mixed with 200–1,000 ng of plasmid DNA(s) per shot in an agitating mixer, to which 4 μL 0.1 M spermidine and 10 μL 2.5 M calcium chloride per 10 μL of the gold solution were subsequently added. The resulting DNA-coated gold particles were collected by centrifugation at 3,300 g for 30 s. The DNA-coated gold particles were then washed once with 70% ethanol, twice with absolute ethanol, and resuspended in absolute ethanol (10 μL per shot). Particle bombardment was performed using a PDS-1000/He system (Bio-Rad Laboratories, USA). The distance between the macro-carrier and target cells was adjusted to approximately 3.0 cm, the helium gas pressure was set to 1,100 psi, and the degree of vacuum was set to at least −25 inHg. For leaf bombardment, we used the leaves of mature N. benthamiana plants, which were taped to a plastic petri dish. The cut ends of the bombarded leaves were covered with a wet wipe and cultured at 25–30°C for 20– 24 h under humid, dark conditions. For pollen bombardment, pollen was collected immediately prior to bombardment and distributed on pollen germination medium solidified with 1% (w/v) NuSieve GTG Agarose (Lonza, Switzerland). Pollen germination media for Nicotiana (Wang and Jiang 2011) and torenia (Okuda et al. 2009) were used as described previously. The same pollen germination medium as that used for Nicotiana was used. After being bombarded, the treated pollen was cultured directly on the medium and observed under an inverted fluorescence microscope (Eclipse Ti2, Nikon; AXIO imager A2, Zeiss). Biolistic delivery into leaf and pollen was examined twice, and gene introduction was evaluated by fluorescence expression.

Detection of Cas9-induced genome editing

In addition to the CRISPR/Cas9 vector described above, plasmid vectors encoding fluorescence protein markers were simultaneously coated with gold particles. Bombarded leaves were observed under a fluorescence microscope (Eclipse Ti2, Nikon; AXIO imager A2, Zeiss) to confirm the efficiency of delivery. An area of approximately 1.5 cm in diameter around the blast center was collected, immersed in a DNA extraction buffer 0.2 M Tris-HCl (pH 8.0), 0.25 M NaCl, 25 mM EDTA, and 0.5% (w/v) SDS, and frozen in liquid nitrogen prior to subsequent analysis. Genomic DNA was extracted from the leaf material using a homogenizer and sonicator. Pollen samples with or without fluorescent signals were collected together into the same DNA extraction buffer and the genomic DNA was then extracted. For PCR analysis, the following steps were performed according to Nekrasov et al. (2013). Genomic DNA was digested with _Mly_I and PCR-amplified with the primer pair _PDSMly_IF and _PDSMlyIR (Nekrasov et al. 2013; Table S2). The PCR product thus obtained was subsequently digested with _Hin_fI and/or used directly as a nested PCR template to remove non-specific DNA fragments. The nested PCR product was cloned into the pCR-BluntII-TOPO vector (Thermo Fisher Scientific, USA) and introduced into _Escherichia coli Mach1 T1R competent cells (Thermo Fisher Scientific). Colonies possessing the fragment of the mutated NbPDS3 gene were identified by colony PCR using primers M13 forward, M13 reverse, and NbPDS3__primer-m (Table S2), of which the 3ʹ end was fully matched to the wild-type _NbPDS3 sequence. Accordingly, for colonies containing mutated NbPDS3 fragments, few or no bands were amplified using NbPDS3__primer-m. A mutation in the _NbPDS3 gene was confirmed by sequence analysis of the plasmid vectors extracted from individual colonies.

Aniline blue staining of pollinated pistils

Wild-type N. benthamiana and N. tabacum pistils were emasculated 2 days prior to pollination. The pollen spread on germination medium that had been hydrated for 15 min was used to pollinate the emasculated pistils using a dissecting needle. At 24 h after pollination, the pollinated flowers were collected, and the remaining petals and sepals were removed. The pistil was fixed with a 3:1 mixture of ethanol and acetic acid (v/v) overnight and treated with 1 N NaOH solution for 1 day. Thereafter, the pistils were stained with 0.1% (w/v) aniline blue in 0.1 M K3PO4 buffer for 1 day. Images were obtained using an Eclipse Ti2 fluorescence microscope under UV light.

Semi-in vivo pollen tube growth assay using N. tabacum

N. tabacum pistils were emasculated 2 days prior to pollination. Plasmid vectors encoding UBQ10p::sGFP and 35Sp::H2B-tdTomato were simultaneously coated on gold particles (mixture 1), as were the vectors encoding LAT52p::mApple and UBQ10p::H2B-mClover (mixture 2). Immediately prior to spreading onto the macro-carrier, mixtures 1 and 2 were mixed and then introduced into pollen via a single bombardment shot. Twenty-four hours after pollination, the pollinated style was excised with a razor 5 mm above the ovary and placed horizontally on the aforementioned pollen germination medium solidified with 1% (w/v) NuSieve GTG Agarose (Lonza), followed by incubation at 25–30°C for 20 h under humid, dark conditions. Pollen tubes emerging from the cut end of the pistil were observed using an Eclipse Ti2 fluorescence microscope.

In vivo experiments using N. tabacum

N. tabacum pistils were emasculated 2 days prior to pollination. Pollen was bombarded with the aforementioned mixture 1 and used immediately thereafter to pollinate the emasculated pistils. At 24 h post-pollination, the pollinated style was cut longitudinally using a razor, and at 24 h and 48 h post-pollination, the ovary wall was removed with forceps to facilitate observation of the pollen tube and ovule within the ovary. The samples were placed in 10% glycerol (v/v) and observed under an Eclipse Ti2 fluorescence microscope.

Article TitleDetection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Abstract

In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.


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