Abstract
Liverworts are key species for studies of plant evolution, occupying a basal position among the land plants. Marchantia polymorpha has emerged as a highly studied model liverwort, and many relevant techniques, including genetic transformation, have been established for this species. Agrobacterium-mediated transformation is widely used in many plant species because of its low cost. Recently, we developed a simplified Agrobacterium-mediated method for transforming M. polymorpha, known as AgarTrap (agar-utilized transformation with pouring solutions). The AgarTrap procedure, which involves culturing the liverwort tissue in various solutions on a single solid medium, yields up to a hundred independent transformants. AgarTrap is a simple procedure, requiring minimal expertise, cost, and time.
Here, we investigated four factors that influence AgarTrap transformation efficiency: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. We adapted the AgarTrap protocol for transforming intact gemmalings, achieving an exceptionally high transformation efficiency of 97%. The improved AgarTrap method will enhance the molecular biological study of M. polymorpha. The present study also provides new possibilities for improving transformation techniques for a variety of plant species.
Introduction
Marchantia polymorpha is a dioecious liverwort, the sister group to all other land plants1. This species has therefore been extensively studied to enhance our understanding of land plant evolution, with research focusing on its taxonomy, development, and physiology; furthermore, its nuclear, chloroplast, and mitochondrial genomes have all been sequenced2–6. The rapidly expanding M. polymorpha research community has recently developed various molecular biology techniques to study this key species, including particle bombardment-and Agrobacterium-mediated transformation, plastid transformation, homologous recombination-mediated gene targeting, and TALEN-and CRISPR/Cas9-mediated genome editing7–16.
Agrobacterium-mediated transformation is widely used for many plant species because it does not require any expensive equipment17. This technique involves three steps: (1) preparation of plant material, (2) co-culture of the material with Agrobacterium tumefaciens containing a recombinant transfer DNA (T-DNA), and (3) antibiotic selection of transgenic cells. During the co-culture step, T-DNA is transferred from the Agrobacterium into the plant cell, where it is integrated into the genome to facilitate the expression of its constituent genes. Previous studies have determined that the co-culture conditions are the most important aspect of transformation efficiency, with the Agrobacterium strain used, duration of co-culture, Agrobacterium density, temperature, co-culture medium, and surfactants used having the greatest impact18–22.
Recently, we developed a simplified Agrobacterium-mediated transformation method for M. polymorpha, which we named AgarTrap (agar-utilized transformation with pouring solutions)23–25. Like the general Agrobacterium-mediated transformation procedure, AgarTrap consists of three steps: (1) pre-culture of M. polymorpha tissue, (2) co-culture of the tissue with Agrobacterium containing recombinant T-DNA, and (3) selection of transgenic cells. A unique feature of AgarTrap is that none of these steps require a liquid medium culture; rather, the appropriate solutions are simply poured onto the solid medium in a single Petri dish (Fig. 1)23–25. We previously developed and optimized AgarTrap for use with M. polymorpha sporelings (S-AgarTrap), intact gemmae/gemmalings (G-AgarTrap), and pieces of mature thallus (T-AgarTrap), achieving transformation efficiencies of approximately 20%, 60%, and 70%, respectively23–25. Despite its low transformation efficiency, S-AgarTrap results in numerous transformants, because spores are produced abundantly, rendering it suitable for the large-scale production of transformants (e.g., T-DNA insertion mutants)23. However, because spores are produced by sexual reproduction, S-AgarTrap transformants do not have a uniform genetic background. G-AgarTrap can be used to produce transformants in a genetically uniform background, because the gemmae develop from single cells asexually generated within the gemma cup on a mature thallus24,26. Similarly, T-AgarTrap results in transformants with uniform genetic backgrounds, because the cells are obtained from mature thalli25; however, fewer individual transformants were obtained using T-AgarTrap than G-AgarTrap despite their respective transformation efficiencies, because the pieces of mature thallus were larger than the gemmae and fewer could be included in a single Petri dish. Thus, of these three methods, G-AgarTrap appears to be the best choice for producing transgenic M. polymorpha; however, because the transformation efficiency of G-AgarTrap was relatively low (approximately 60%), this approach needed improvement. As the co-culture step is the most critical for efficient transformation18,22, the transformation efficiency of G-AgarTrap would likely be improved by optimizing this step.
