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Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.\" } { \"@context\": \"http://schema.org\", \"@type\": \"BreadcrumbList\", \"itemListElement\": [ { \"@type\": \"ListItem\", \"position\": \"1\", \"item\": { \"@id\": \"https://f1000research.com/\", \"name\": \"Home\" } }, { \"@type\": \"ListItem\", \"position\": \"2\", \"item\": { \"@id\": \"https://f1000research.com/browse/articles\", \"name\": \"Browse\" } }, { \"@type\": \"ListItem\", \"position\": \"3\", \"item\": { \"@id\": \"https://f1000research.com/articles/9-356\", \"name\": \"High transformation efficiency in Arabidopsis using extremely low...\" } } ] } Home Browse High transformation efficiency in Arabidopsis using extremely low... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Wang Y, Yaghmaiean H and Zhang Y. High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.12688/f1000research.23449.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report Revised High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] Yiran Wang 1 , Hoda Yaghmaiean 1 , Yuelin Zhang https://orcid.org/0000-0002-3480-5478 1 Yiran Wang 1 , Hoda Yaghmaiean 1 , Yuelin Zhang https://orcid.org/0000-0002-3480-5478 1 PUBLISHED 16 Sep 2020 Author details Author details 1 Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada Yiran Wang Roles: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation Hoda Yaghmaiean Roles: Conceptualization, Investigation, Methodology Yuelin Zhang Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS Abstract Agrobacterium -mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600 ≈2.0) in large cultures and resuspended in inoculation medium to OD 600 ≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600 =0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel. READ ALL READ LESS Keywords Arabidopsis, floral dip, transformation Corresponding Author(s) Yuelin Zhang ( [email protected] ) Close Corresponding author: Yuelin Zhang Competing interests: No competing interests were disclosed. Grant information: This work was supported by the Natural Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2020 Wang Y et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Wang Y, Yaghmaiean H and Zhang Y. High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.12688/f1000research.23449.2 ) First published: 13 May 2020, 9 :356 ( https://doi.org/10.12688/f1000research.23449.1 ) Latest published: 16 Sep 2020, 9 :356 ( https://doi.org/10.12688/f1000research.23449.2 ) Revised Amendments from Version 1 The updated version of this manuscript includes additional references on floral dip transformation in the introduction section, clarification of data in Figure 1 in the legend, and addition of the OD 600 value of the Agrobacterium cell density at the time of harvest in the Method section. The updated version of this manuscript includes additional references on floral dip transformation in the introduction section, clarification of data in Figure 1 in the legend, and addition of the OD 600 value of the Agrobacterium cell density at the time of harvest in the Method section. See the authors' detailed response to the review by Zhanyuan J Zhang See the authors' detailed response to the review by Ying Wang and Shelley Hepworth READ REVIEWER RESPONSES Introduction Plant transformation integrates foreign genes into the plant nuclear genome. The development of different transformation protocols in various plants has enabled advances in plant molecular biology and crop improvements ( Saifi et al ., 2020 ). Agrobacterium is routinely used as a plant gene transformation vehicle as it naturally possesses the ability to transfer a segment of its plasmid DNA (T-DNA) into its host nucleus, which ultimately leads to integration of the T-DNA into the nuclear genome ( Tzfira et al ., 2004 ). During 1980s and early 1990s, generating transgenic plants by leaf disc-based Agrobacterium -mediated transformation required laborious plant tissue culture and regeneration steps. In 1993, a simple floral vacuum infiltration method was developed in Arabidopsis for stable transformation, overcoming the tedious tissue culture requirements ( Bechtold et al ., 1993 ). Later, the vacuum infiltration step was replaced by floral dipping where the developing floral tissues are dipped into a solution containing Agrobacterium , sucrose and the surfactant Silwet L-77 ( Clough & Bent, 1998 ). Because of the simplicity and reliability of this floral dip method, it is now the commonly used transformation method in Arabidopsis. This protocol has also been shown to work in certain Brassicaceae plants ( Bent, 2006 ). Floral dip transformation may be feasible in plants such as wheat, maize, tomato, flax, Medicago truncatula and Setaria viridis ( Agarwal et al ., 2009 ; Bastaki & Cullis, 2014 ; Martins et al ., 2015 ; Mu et al ., 2012 ; Trieu et al ., 2000 ; Yasmeen et al ., 2009 ). In