F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.23449.2 Brief Report Articles High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

[version 2; peer review: 1 approved, 1 approved with reservations]

 Wang Yiran Data Curation Formal Analysis Investigation Writing – Original Draft Preparation 1 Yaghmaiean Hoda Conceptualization Investigation Methodology 1 Zhang Yuelin Conceptualization Funding Acquisition Methodology Project Administration Supervision Writing – Review & Editing https://orcid.org/0000-0002-3480-5478 a 1 Department of Botany, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada yuelin.zhang@ubc.ca

No competing interests were disclosed.

 16 9 2020 2020 9 356 8 9 2020 Copyright: © 2020 Wang Y et al. 2020 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 OD600≥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.

 Arabidopsis floral dip transformation Natural Sciences and Engineering Research Council of Canada 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. 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.

 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 <italic toggle="yes">Agrobacterium tumefaciens</italic>

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.

The effect of <italic toggle="yes">Agrobacterium</italic> 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).

 Agarwal S Loar S Steber C : Floral transformation of wheat. Methods Mol Biol. Humana Press.2009;478:105–113. 19009441 10.1007/978-1-59745-379-0_6 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. 25549243 10.3791/52189 4396936 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. 16988336 10.1385/1-59745-130-4:87 Clough SJ Bent AF : Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16(6):735–743. 10069079 10.1046/j.1365-313x.1998.00343.x Gao M Wang X Wang D : Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe. 2009;6(1):34–44. 19616764 10.1016/j.chom.2009.05.019 Martins PK Nakayama TJ Ribeiro AP : Setaria viridis floral-dip: a simple and rapid Agrobacterium-mediated transformation method. Biotechnol Rep (Amst). 2015;6:61–63. 28435809 10.1016/j.btre.2015.02.006 5374283 Mu G Chang N Xiang K : Genetic transformation of maize female inflorescence following floral dip method mediated by Agrobacterium. Biotechnology. 2012;11(3):178–183. 10.3923/biotech.2012.178.183 Saifi SK Passricha N Tuteja R : In planta transformation: A smart way of crop improvement. Advancement in Crop Improvement Techniques. Duxford: Woodhead Publishing.2020;351–362. 10.1016/B978-0-12-818581-0.00021-8 Trieu AT Burleigh SH Kardailsky IV : Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J. 2000;22(6):531–541. 10886773 10.1046/j.1365-313x.2000.00757.x Tzfira T Li J Lacroix B : Agrobacterium T-DNA integration: molecules and models. Trends Genet. 2004;20(8):375–383. 15262410 10.1016/j.tig.2004.06.004 Van Larebeke N Engler G Holsters M : Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 1974;252(5479):169–170. 4419109 10.1038/252169a0 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 : In planta transformation of tomato. Plant Mol Biol Report. 2009;27(1):20–28. 10.1007/s11105-008-0044-5 Zhang X Henriques R Lin SS : Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2006;1(2):641–6. 17406292 10.1038/nprot.2006.97 10.5256/f1000research.25880.r63860 Reviewer response for version 1 Zhang Zhanyuan J 1 Referee Plant Biotechnology Innovation Laboratory, Division of Plant Sciences, University of Missouri, Columbia, MO, USA

Competing interests: No competing interests were disclosed.

 27 5 2020 Copyright: © 2020 Zhang ZJ 2020 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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

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

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

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.

Zhang Yuelin University Golf Club, Canada

Competing interests: No competing interests.

 4 9 2020

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.

10.5256/f1000research.25880.r63373 Reviewer response for version 1 Hepworth Shelley 1 Referee https://orcid.org/0000-0002-6496-3792 Wang Ying 1 Co-referee Department of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada

Competing interests: No competing interests were disclosed.

 26 5 2020 Copyright: © 2020 Wang Y and Hepworth S 2020 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

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

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

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

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.

Zhang Yuelin University Golf Club, Canada

Competing interests: No competing interests.

 4 9 2020

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"