In our previous study, we optimized several factors of AgarTrap transformation, including the pre-culture period of M. polymorpha tissue, the duration of co-culture, Agrobacterium density (OD600 in transformation buffer), acetosyringone concentration in the transformation buffer, medium composition, and Agrobacterium culture conditions23–25. In the present study, we investigated four additional co-culture factors: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. We also fine-tuned the pre-culture period, ultimately achieving an exceptionally high transformation efficiency for the G-AgarTrap procedure, of nearly 100%.
Results
Humidity conditions during co-culture
In our previous study, gemmalings (BC3-38) were pre-cultured for one day and co-cultured with Agrobacterium for three days on ½ B5 medium supplemented with 1% sucrose, which resulted in a median transformation efficiency of 57.0% (mean: 59.2%) (Fig. 2a)24. Permeable microporous tape was used to seal the Petri dishes containing the solid medium; therefore, the humidity to which the plants were exposed depended on the humidity of the culture room. The humidity of the culture room was kept at approximately 40% with a humidifier, as in our previous study24. In the present study, we tested whether humidity differences in the co-culture step influence transformation efficiency. Without the humidifier, the humidity in the culture room decreased to approximately 20%. When gemmalings were co-cultured with Agrobacterium at 20% humidity, the median transformation efficiency was decreased by 8.1%, and the mean efficiency decreased by 10.5% (Fig. 2a, see also Supplementary Table S1). These results suggested that higher humidities during the co-culture step increase transformation efficiency; however, it can be challenging to control the humidity in culture rooms precisely, because humidity fluctuates depending on the location and/or season.
To maintain a high humidity in the Petri dishes during co-culture, we sealed the dishes with Parafilm, which is more airtight than microporous tape. When using Parafilm, almost all gemmalings co-cultured for three days suffered from an overgrowth of Agrobacterium such as Supplementary Fig. S1, suggesting that the growth of this bacterium is enhanced by high humidity. Since it was difficult to completely eliminate the bacteria in the subsequent selection step when they were overgrown, the co-culture period was shortened to two days when using Parafilm, which increased the median transformation efficiency to 62.3% (mean: 59.6%) in the 20% humidity condition (Fig. 2a, see also Supplementary Table S1). These results indicate that the high humidity in Parafilm-sealed Petri dishes during the co-culture step increases the transformation efficiency, while shortening the required duration of the co-culture period from three days (at 40% humidity when sealed with microporous tape) to two days.
Next, we investigated the pre-culture period of gemmae/gemmalings required when sealing the dishes with Parafilm during the co-culture step. The gemmalings were pre-cultured for 0, 1, 2, 3, and 4 days in a Petri dish sealed with microporous tape, then co-cultured for two days in a Petri dish sealed with Parafilm, which led to median transformation efficiencies of 0% (mean: 0.6%), 74.1% (mean: 62.6%), 74.1% (mean: 70.3%), 47.4% (mean: 45.8%), and 9.1% (mean: 12.2%), respectively (Fig. 2b, see also Supplementary Table S2). These results indicate that pre-culture periods of one and two days are optimal.
The use of Parafilm-sealed Petri dishes shortened the period required for the AgarTrap co-culture step. For the following investigations, we used fixed conditions of a two-day pre-culture, a two-day co-culture with Agrobacterium strain GV2260 in the light in Petri dishes sealed with Parafilm, and no surfactant in the transformation buffer. These conditions were varied as described below, to investigate their impact on transformation efficiency.
Surfactants in transformation buffer
In previous studies of Agrobacterium-mediated transformation, it was reported that the use of surfactants in the co-cultivation medium during co-culture increased the transformation efficiency27,28. We therefore examined whether surfactants in the transformation buffer influenced the efficiency of G-AgarTrap.