Agrobacterium -mediated transformation protocols, the concentration of bacterial inoculum has been considered crucial to the success of plant transformation. In the commonly used floral dip protocol, bacterial cells are grown to stationary phase (OD 600 =2.0), pelleted and resuspended in inoculation medium to OD 600 ≥0.8 ( Clough & Bent, 1998 ; Zhang et al ., 2006 ). Here, we tested whether a low concentration of Agrobacterium inoculum affects the plant transformation rate. Our data showed that, contrary to our expectation, using an extremely low density of Agrobacterium inoculum (OD 600 =0.002) in the floral dip method still warrants relatively high transformation rate in Arabidopsis. Method Plant materials and growth conditions Arabidopsis Col-0 wild type plants were grown in a growth room under long day (16 h light/8 h dark cycle) at 23°C. Seedlings were grown at a density of 30–40 per 64 cm 2 (8 cm × 8 cm) pot in moistened potting soil initially and transplanted to 64 cm 2 pots with eight plants per pot when they were two weeks old. After plants bolted and floral buds are formed (~30-day-old), they were used in floral dip transformation. Culture of Agrobacterium tumefaciens The plasmid pCambia1305-3flag-NOS was transformed into Agrobacterium tumefaciens strain GV3101 ( Van Larebeke et al ., 1974 ) by mixing the plasmid DNA with the bacterial cells in a 1mm gap cuvette (BTX, #45-0124) followed by electroporation for 5 millisecond at 1,500 volts using the ECM 399 Electroporation System (BTX, #45-0000) ( Gao et al ., 2009 ). The resulting strain was used in the plant transformation experiments. Bacteria were grown overnight in sterilized 4 ml LB media (Bio Basic Inc., #SD7002) with kanamycin, gentamicin and rifampicin antibiotics (50 μg/ml each, Bio Basic Inc. #KB0286, #GB0217, #RB0808) in a 28°C shaker (New Brunswick Scientific Co G25 Controlled Environment Incubator Shaker). Then the overnight culture was diluted into 100 ml LB media with kanamycin (50 μg/ml) and allowed to grow further for 8 h (OD 600 =1.5~1.8) in the same shaker. The bacteria were collected by centrifugation (Thermo Scientific, Sorvall Legend X1R) at 6000 g for 10 min at room temperature and then resuspended in 100 ml floral dip medium to final OD 600 of 1, 0.1, 0.01, and 0.002 (measured by BioSpec-1601 UV-visible spectrophotometer from SHIMADZU) prior to use. The floral dip medium contained 5.0% (w/v) sucrose (Bio Basic Inc. #SB0498) and 0.01% (v/v) Silwet L-77 (PhytoTechnology Laboratories #S7777) in distilled water. Floral dip transformation For floral dip, pots were tilted and floral buds were submerged in bacterial suspension with 30 sec of gentle agitation. The dipped plants were then covered with a tall clear-plastic dome to maintain humidity. Plants were placed in a dark room overnight before being moved back to the growth room. The domes were removed approximately 48 h after the floral dip treatment. Plants were grown for another 30–32 days until siliques became brown and dry. Each pot with 8 plants were transformed separately. For each concentration of Agrobacterium inocula, 4–6 pots of plants were transformed depending on the number of plants available for transformation in each experiment, this varied mainly due to the uneven germination of the seeds in each experiment. About 6000 seeds were bulk harvested from the plants grown in a pot. Seeds were harvested by gentle stripping of dried inflorescences by fingers above a piece of clean paper. The debris from the stem and pods was removed from the seeds by gentle blowing. Seeds were kept in a 37°C incubator for two days for desiccation. Selection of transformants Prior to selection, seeds were surface sterilized with 20% (v/v) bleach (Clorox Regular Bleach) containing 0.1% (v/v) Tween20 (Sigma-Aldrich #P1379) for 1min, followed by three times rinse with sterile water. The sterilized seeds were suspended in 0.1% (w/v) sterile agar (Bio Basic Inc. #FB0010) and plated on hygromycin selection plates (1/2 MS medium, Murashige & Skoog Basal Medium with Vitamins from PhytoTechnology Laboratories #M519 and 50 μg/ml hygromycin, Bio Basic Inc. #HD0230) at a density of approximately 3000 seeds (0.06 gram by weight) per 92×12mm (diameter×height) petri plate (Sarstedt #82.1473.001). Seeds collected from each pot (4–6 pots for each concentration of Agrobacterium inocula) were plated on a separate selection plate. Plates were placed in 4°C refrigerator for two days before moved to a plant growth chamber (16 h light/8 h dark cycle, Conviron Model A1000). The plants were grown at 23°C for 10 days before transformants were identified as hygromycin-resistant seedlings that produced green leaves and well-established roots grown on the selective medium. The experiment was repeated three times by transforming independently grown plants with different concentrations of Agrobacterium inocula. Statistical analysis Analysis of statistical