Plant materials

Tomato (Solanum lycopersicum L.) plants of cv. ‘Ailsa Craig’ were grown in a greenhouse at Yunnan Agricultural University (Kunming, China). Greenhouse temperature was maintained at 26 °C during the day (16 h) and at 18 °C during the night (8 h). Tomato flower buds were removed from the plants at flowering period and placed in an ice box. Anthers were collected under low temperatures, quickly treated with liquid nitrogen and stored at − 80 °C.

Cloning and characterization of the tomato SlAMS gene

Total RNA was extracted from tomato anthers using Trizl Kits (TransGen Biotech. Co., Ltd., Beijing, China). The cDNA of the tomato was synthesized using High Fidelity Prime Script RT-PCR Kits (Takara Biomedical Technology Co., Ltd. Beijing, China), following the manufacturer’s instructions, and amplified using PCR with the primers AMSad1 and AMSad2 (Additional file 13). Primers were designed based on the previously-published tomato AMS gene sequence (GenBank ID. LOC101253608) using Primer 5.0. The PCR volume contained 2 μL cDNA, 1 μL each primer (10 μmol/μL), 12.5 μL of 2 × EasyTaq PCR SuperMix, and ddH2O to make 25 μL. The PCR cycling conditions were as follows: pre-denaturation at 95 °C for 3 min; 35 cycles of denaturation at 94 °C for 50 s, annealing at 50 °C for 1 min, and extension at 72 °C for 3 min; a final extension at 72 °C for 10 min; and an indefinite hold at 4 °C. The target DNA fragment was extracted from the agarose gel using by gel filtration, and the purified using a TIANgen Midi Purification Kit (TIANGEN Biotech. Co., Ltd., Beijing, China) following the manufacturer’s instructions. The target fragment was ligated to the pMDT-18 cloning vector (0.5 μL of vector, 4 μL of PCR product, 5 μL of Solution I, and 0.5 μL of T4 DNA ligase) to form pMDT-18-SlAMS, incubated at 16 °C for 3 h, and then transformed into Escherichia coli DH competent cells. The transformed cells were cultured overnight at 37 °C. Six single white colonies were selected, and the length of the inserted fragment was confirmed using PCR. Recombinant plasmid DNA was isolated using TIANprep Mini Plasmid Kits (Yunnan Morning Green Biotechnology Co., Ltd. Yunnan, China) and sequenced by the Shuo Qing Biotechnology Co., Ltd. (Kunming, China).

The homology of the cloned SlAMS gene sequence was investigated using DNAMAN 6.0 and NCBI BLAST. To compare the predicted tomato SlAMS protein with other AMS homologs, both within the Solanaceae and among other closely-related families (i.e., the Compositae, Cruciferous, Leguminosae, and Cucurbitaceae), we constructed a phylogeny of the AMS proteins from 25 plant species (Additional file 14) using the neighbor-joining method in MEGAX10.1.5. A subcellular localization analysis was performed using PSORT II predict. The primary structure of the encoded SlAMS protein was predicted using PredictProte; the secondary structure was predicted using SOPMA; and the tertiary structure was predicted using SWISS-MODEL. The websites for the online tools are given in Additional file 15.

Obtaining the SlAMS-silenced plants by VIGS and morphological observation of pollen

We designed the primers AMS-F and AMS-R (Additional file 13) to amplify the SlAMS-interference fragment which was 274 bp long (Additional file 1), using the SNG VIGS TOOL (https://vigs.solgenomics.net/) and pTRV2 as a basic VIGS vector (Additional file 9), we amplified the interference fragment from cloning vector pMDT-18-SlAMS as a template. We chose tobacco brittle virus (TRV) as gene silencing vector [34, 35]. The TRV genome contains two RNA chains, RNA1 and RNA2, which form binary vectors. TRV vector-mediated gene silencing requires the simultaneous action of the RNA1 and RNA2 chains [36]. The pTRV2 vector was double-digested with Sac I and Bam HI, and then ligated to the SlAMS-interference fragment to form the pTRV2-SlAMS carrier. The recombinant SlAMS products were then transformed into E. coli DH and Agrobacterium tumefaciens GV310. SlAMS clones were verified using colony PCR with the primers AMS-R and pTRV2-seqE (Additional file 13) and then sequenced. The positive clones were fully mixed with 50% glycerin (a glycerin: bacterial culture ratio of 1:1) and stored at − 80 °C.