To determine a suitable surfactant for M. polymorpha transformation, a survival test was performed using three surfactants, Silwet L-77, Triton X-100, and Tween 20. We added various concentrations of these surfactants to the transformation buffer and treated the pre-cultured gemmalings with this buffer. After two days of co-culture, the survival rates of the gemmalings were estimated. Four concentrations of Silwet L-77 (0.01%, 0.02%, 0.05%, and 0.1%) were analyzed, resulting in mean survival rates of 100%, 100%, 98.8%, and 11.7%, respectively (Fig. 3a). When 0.01%, 0.02%, 0.05%, or 0.1% Triton X-100 was used, the mean survival rates of the gemmalings were 100%, 100%, 99.2%, and 63.5%, respectively (Fig. 3b). Because gemmalings could not survive in the higher concentrations of Silwet L-77 and Triton X-100, these surfactants may be toxic to M. polymorpha. By contrast, when Tween 20 concentrations of 0.01%, 0.02%, 0.05%, and 0.1% were tested, the mean survival rate was 100% for all concentrations (Fig. 3c). Tween 20 seemed to have no effect on gemmaling growth, and was therefore selected for use as a surfactant.
We assessed whether the use of Tween 20 in the transformation buffer increased the efficiency of G-AgarTrap. Tween 20 concentrations of 0%, 0.01%, 0.02%, 0.05%, and 0.1% resulted in median transformation efficiencies of 57.6% (mean: 59.3%), 80.0% (mean: 74.1%), 77.4% (mean: 73.8%), 70.6% (mean: 65.8%), and 54.8% (mean: 61.1%), respectively (Fig. 3d, see also Supplementary Table S3). These results showed that the use of 0.01–0.02% Tween 20 in the transformation buffer slightly increased the efficiency of G-AgarTrap transformation; however, the differences were not statistically significant. Nevertheless, when the gemmalings were co-cultured in transformation buffer, the solutions lacking surfactant were often repelled by the plants, requiring careful manipulation to ensure proper coverage. When surfactants such as Tween 20 were added to the transformation buffer, this hydrophobicity was counteracted; therefore, the addition of surfactants improves the ease of performing G-AgarTrap transformations.
Agrobacterium strain
Agrobacterium strains influence the efficiency of Agrobacterium-mediated transformations in other plant species, with the most effective strain being dependent on the plant species or transformation method used29–33. For the transformation of M. polymorpha above, and in the previous G-AgarTrap study, the GV2260 strain was used24. To assess the best strain for G-AgarTrap transformation, we compared the efficiencies of the technique using five Agrobacterium strains, GV2260, EHA101, EHA105, LBA4404, and MP9034–38. The median transformation efficiencies using these strains were 61.0% (mean: 57.6%), 96.7% (mean: 93.8%), 47.6% (mean: 47.2%), 28.3% (mean: 26.2%), and 9.2% (mean: 18.1%), respectively (Fig. 4a, see also Supplementary Table S4). The use of Agrobacterium strain EHA101 resulted in over a 90% efficiency in eight out of 10 transformations, and 100% efficiency on four occasions (Fig. 4a, see also Supplementary Table S4). EHA101 was therefore the superior strain for AgarTrap, contributing consistently high levels of transformation efficiency (Fig. 4a, see also Supplementary Table S4), which also resulted in the presence of many transformed cells within each gemmaling (Fig. 4b). Conversely, MP90 was not suitable for AgarTrap, as its use resulted in a 0% efficiency for two of 10 transformations, and only ever resulted in one or a few transformed cells within a single gemmaling (Fig. 4a, c, see also Supplementary Table S4).