differences between transformation rates from different concentrations of Agrobacterium inocula was performed by one-way ANOVA using Microsoft® Office Excel version 16.35 (20030802). Results and discussion Four Agrobacterium inocula from high to low concentrations (OD 600 =1, 0.1, 0.01, 0.002) were used in floral dip transformation to test the effect of bacterial concentration on the transformation rate. As shown in Figure 1 (Underlying data ( Wang, 2020 )), similar transformation rate (approximately 0.60%) was observed under all tested bacterial concentration. Notably, the transformation efficiency remains unchanged even though the Agrobacterium inoculum was diluted 500 times form OD 600 = 1 to OD 600 = 0.002. Therefore, it is feasible to dramatically reduce the Agrobacterium inoculum concentration in the floral dip method. Regardless of the inoculum concentration, transforming eight Arabidopsis plants grown in a single pot produced about 36 T1 transgenic lines on average, which is sufficient for most studies. Figure 1. The effect of Agrobacterium concentration on transformation rate in floral dip method. Transformation rates were calculated as [(# of hygromycin-resistant seedlings) / (total # seedlings tested)] × 100%. The data are shown as mean ± SE from six independent repeats in one representative experiment. The same letters denote no statistically significant difference according to one-way ANOVA (p<0.05). Standard floral dip protocols use high concentrations of Agrobacterium inoculum, which requires growing large bacterial cultures ( Clough & Bent, 1998 ; Zhang et al ., 2006 ). Our study showed that Agrobacterium inoculum can be diluted to as low as OD 600 = 0.002 without sacrificing the transformation efficiency. Thus, the volume of bacterial culture used in each transformation experiment could be greatly reduced. For example, diluting 0.1 ml of overnight culture (OD 600 = 2) to OD 600 = 0.002 gives ~100 ml bacterial inoculum, which is sufficient in most transformation experiments. Such improvement allows researchers to culture small volume of a large number of Agrobacterium strains in parallel and use the diluted cultures to carry out high-throughput transformation of a large number of different constructs into Arabidopsis plants. Data availability Open Science Framework: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum project. https://doi.org/10.17605/OSF.IO/YF6AE ( Wang, 2020 ) This project contains the following underlying data: Transformation efficiency.xlsx (raw data of results from transformation using different Agrobacterium concentrations) Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Faculty Opinions recommended References Agarwal S, Loar S, Steber C, et al. : Floral transformation of wheat. Methods Mol Biol. Humana Press. 2009; 478 : 105–113. PubMed Abstract | Publisher Full Text Bastaki NK, Cullis CA: Floral-dip transformation of flax ( Linum usitatissimum ) to generate transgenic progenies with a high transformation rate. J Vis Exp. 2014; (94): 52189. PubMed Abstract | Publisher Full Text | Free Full Text Bechtold N, Ellis J, Pelletier G: In-planta Agrobacterium -mediated gene transfer by infiltration of adult Arabidopsis-thaliana plants. Comptes Rendus De l Academie Des Sciences Serie Iii-Sciences De La Vie-Life Sciences. 1993; 316 (10): 1194–1199. Reference Source Bent A: Arabidopsis thaliana floral dip transformation method. Methods Mol Biol. Humana Press. 2006; 343 : 87–103. PubMed Abstract | Publisher Full Text Clough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana . Plant J. 1998; 16 (6): 735–743. PubMed Abstract | Publisher Full Text Gao M, Wang X, Wang D, et al. : Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis . Cell Host Microbe. 2009; 6 (1): 34–44. PubMed Abstract | Publisher Full Text Martins PK, Nakayama TJ, Ribeiro AP, et al. : Setaria viridis floral-dip: a simple and rapid Agrobacterium -mediated transformation method. Biotechnol Rep (Amst). 2015; 6 : 61–63. PubMed Abstract | Publisher Full Text | Free Full Text Mu G, Chang N, Xiang K, et al. : Genetic transformation of maize female inflorescence following floral dip method mediated by Agrobacterium. Biotechnology. 2012; 11 (3): 178–183. Publisher Full Text Saifi SK, Passricha N, Tuteja R, et al. : In planta transformation: A smart way of crop improvement. Advancement in Crop Improvement Techniques. Duxford: Woodhead Publishing. 2020; 351–362. Publisher Full Text Trieu AT, Burleigh SH, Kardailsky IV, et al. : Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium . Plant J. 2000; 22 (6): 531–541. PubMed Abstract | Publisher Full Text Tzfira T, Li J, Lacroix B, et al. : Agrobacterium T-DNA integration: molecules and models. Trends Genet. 