Tomato plants were randomly selected and allocated among four groups: plants in the silencing group were infected with A. tumefaciens carrying pTRV2-SlAMS and pTRV1; plants in the albefaction group (mock-infection) were infected with A. tumefaciens carrying pTRV2-PDS and pTRV1; plants in the negative control group were infected with A. tumefaciens carrying pTRV2 and pTRV1; and plants in the blank control group were uninfected wild type plants. Each group was represented by three replicates, and each replicate was comprised of 10 plants. To activate Agrobacterium, 20 μL of glycerol-stored Agrobacterium carrying pTRV2-SlAMS, pTRV1, pTRV2, or pTRV2-PDS were added to 1 ml of Luria-Bertani medium (supplemented with 50 μg/ml kanamycin and 25 μg/ml rifampicin), and incubated overnight at 28 °C with shaking at 200 rpm. After incubation, the bacterial solution was added to 10 ml of LB medium and cultured overnight at 28 °C with shaking at 200 rpm. The bacterial solution was then centrifuged at 800 g for 15 min. Each A. tumefaciens pellet was resuspended in sufficient infection solution to make an optical density (OD) at 600 nm of 1.0. Then, each A. tumefaciens solution (carrying pTRV2-SlAMS, pTRV2, or pTRV2-PDS) was separately mixed with pTRV1 at a volume ratio of 1:1. The A. tumefaciens mixtures were cultured at 28 °C with shaking at 50 rpm for 3 h. After two euphyllas had completely unfolded, 30 plants per group were injected with 1 ml of the appropriate A. tumefaciens solution; the plants in the blank control group were injected with sterile water. After injection, plants were incubated in the dark at 21 °C for 24 h, and then cultivated in the greenhouse, covered with nylon nets to avoid cross-contamination and insect-mediated virus transfer, until flowering.

Pollen was collected from five flowers from each plant in the wild type, negative control, and SlAMS-silenced groups. Pollen viability was measured using the blue-ink staining method. In brief, a small amount of pollen was placed on a glass slide, and a drop of distilled water was added. The pollen grains were spread out with tweezers, and then a drop of blue ink solution was added. Viable and non-viable pollen grains were counted in three different fields of view, and the final percent viable was calculated as the average of the three counts; unstained pollen were viable, while blue-stained pollen were abortive. The significance analysis of non-viability pollen percentage was implemented using t test method by statistical software SPSS 25.0. The morphological characters of the pollen grains were then observed under a scanning electron microscope (SEM) with an acceleration voltage (EHT) of 3.0 kv and a magnification (MAG) of 1.2 k.

pCRISPR/Cas 9-mediated SlAMS knockout and mutation analysis

A pCRISPR-SlAMS vector was constructed using a Cas9/gRNA plasmid construction kit (VK005–08, Beijing Viewsolid Biotech., Co., Ltd., Beijing, China), following the manufacturer’s instructions. In brief, the target gRNA sequence was designed based on the cloned SlAMS gene sequence using an online CRISPR design tool (crisPr.mit.edu). We then designed sense and anti-sense primers (pCRISPR-Sens and pCRISPR-Anti, respectively; Additional file 13) based on the target sequence and synthesized the oligomer. The oligomer was inserted into the Cas9/gRNA vector and transformed into E. coli DH (Additional file 10). Then, colonies were randomly selected and sequenced to identify positive clones using the primers pCRISPR-SeqE (Additional file 13). The identified pCRISPR-SlAMS vector was transformed into A. tumefaciens GV3101. Then, we used Agrobacterium-mediated method to transform the vector into tomato genome [37]. We conducted three batches of Agrobacterium infection using cotyledons (i.e 3 replicates), which comprised 80 ~ 100 explants in each batch.

