The dataset contains all the source data and original raw files for the manuscript: Strain, procedures and tools for reproducible genetic transformation and genome editing of Spirodela polyrhiza (L.) Schleid. Verónica Barragán-Borrero1, Amanda de Santana Lopes2, Enrico Diniz Rodrigues Batista2, Martin Höfer2, Rana Elias1, Abhisek Chakraborty2, Arturo Ponce-Mañe1, Clotilde Descombes1,3, Laura Diezma-Navas1, Lydia Petraki1,4, Meret Huber2**, Shuqing Xu2**and Arturo Marí-Ordóñez1**. 1 Gregor Mendel Institute of Molecular Plant Biology (GMI) of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria. 2 Institute of Organismic and Molecular Evolution (IomE), Johannes Gutenberg University, Mainz Biozentrum I, Hanns-Dieter-Hüsch-Weg 15 D-55128, Mainz. Germany. 3 Current address: Institute of Plant Sciences Paris‐Saclay (IPS2), Centre National de la Recherche Scientifique, Université Paris‐Saclay, Gif Sur Yvette 91190, France. 4 Current address: Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece. ** The authors responsible for correspondence and distribution of materials integral to the findings presented in this article are: Arturo Marí-Ordóñez (arturo.mari-ordonez@gmi.oeaw.ac.at) Shuqing Xu (shuqing.xu@uni-mainz.de) Meret Huber (meret.huber@uni-mainz.de) Original images, protein and RNA blots, qPCR data, Sanger sequencing files, and any other type of source data, are sorted by figure and figure panel. NGS data has been deposited to NCBI, accession numbers of datasets used in each figure are listed accordingly in this document. Contribution of each author to the source data presented here is shown at the end of the document. The content of each file is: FIGURES: FIGURE 1: - 1B: Table with callus induction rate raw measurements of 4 S. polyrhiza clones under four tested protocols. - 1C: Original pictures of S. polyrhiza callus induction. - 1D: Table with callus induction raw rate measurements of S. polyrhiza clones. - 1E: Original callus induction pictures of several S. polyrhiza clones. - 1F-G: Callus growth rates raw measurement table and original pictures of S. polyrhiza SP162 and 9509 clones during regeneration. FIGURE 2: - 2B: Original pictures of S. polyrhiza callus induction at day 0. - 2C: Original pictures of S. polyrhiza callus induction after 4 weeks. - 2D: Original pictures of S. polyrhiza dark compact calli derived from root and pocket after 4 weeks and a callus after 10 weeks. - 2E: Original pictures of S. polyrhiza pale green calli after 12 and 16 weeks. - 2F: Raw calli growth rates measurement table and original side by side picture of S. polyrhiza SP162 dark and pale callus. - 2I: Original picture of SP162 callus after 4 weeks of regeneration. - 2J: Original pictures of SP162 callus acclimatation in magenta and dishes and close view of regenerating calli. - 2K: Original pictures of SP162 regenerating frond. FIGURE 3: - 3A: Table with raw survival rate data of SP162 calli under different selection media. - 3B: Images of SP162 calli under different antibiotic selection strength at start time, one and two weeks including replicates. FIGURE 4: - 4B: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 2-, 7- and 14-days post transformation under white and UV light. - 4C: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 3- and 4-weeks post transformation under white and UV light. - 4D: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 5- and 6-weeks post transformation under white and UV light. - 4E: Original pictures of SP162 callus transformed with pZmUBQ::eGFP at 10-weeks post transformation under white and UV light. - 4F: Raw Southern blot autoradiogram and EtBr gel staining prior to membrane transfer for the detection of pZmUBQ::eGFPT-DNA integration events in SP162 calli. FIGURE 5: - 5A: Original pictures of SP162 callus transformed with pZmUBQ::eGFP after 4-weeks of regeneration under white and UV light. - 5B: Original pictures of SP162 callus transformed with pZmUBQ::eGFP after 6-weeks of regeneration under white and UV light. - 5C: Original pictures of SP162 regenerated fronds transformed with pZmUBQ::eGFP after 12-weeks of