We assessed the combined use of the most efficient Agrobacterium strain, EHA101, and 0.01–0.02% Tween 20 as a surfactant. When gemmalings were transformed with EHA101 in the presence of 0.01% Tween 20, the median transformation efficiency was 95.5% (mean: 93.1%), which was similar to the efficiency of EHA101-mediated transformations without a surfactant (Supplementary Fig. S2, see also Supplementary Table S5). The median transformation efficiency of EHA101 using 0.02% Tween 20 as a surfactant decreased to 17.6% (mean: 40.1%) (Supplementary Fig. S2, see also Supplementary Table S5). Thus, when using EHA101, 0.01% Tween 20 yields better results than 0.02% Tween 20.
Light/dark condition during co-culture
In previous studies of Agrobacterium-mediated transformation, light and dark conditions were reported to influence the transformation efficiency39–41. All previous studies of AgarTrap were performed under continuous white light conditions (75 µmol photons m−2 s−1) 23–25. When M. polymorpha was co-cultured with Agrobacterium strain GV2260, the median transformation efficiencies under light and dark conditions were 61.5% (mean: 61.3%) and 97.1% (mean: 95.3%), respectively (Fig. 5a, see also Supplementary Table S6). Additionally, the combined use of the most efficient Agrobacterium strain, EHA101, and dark conditions during the co-culture period resulted in a median transformation efficiency of 100% (mean: 97.0%), which was the highest efficiency observed in this study. Of the seven transformations performed in darkness using EHA101, a transformation efficiency of 100% was achieved five times (Fig. 5a, b, see also Supplementary Table S6). Numerous cells in each gemmaling were transformed under the dark condition when using either GV2260 or EHA101 (Fig. 5c, d). Thus, for the G-AgarTrap transformation of M. polymorpha, the transformation efficiency when gemmalings were co-cultured with Agrobacterium under dark conditions was higher than that under light conditions.
Discussion
To improve the efficiency of the G-AgarTrap transformation of M. polymorpha, we focused on optimizing the co-culture step for the following four factors: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. Among these factors, humidity, Agrobacterium strain, and light/dark condition contributed to increases in transformation efficiency.
Because AgarTrap is performed on a solid medium, we predicted that humidity might influence the transformation efficiency. We found that high humidities during co-culture promoted transformation efficiency, and that sealing the Petri dishes with Parafilm instead of microporous tape could overcome the problem of low culture room humidity. The high humidity also enhanced Agrobacterium growth, suggesting that this bacterium is sensitive to drying out. Sealing the Petri dishes with Parafilm might better maintain a high internal humidity than sealing the Petri dishes with microporous tape. The enhancement of Agrobacterium observed in Petri dishes sealed with Parafilm might promote transformation efficiency; however, the overgrown bacteria were difficult to completely eliminate in the subsequent selection step of G-AgarTrap. When Parafilm was used to seal the Petri dishes during two days of co-culture, efficient pre-culture periods were one and two days. This result was consistent with our previous study using microporous tape-sealed Petri dishes, in which the humidity was approximately 40%24. This suggests that the gemmaling cell states arising after 1–2 days of pre-culture might be the most suitable for transformation.
In the Agrobacterium-mediated transformation of Arabidopsis thaliana, the use of a surfactant, Silwet L-77, increases the transformation efficiency by reducing the surface tension of the aqueous solution27,42. In the present study, we did not find any significant improvement in transformation efficiency when using a range of surfactants; however, the addition of surfactants simplified the procedure by reducing the hydrophobicity of the gemmalings, which otherwise repelled the transformation solution. When 0.05% and 0.1% Tween 20 were used, the transformation efficiency using Agrobacterium GV2260 was decreased relative to the efficiency when using 0.01% and 0.02% Tween 20 solutions, even though ∼1% Tween 20 did not cause damage to M. polymorpha gemmalings. The solutions did not appear to affect the survival rate of M. polymorpha; therefore, the higher concentrations (0.05% and 0.1%) of Tween 20 might affect the bacterium itself. The inclusion of Tween 20 when using the more effective EHA101 strain also requires caution, because the transformation efficiency was greatly decreased with a 0.02% concentration of Tween 20 in the transformation solution. EHA101 might therefore be more sensitive to Tween 20 than GV2260.