After the co-culture, selection culture of the infected cotyledons, the regenerated plants were obtained. The genomic DNA were extracted from T0 progeny regenerated plants using the CTAB method. Regenerated but untransformed plants were used as controls. We detected transgenic plants via the PCR amplification of the Npt II selection gene. The PCR volume included 1 μL DNA; 2 μL 10 × PCR buffer, 0.4 μL dNTP mixture (2 mmol/L each), 0.2 μL forward and reverse primers (10 μmol/L), 0.2 μL Taq DNA polymerase (1 U/μL); and ddH2O to make 20 μL. The thermal cycling procedure was as follows: 94 °C for 3 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min. In the CRISPR/Cas9 system, the Cas cleavage site occurs mostly 3 bp upstream of the protospacer, thus the insertion and deletion mutations around 3 bp upstream of the protospacer were considered to be mutations induced by Cas9. To identify the mutations, we designed the primers (AMS-F2/AMS-R2, Additional file 13) based on the gene editing site of SlAMS, and extracted the flower DNA of 33 individual Npt II positive plants. DNA extraction and PCR cycling conditions were the same as the Npt II ampification. The 267 PCR products were sequenced and confirmed for mutation (ShuoQing Biotech., Co., Ltd., Kunming, China).

The positive transgenic plants and the untransformed wild type plants were then cultivated in the greenhouse until flowering (covered in a nylon net to avoid cross-contamination as above). Pollen viability ratios in the control and transgenic plants were assessed as described above. The methods of statistic analysis and morphological observation of pollen grains were conducted as described above.

Agrobacterium-mediated SlAMS overexpression

Primers 2301-AMSF and 2301-AMSR (Additional file 13) were designed to amplify the SlAMS gene, using clone vector pMDT-18-SlAMS as a template. The SlAMS target gene fragment was ligated to the pCAMBIA2301 vector after digestion with Sac I and Bam HI to construct the overexpression vector pCAMBIA2301-SlAMS (Additional file 11). The recombinant product was transformed into E. coli DH and A. tumefaciens GV3101. After verification of a single colony using PCR and sequencing, the positive clones were fully mixed with 50% glycerin (a glycerin: bacterial culture ratio of 1:1) and stored at − 80 °C.

Agrobacterium-mediated transformation was performed using cotyledons as target infection explants as described above. Three batches of Agrobacterium infection using cotyledons were conducted. The transgenic plants were then cultivated until flowering as described above. The genomic DNA of T0 progeny transformed plants were extracted by CTAB method and then detected transgenic plants via the PCR amplification of the Npt II gene contained in the overexpression vector pCAMBIA2301. Pollen viability and morphology were compared among the positive transgenic plants and the control plants as described above. The methods of statistic analysis and morphological observation of pollen grains were the same as above.

Anther RNA extraction and qRT-PCR analyses of SlAMS

We collected the anthers of SlAMS-silenced, −knockouted, −overexpressed plants and wild type plants during the flowering period at two pollen formation stages (tetrad and maturity). Total RNA was isolated from anthers of two flower buds using Trizol Kits (TransGen Biotech. Co., Ltd., Beijing, China) following the manufacture’s instructions, and the cDNA was synthesized via performing the reverse transcription as described in the cloning of SlAMS gene. For SlAMS gene expression level detection, the specific primers (qAMS-F/qAMS-R) for qRT-PCR were designed based on the cloned SlAMS. We used Actin (GQ337966.1) as the internal control gene for its stable expression level in different plant tissues (Actin-F/Actin-R). The primers used were listed in Additional file 13.

The qRT-PCR was conducted using TB Green® PrimeScript™ RT-PCR Kit (TaKaRa Bio. Co., Beijing, China) following the manufacture protocol on ABI 7500 real-time PCR system (Applied Biosystems, USA). The 10 μL qRT-PCR volume contained 0.2 μL each primer (10 μL mol/μL), 5 μL of 2XTB Green RT-PCR Buffer, 1 μL cDNA (200 ng), 3.6 μL ddH2O. Cycling reaction conditions were 95 °C for 3 min, followed by 40 cycles of 10 s at 95 °C, 15 s at 60 °C, and 20 s at 72 °C. Three biological replicates (at least three plants for each replicate) were performed in qRT-PCR. The relative expression levels of SlAMS were calculated by the 2Ct method. The difference significant analysis was conducted via t test using statistic software SPSS 25.0.

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