regeneration under white and UV light. - 5D: Raw Southern blot autoradiogram and EtBr gel staining prior to membrane transfer for the detection of pZmUBQ::eGFPT-DNA integration events in SP162 regenerated lines. - 5E: Original image and raw biomolecular scanner files of SP162 pZmUBQ::eGFP regenerated fronds. - 5F: Raw qPCR data for eGFP expression in SP162 pZmUBQ::eGFP regenerated lines. - 5G: Western blot and Coomassie staining raw image files for the detection of GFP from SP162 pZmUBQ::eGFP regenerated lines. - 5H: Original pictures of SP162 callus and regenerated fronds transformed with pZmUBQ::RUBY. - 5I: Original pictures of SP162 regenerated fronds transformed with pZmUBQ::DsRed2 under white and UV light. FIGURE 6: - 6B: SpZMET gDNA, intron-exon annotated gDNA, protein, and protein domains annotated sequences in fasta(.fas) and genebank (.gbk) formats. - 6C: Original pictures of non-transgenic SP162 callus and transformed with pZmUBQ::eGFP or pZmUBQ::zCas9i:P2A:eGFP under white and UV light. - 6D: Western blot and Coomassie staining raw image files for the detection of Cas9 and GFP from SP162 pZmUBQ::eGFPand pZmUBQ::zCas9i:P2A:eGFP calli. - 6E: Original image and raw biomolecular scanner files of SP162 pZmUBQ::eGFP regenerated lines. - 6F: Western blot and Coomassie staining raw image files for the detection of Cas9 and GFP from SP162 pZmUBQ::zCas9i:P2A:eGFP regenerated fronds. - 6G: Original image of EtBr staining of agarose gel electrophoresis of SpZMET locus PCR in SP162 and pZmUBQ::zCas9i:P2A:eGFP regenerated fronds. - 6H/J: See Figure S8 source files. FIGURE 7: - 7A: Original raw biomolecular scanner files of SP162 infiltrated with pZmUBQ::eGFP. Also used for Supplemental Figure S12. - 7B: Original pictures of SP162 infiltrated without or with pZmUBQ::eGFP Agrobacterium 21 d.p.i. - 7C: Original scanner images of bacteria titration plates and table with estimated cfu/frond extracted from individual fronds 21dpi. - 7D: Raw qPCR data and statiscal analysis report for eGFP expression in SP162 after infiltration of pZmUBQ::eGFP. - 7E: Western blot and Coomassie staining raw image files for the detection of GFP protein from SP162 infiltrated with pZmUBQ::eGFP. - 7H: Original pictures of SP162 infiltrated with pZmUBQ::eGFP or pUBQ10::sXVE::eGFP under white and UV light with or without estradiol. - 7H: Northern blots (autoradiography) raw image files for the detection of GFP- derived, SpTAS3 and snoRNA U6 + miR159controls in SP162 infiltrated with pZmUBQ::eGFP or pZmUBQ::FPPFhp. SUPPLEMENTAL FIGURE S1: - S1A: Original pictures of S. polyrhiza SP162 fronds grown in a beaker in SH-media and N-media at different times (weeks) of turion induction. - S1B: Original pictures of S. polyrhiza SP162 developing turions in fronds grown in N-media and released turions. - S1C: Original pictures of a turion induction culture in N-media before turion harvest. - S1D: Original pictures of turions in storage. - S1G: Original pictures of germinating turions. - S1E-F: Table with turion germination rates after different times of stratification. SUPPLEMENTAL FIGURE S2: - Original pictures of S. polyrhiza SP162 fronds after 1, 2, 3 or 4 weeks in callus induction media. SUPPLEMENTAL FIGURE S3: - S3A: Original pictures of S. polyrhiza SP162 calli after 1 week in frond regeneration media containing TDZ or Zeatin. - S3B: Original pictures of S. polyrhiza SP162 calli after 2 weeks in frond regeneration media containing TDZ or Zeatin. SUPPLEMENTAL FIGURE S4: - S4A: Original pictures of S. polyrhiza SP162 calli after 6 weeks of regeneration initiated with TDZ or Zeatin. - S4B: Original pictures of S. polyrhiza SP162 calli after 12 weeks of regeneration initiated with TDZ or Zeatin. SUPPLEMENTAL FIGURE S5: - For source data see Figure 3B SUPPLEMENTAL FIGURE S6: - S6A: Original pictures of S. polyrhiza SP162 regenerated non transgenic fronds and expressing GFP or RUBY at the Mari-Ordonez Lab. - S6B: Original pictures of S. polyrhiza SP162 calli and regenerated transgenic fronds expressing dsRED or GFP selected with Hygromycin or Spectomycin at the Hubert Lab. SUPPLEMENTAL FIGURE S8: - S8A-E: Raw sanger sequencing trace data ab1 files for analysis of CRISPR/Cas9 editing results in Figure S6 and Figures 6H and 6J. SUPPLEMENTAL FIGURE S9: - S9A: Original pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing GFP or RUBY. - S9B: Source images, frond length measurement tables and statistical analysis results of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing GFP or RUBY (the images correspond to day 8 of the growth curve perform in Figure S8). SUPPLEMENTAL FIGURE S10: - S10A: Original and processed pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated lines expressing GFP or RUBY for Ilastik pixel and object classification of live and senescent frond tissue areas during growth time-course. Ilastik pixel classification probabilities and object classification output images for each picture are also contained in the folder. - S10B-D: Data summary tables of total, relative and senescent areas in pixel and cm2 obtained from Ilastik analysis. - S10F: Summary report of curve fitting for relative surface area fold-change (Figure S8C) data. SUPPLEMENTAL FIGURE S11: - S11A: Original pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing pZmUBQ::zCas9i:P2A:eGFP_4X_sgRNA@ZMET. - S11B: Source images, frond length measurement tables and statistical analysis results of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated fronds expressing pZmUBQ::zCas9i:P2A:eGFP_4X_sgRNA@ZMET (the images correspond to day 7 of the growth curve perform in Figure S9C-F). - S11C: Original and processed pictures of S. polyrhiza SP162 WT, regenerated non transgenic, and independently regenerated lines expressing pZmUBQ::zCas9i:P2A:eGFP_4X_sgRNA@ZMET for Ilastik pixel and object classification of live and senescent frond tissue areas during growth time-course. Ilastik pixel classification probabilities and object classification output images for each picture are also contained in the folder. - S11D-F: Data summary tables of total, relative and senescent areas in pixel and cm2 obtained from Ilastik analysis. SUPPLEMENTAL FIGURE S12: - S12A: FACS report and raw data files for 1C estimation of regenerated fronds. - S12B: Table with estimation of the genome size (1C) of regenerated fronds based on FACS data. SUPPLEMENTAL FIGURE S13: - See Figure 7A source data. SUPPLEMENTAL FIGURE S14: - Original images of transiently expressed GFP at 3, 6 and 10 dpi. SUPPLEMENTAL FIGURE S15: - S15A: Original images of estradiol-inducible GFP in calli. - S15B: Original images of estradiol-inducible GFP in regenerated fronds. SUPPLEMENTAL FIGURE S16: - S16D: Arabidopsis and Spirodela miR390 sequences. - S16E: SpTAS3 sequence in fasta format between both miR390 target sites. NGS DATASETS: All the NGS data generated for this study can be found under the bio-project number PRJNA1308930. The genome assembly and annotation have been deposited on Figshare: https://figshare.com/articles/dataset/Genome_assembly_and_annotation_of_i_Spirodela_polyrhiza_i_strain_SP162_ID_5676_/29942036. The data was used to generate the following figure panels: - Supplemental Figure S7. Additionally, publicly available small RNA sequencing data from SRA BioProject ID PRJNA1164696 was used to generate Supplementary Figure S16E. AUTHOR DATA AND FIGURE CONTRIBUTION: FIGURE PANEL DATA/ANALYSIS CONTRIBUTED BY 1 1B MHO 1C MHO, RE 1D MHO, RE 1E MHO 1F MHO, RE 1G VBB, RE 2 2B RE 2C RE 2D RE, VBB 2E RE, VBB 2F RE, VBB 2I VBB 2J VBB 2K VBB 3 3A ERB 3B ASL 4 4B VBB 4C VBB 4D VBB 4E VBB 4F VBB 5 5A VBB 5B VBB 5C VBB 5D VBB 5E VBB, AMO 5F VBB 5G VBB 5H VBB 5I ERB 6 6A VBB, CD 6B CD, AMO 6C CD, VBB 6D VBB 6E VBB, AMO 6F VBB 6G VBB 6H VBB, AMO 6J VBB, AMO 7 7A APM 7B APM 7C APM, AMO 7D APM 7E APM 7F ERB 7H APM S1 S1A LP, LD S1B LP, LD S1C LP, LD S1D LP, LD S1E LP, LD S1E LP, LD S1F LP, LD S2 S2 RE S3 S3A VBB S3B VBB S4 S4A VBB S4B VBB S5 S5 ASL S6 S6A VBB, AMO S6B ERB, ASL S7 S7 AC, SX S8 S8A-D VBB, AMO S9 S9A VBB, AMO S9B VBB, AMO S10 S10A VBB, AMO S10B AMO S10C AMO S10D AMO S10F AMO S11 S11A VBB, AMO S11B AMO S11C VBB, AMO S11D AMO S11E AMO S11F AMO S12 S12A RE S12C RE S13 S13 APM S14 S14 ASL S15 S15 ASL S16 S16 AMO