The transformation efficiency of G-AgarTrap varied significantly with the use of different Agrobacterium strains; the strains yielding the highest and lowest efficiencies were EHA101 and MP90, respectively. In a previous study using tomato (Solanum lycopersicum), it was suggested that differences in transformation efficiency using different Agrobacterium strains was caused by variations in the plant tissue mortality32, which might also be the case in the present study. Additionally, for many methods using Agrobacterium-mediated plant transformation, the co-culture medium was optimized for transformation, but was also used for the culture of both plant material and Agrobacterium. By contrast, in AgarTrap, the co-culture was performed on a solid medium (½ B5 supplemented with 1% sucrose in agar) optimized for the growth of M. polymorpha, but not optimized for Agrobacterium. Thus, the solid medium might negatively affect Agrobacterium, leading to differences in transformation efficiency as a result of differences in the adaptability of the Agrobacterium strains to the medium.
It was previously reported that the EHA101 and EHA105 strains are genetically almost identical, as EHA105 was developed by the removal of a kanamycin resistance gene from EHA10137; however, in G-AgarTrap, we found a remarkable difference in transformation efficiency when using EHA101 in comparison with EHA105. This might suggest that they are less genetically similar than previously thought. This possibility remains to be investigated.
Previous reports using intact tobacco (Nicotiana tabacum) seedlings, A. thaliana root segments, and tepary bean (Phaseolus acutifolius) calli suggested that light enhanced transformation efficiency39,41, but another report using carnation (Dianthus caryophyllus) stem explants reported that dark conditions resulted in a higher proportion of transformants40. No significant differences in transformation efficiency were observed between light and dark conditions in garlic (Allium sativum)43. These differences suggest that the effects of light on transformation efficiency might depend on the plant species or tissue used. In the present study, we found that performing the co-culture in darkness significantly enhanced the transformation efficiency. The dark-mediated improvement in transformation efficiency for carnation stem explants was previously suggested to be caused by an increased susceptibility to infection in the etiolated tissue, and/or by an enhanced Agrobacterium activation40. A subsequent report confirmed that Agrobacterium activation is greater in darkness44. Plants are more susceptible to infection by pathogens at night, because the reactive oxygen species produced by photosynthesis enhance their resistance to attack45. Taken together, we hypothesize that the dark-mediated activation of Agrobacterium and the increased susceptibility to infection in the gemmaling cells in darkness result in the observed improvement in transformation efficiency when performing the AgarTrap co-culture in the dark compared with the light condition.
In this study, we successfully developed a highly efficient G-AgarTrap procedure by making several modifications (high humidity, darkness, Agrobacterium strain EHA101) to the co-culture step. The improved G-AgarTrap technique will benefit future molecular biology studies of M. polymorpha. These improved conditions may also be applicable to other AgarTrap methods (S-and T-AgarTrap). Furthermore, understanding the biological mechanisms underpinning the benefits of these improvements may contribute to the enhancement of the many other transformation technologies using Agrobacterium applied to various plant species.
Methods
Plant materials and growth conditions
Marchantia polymorpha (L.) gemmae/gemmalings of BC3-38, the female line of the third backcross generation created in the crossing of Takaragaike-1 (male line) and Takaragaike-2 (female line), were used in this study. BC3-38 was provided by Dr. Takayuki Kohchi (Kyoto University, Kyoto, Japan). The plants were maintained on half-strength Gamborg’s B5 (½ B5) medium46,47 containing 1% agar (BOP; SSK Sales Co., Ltd., Shizuoka, Japan), pH 5.5, in a 90-mm disposable sterile Petri dish. M. polymorpha tissues were illuminated with 75 µmol photons m−2 s−1 continuous white light (FL40SW; NEC Corporation, Tokyo, Japan) in a culture room maintained at around 22°C with air conditioning. The gemmae/gemmalings subjected to G-AgarTrap transformation were obtained from one-to two-month-old thalli.