2004; 20 (8): 375–383. PubMed Abstract | Publisher Full Text Van Larebeke N, Engler G, Holsters M, et al. : Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 1974; 252 (5479): 169–170. PubMed Abstract | Publisher Full Text Wang Y: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum. 2020. http://www.doi.org/10.17605/OSF.IO/YF6AE Yasmeen A, Mirza B, Inayatullah S, et al. : In planta transformation of tomato. Plant Mol Biol Report. 2009; 27 (1): 20–28. Publisher Full Text Zhang X, Henriques R, Lin SS, et al. : Agrobacterium -mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006; 1 (2): 641–6. PubMed Abstract | Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 13 May 2020 ADD YOUR COMMENT Comment Author details Author details 1 Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada Yiran Wang Roles: Data Curation, Formal Analysis, Investigation, Writing – Original Draft Preparation Hoda Yaghmaiean Roles: Conceptualization, Investigation, Methodology Yuelin Zhang Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This work was supported by the Natural Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (2) version 2 Revised Published: 16 Sep 2020, 9:356 https://doi.org/10.12688/f1000research.23449.2 version 1 Published: 13 May 2020, 9:356 https://doi.org/10.12688/f1000research.23449.1 Copyright © 2020 Wang Y et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Wang Y, Yaghmaiean H and Zhang Y. High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.12688/f1000research.23449.2 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 13 May 2020 Views 0 Cite How to cite this report: Zhang ZJ. Reviewer Report For: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.5256/f1000research.25880.r63860 ) The direct URL for this report is: https://f1000research.com/articles/9-356/v1#referee-response-63860 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 27 May 2020 Zhanyuan J Zhang , Plant Biotechnology Innovation Laboratory, Division of Plant Sciences, University of Missouri, Columbia, MO, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.25880.r63860 The work showed an interesting result that is contrary to the common practice in Arabidopsis floral dip transformation. The authors demonstrated that the use of an unusually low concentration of Agrobacterium inoculum (OD600=0.002) could achieve the same transformation rate as ... Continue reading READ ALL The work showed an interesting result that is contrary to the common practice in Arabidopsis floral dip transformation. The authors demonstrated that the use of an unusually low concentration of Agrobacterium inoculum (OD600=0.002) could achieve the same transformation rate as a much higher concentration (OD600=1) does. They also discover a practical implication by using a small amount of Agrobacterium culture. The manuscript is well-written and results are convincing with a sound conclusion. The statistical method was appropriate as well. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium . If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. \" Agrobacteria \" should be \" Agrobacterium \" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. The other reviewer has pointed out all the written and grammatical issues that I agree with, so no need for me to raise them again. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Plant tissue culture and transformation and plant molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Zhang ZJ. Reviewer Report For: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.5256/f1000research.25880.r63860 ) The direct URL for this report is: https://f1000research.com/articles/9-356/v1#referee-response-63860 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Sep 2020 Yuelin Zhang , Department of Botany, University of British Columbia, Vancouver, V6T 1Z4, Canada 10 Sep 2020 Author Response We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor ... Continue reading We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. This is an excellent point. The OD600 value of the Agrobacterium cell density at the time of harvest is now added to the Method section. They were between 1.5-1.8, which was before reaching the stationary phase. \"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. Corrected as suggested. We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. This is an excellent point. The OD600 value of the Agrobacterium cell density at the time of harvest is now added to the Method section. They were between 1.5-1.8, which was before reaching the stationary phase. \"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. Corrected as suggested. Competing Interests: No competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Sep 2020 Yuelin Zhang , Department of Botany, University of British Columbia, Vancouver, V6T 1Z4, Canada 10 Sep 2020 Author Response We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor ... Continue reading We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. This is an excellent point. The OD600 value of the Agrobacterium cell density at the time of harvest is now added to the Method section. They were between 1.5-1.8, which was before reaching the stationary phase. \"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. Corrected as suggested. We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. This is an excellent point. The OD600 value of the Agrobacterium cell density at the time of harvest is now added to the Method section. They were between 1.5-1.8, which was before reaching the stationary phase. \"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. Corrected as suggested. Competing Interests: No competing interests. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Wang Y and Hepworth S. Reviewer Report For: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.5256/f1000research.25880.r63373 ) The direct URL for this report is: https://f1000research.com/articles/9-356/v1#referee-response-63373 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 26 May 2020 Ying Wang , Department of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada Shelley Hepworth , Department of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada Approved VIEWS 0 https://doi.org/10.5256/f1000research.25880.r63373 The floral dip method for the transformation of Arabidopsis plants has been a revolutionary tool in plant biology. Several refinements to the popular protocol of Clough and Bent (1998) have been published. This report shows that Agrobacterium ... Continue reading READ ALL The floral dip method for the transformation of Arabidopsis plants has been a revolutionary tool in plant biology. Several refinements to the popular protocol of Clough and Bent (1998) have been published. This report shows that Agrobacterium cultures diluted to as low as OD600=0.002 yielded a transformation rate similar to infection with regular-density suspensions (OD600=1). Down-scaling in this way can save time, space, and expense -- especially important for high-throughput transformation experiments. The article is well-written and the data convincing. Other steps of the transformation process such as plant preparation and downstream selection remain labor intensive, so follow-up experiments are important. The Arabidopsis Col-0 ecotype used in this study is relatively easy to transform. It will be interesting to see if low Agro concentrations are equally suitable for other ecotypes / mutant genetic backgrounds or if the method can be combined with other efficiencies like making the Agrobacterium solution directly from plates to further avoid culturing and sub-culturing steps. Minor comments: Please see attached PDF for small corrections in grammar. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Unable to access the underlying raw dataset - check the file is attached to the link. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Plant development, Arabidopsis thaliana, molecular genetics, flowering, meristems, transcription factors, gene expression, transgenic plants We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Wang Y and Hepworth S. Reviewer Report For: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.5256/f1000research.25880.r63373 ) The direct URL for this report is: https://f1000research.com/articles/9-356/v1#referee-response-63373 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Sep 2020 Yuelin Zhang , Department of Botany, University of British Columbia, Vancouver, V6T 1Z4, Canada 10 Sep 2020 Author Response We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please ... Continue reading We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please see attached PDF for small corrections in grammar. Thanks a lot for your suggestion. We have made the suggested corrections. 2. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). References for floral dip transformation in species other than Arabidopsis have been added to the revised manuscript as suggested. 3. Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Figure 1 is one representative experiment of six pots of plants. This is clarified in the revised legend for figure 1. 4. Unable to access the underlying raw dataset - check the file is attached to the link. We checked the access to the raw dataset, there is no problem with the link. The data is under “Archive of OSF Storage” inside the “File” sign shown on the left side of the page. To access the raw data: Click the link provided in the paper, then click \"file”on the left side of the page, select “Archive of OSF Storage\" We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please see attached PDF for small corrections in grammar. Thanks a lot for your suggestion. We have made the suggested corrections. 2. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). References for floral dip transformation in species other than Arabidopsis have been added to the revised manuscript as suggested. 3. Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Figure 1 is one representative experiment of six pots of plants. This is clarified in the revised legend for figure 1. 4. Unable to access the underlying raw dataset - check the file is attached to the link. We checked the access to the raw dataset, there is no problem with the link. The data is under “Archive of OSF Storage” inside the “File” sign shown on the left side of the page. To access the raw data: Click the link provided in the paper, then click \"file”on the left side of the page, select “Archive of OSF Storage\" Competing Interests: No competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Sep 2020 Yuelin Zhang , Department of Botany, University of British Columbia, Vancouver, V6T 1Z4, Canada 10 Sep 2020 Author Response We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please ... Continue reading We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please see attached PDF for small corrections in grammar. Thanks a lot for your suggestion. We have made the suggested corrections. 2. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). References for floral dip transformation in species other than Arabidopsis have been added to the revised manuscript as suggested. 3. Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Figure 1 is one representative experiment of six pots of plants. This is clarified in the revised legend for figure 1. 4. Unable to access the underlying raw dataset - check the file is attached to the link. We checked the access to the raw dataset, there is no problem with the link. The data is under “Archive of OSF Storage” inside the “File” sign shown on the left side of the page. To access the raw data: Click the link provided in the paper, then click \"file”on the left side of the page, select “Archive of OSF Storage\" We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please see attached PDF for small corrections in grammar. Thanks a lot for your suggestion. We have made the suggested corrections. 2. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). References for floral dip transformation in species other than Arabidopsis have been added to the revised manuscript as suggested. 3. Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Figure 1 is one representative experiment of six pots of plants. This is clarified in the revised legend for figure 1. 4. Unable to access the underlying raw dataset - check the file is attached to the link. We checked the access to the raw dataset, there is no problem with the link. The data is under “Archive of OSF Storage” inside the “File” sign shown on the left side of the page. To access the raw data: Click the link provided in the paper, then click \"file”on the left side of the page, select “Archive of OSF Storage\" Competing Interests: No competing interests. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 13 May 2020 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 2 (revision) 16 Sep 20 Version 1 13 May 20 read read Ying Wang , Carleton University, Ottawa, Canada Shelley Hepworth , Carleton University, Ottawa, Canada Zhanyuan J Zhang , University of Missouri, Columbia, USA Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2020 Zhang Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 27 May 2020 | for Version 1 Zhanyuan J Zhang , Plant Biotechnology Innovation Laboratory, Division of Plant Sciences, University of Missouri, Columbia, MO, USA 0 Views copyright © 2020 Zhang Z. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The work showed an interesting result that is contrary to the common practice in Arabidopsis floral dip transformation. The authors demonstrated that the use of an unusually low concentration of Agrobacterium inoculum (OD600=0.002) could achieve the same transformation rate as a much higher concentration (OD600=1) does. They also discover a practical implication by using a small amount of Agrobacterium culture. The manuscript is well-written and results are convincing with a sound conclusion. The statistical method was appropriate as well. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium . If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. \" Agrobacteria \" should be \" Agrobacterium \" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. The other reviewer has pointed out all the written and grammatical issues that I agree with, so no need for me to raise them again. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Plant tissue culture and transformation and plant molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 10 Sep 2020 Yuelin Zhang, Department of Botany, University of British Columbia, Vancouver, V6T 1Z4, Canada We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor revisions will be needed, though: Please indicate the Agrobacterium cell density (OD600 value (values)) at the time of harvest before the Agrobacterium cells were resuspended to OD600=0.001-1.0. This is an important growth parameter for Agrobacterium. If other users would harvest Agrobacterium cells at a too early stage, say, well before the log growth phase, with the same cell density of OD600=0.002, they may not obtain the same transformation rate. In other words, the description of the Agrobacterium growth phase (lag, log, stationary, etc.) to be harvested will be important. This is an excellent point. The OD600 value of the Agrobacterium cell density at the time of harvest is now added to the Method section. They were between 1.5-1.8, which was before reaching the stationary phase. \"Agrobacteria\" should be \"Agrobacterium\" when it is used as an adjective such as Agrobacterium concentration. Please make corrections throughout the paper. Corrected as suggested. View more View less Competing Interests No competing interests. reply Respond Report a concern Zhang ZJ. Peer Review Report For: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.5256/f1000research.25880.r63860) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/9-356/v1#referee-response-63860 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2020 Hepworth S et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 26 May 2020 | for Version 1 Ying Wang , Department of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada Shelley Hepworth , Department of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada 0 Views copyright © 2020 Hepworth S et al. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The floral dip method for the transformation of Arabidopsis plants has been a revolutionary tool in plant biology. Several refinements to the popular protocol of Clough and Bent (1998) have been published. This report shows that Agrobacterium cultures diluted to as low as OD600=0.002 yielded a transformation rate similar to infection with regular-density suspensions (OD600=1). Down-scaling in this way can save time, space, and expense -- especially important for high-throughput transformation experiments. The article is well-written and the data convincing. Other steps of the transformation process such as plant preparation and downstream selection remain labor intensive, so follow-up experiments are important. The Arabidopsis Col-0 ecotype used in this study is relatively easy to transform. It will be interesting to see if low Agro concentrations are equally suitable for other ecotypes / mutant genetic backgrounds or if the method can be combined with other efficiencies like making the Agrobacterium solution directly from plates to further avoid culturing and sub-culturing steps. Minor comments: Please see attached PDF for small corrections in grammar. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Unable to access the underlying raw dataset - check the file is attached to the link. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Plant development, Arabidopsis thaliana, molecular genetics, flowering, meristems, transcription factors, gene expression, transgenic plants We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 10 Sep 2020 Yuelin Zhang, Department of Botany, University of British Columbia, Vancouver, V6T 1Z4, Canada We sincerely appreciate the support and constructive reviews from the reviewers. We have revised our manuscript according to the comments. Our point-to-point responses to comments are listed below. Minor comments: 1. Please see attached PDF for small corrections in grammar. Thanks a lot for your suggestion. We have made the suggested corrections. 2. Floral dip transformation is feasible in quite a few other species besides the ones listed (e.g. Bastaki and Cullis, 2014, references therein). References for floral dip transformation in species other than Arabidopsis have been added to the revised manuscript as suggested. 3. Clarify the figure legend of Figure 1. Was this one representative experiment of six pots of plants? Or an average of the three independent trials mentioned in the materials and methods. Figure 1 is one representative experiment of six pots of plants. This is clarified in the revised legend for figure 1. 4. Unable to access the underlying raw dataset - check the file is attached to the link. We checked the access to the raw dataset, there is no problem with the link. The data is under “Archive of OSF Storage” inside the “File” sign shown on the left side of the page. To access the raw data: Click the link provided in the paper, then click \"file”on the left side of the page, select “Archive of OSF Storage\" View more View less Competing Interests No competing interests. reply Respond Report a concern Wang Y and Hepworth S. Peer Review Report For: High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum [version 2; peer review: 1 approved, 1 approved with reservations] . F1000Research 2020, 9 :356 ( https://doi.org/10.5256/f1000research.25880.r63373) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/9-356/v1#referee-response-63373 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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