G-AgarTrap
The basic procedure of G-AgarTrap was previously reported24. Gemmae were sown on approximately 10 mL ½ B5 solid medium (1% agar) supplemented with 1% sucrose, pH 5.5, in a 60-mm disposable sterile Petri dish, and pre-cultured into gemmalings. For the co-culture, 1–3 mL transformation buffer (10 mM MgCl2; 10 mM MES-NaOH, pH 5.7; 150 µM acetosyringone; Agrobacterium OD600 = 0.5) was poured over the gemmalings, with the excess being removed after 1 min using an aspirator or micropipette. Four factors were considered, including sealing of the Petri dish with Parafilm, the Agrobacterium strain used, the addition of a surfactant (0.01–0.1% Tween 20) in the transformation buffer, and dark treatment during the co-culture period. After co-cultivation, the Agrobacterium was twice washed from the gemmalings and solid medium with 1–4 mL sterile water, and then 1 mL selection buffer containing antibiotics (100 µg hygromycin B and 1 mg Claforan) was poured over the gemmalings and the solid medium. After culturing for a few weeks, the transformed cells had grown and the non-transgenic cells had died24.
Agrobacterium preparation for G-AgarTrap
Agrobacterium tumefaciens harboring the pMpGWB103-Citrine vector, which encodes bacterial aminoglycoside resistance (aadA), was stored in 30% glycerol at –80°C. On the same day that the gemmae were sown on the ½ B5 medium (the first step in the G-AgarTrap procedure), Agrobacterium was streaked onto Luria-Bertani (LB) solid medium (1% agar) supplemented with 100 mg L−1 spectinomycin and incubated at 28°C for 2–3 days (Supplementary Fig. S1a). The Agrobacterium was then suspended in transformation buffer at OD600 = 0.5 (Supplementary Fig. S1b). Surfactant (0.01–0.1% Silwet L-77, Triton X-100, or Tween 20) was included in the transformation buffer. A 1-mL aliquot of transformation buffer was poured onto each Petri dish during the co-culture step.
Microscopy observation
M. polymorpha gemmalings were observed using a MZ16F stereo fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Chlorophyll fluorescence and Citrine fluorescence (in transgenic cells) were determined using a fluorescence module (excitation filter: 480/40 nm; barrier filter: LP 510 nm). Images were taken using a DP73 digital camera (Olympus, Tokyo, Japan).
Transformation efficiency
The transformation efficiency was evaluated using the binary vector pMpGWB103-Citrine, which was transformed into Agrobacterium as described previously23–25. The T-DNA of pMpGWB103-Citrine possessed two marker genes encoding hygromycin B phosphotransferase and Citrine fluorescent protein23–25. To identify stable transformants, M. polymorpha gemmalings were selected for their ability to grow on the antibiotic hygromycin B (10 µg mL−1), and their yellow fluorescence was observed using fluorescence microscopy more than two weeks after the selection buffer was poured (transient expression of Citrine has not been observed after this time)23–25. A gemmaling containing one or more transformed cells was considered transformed24. The transformation efficiency (%) was calculated as the number of transformed gemmalings divided by the total number of gemmalings, multiplied by 100. Approximately 10–50 gemmalings per Petri dish were served for transformation. The median transformation efficiency was considered to be representative, and the mean was also reported to facilitate comparisons with previous studies. Statistics were analyzed by t-test, Tukey’s test, or Tukey-Kramer’s test.
Author contributions
S.T., S.N., H.E. and Y.K. designed the research. S.T. and Y.K. wrote the paper. S.T. performed the examinations.
Additional information
Competing financial interests: The authors declare no competing financial interests.
Acknowledgments
This work was supported by Grant-in-Aid for JSPS Fellows Grant Number 15J09907 (S.T.-T.), the Plant Transgenic Design Initiative of the University of Tsukuba (S.N., H.E., and Y.K.), and the JST-ERATO Numata Organelle Reaction Cluster Grant Number JPMJER1602 (Y.K.).