Regeneration and transient gene expression in protoplasts of Draparnaldia (chlorophytes), an emerging model for comparative analyses with basal streptophytes
Green plants comprise two lineages: (1) the streptophytes that colonised land and (2) the chlorophytes that have adaptations to land but remained mostly aquatic. To better understand what made streptophytes so successful, we are currently establishing the chlorophyte alga Draparnaldia sp. (Chaetophorales, Chlorophyceae) as a model for comparative analyses between these two lineages. However, establishing Draparnaldia as a valuable model requires that it can be transformed. Thus, our goal is to develop a transformation protocol for this alga.
We have established the first transformation protocol for Draparnaldia. This protocol is based on protoplast transformation by electroporation. It includes instructions on protoplast isolation, regeneration and transient transfection. It also provides a list of the effective selective agents for future Draparnaldia transformations.
Our protocol opens a way for Draparnaldia functional genomics analyses. Moreover, it also provides an important base for establishment of stable transformation.
KeywordsChlorophytes Colonization of land Draparnaldia Land plants Model organism Protoplasts Streptophytes Transformation
Colonization of land by plants was a major transition on Earth. Although it is generally accepted that land plants evolved from freshwater streptophyte algae, their key properties enabling such a transition are still poorly understood [1, 2, 3, 4, 5 and citations therein]. To examine these properties several basal land plant and streptophyte algal models, such as Anthoceros , Chara , Closterium , Klebsormidium , Marchantia , Mougeotia  and Physcomitrella  are (or are currently being) established. However, there are also many chlorophytes (a sister lineage to streptophyte algae and land plants) that moved to terrestrial habitats and morphologically even resemble mosses. This raises the important question of why no land plants have evolved from chlorophytes?
Draparnaldia transcriptome has recently been sequenced and will be published in a separate paper. In addition, there is a plan for genome sequencing. However, establishing Draparnaldia as a valuable model also requires that it is genetically transformable. From the variety of methods for plant and algal transformation [11, 16, 17, 18, 19, 20, 21] we focused on transformation via protoplasts. The reasons for this choice were threefold: (1) It permits regeneration of the alga from a single cell, which is crucial for developmental studies. (2) Exogenous DNA can be delivered into the cell using different methods, such as electroporation [22, 23] or polyethylene glycol (PEG)-mediated transformation [24, 25]. (3) A few reports about protoplast isolation and regeneration in filamentous chlorophytes exist [26, 27, 28], suggesting that protoplast transformation might be possible. Here, we present the first protocol for protoplast transformation of Draparnaldia. The protocol consists of four parts: protoplast isolation, regeneration, transient transfection via electroporation, and identification of effective selective agents for future Draparnaldia transformations.
Chemicals and equipment
All chemicals were of highest purity grade and were purchased from Bayer, Duchefa Biochemie, Merck, New England BioLabs, Roth, Serva, Sigma-Aldrich or Thermo Fisher Scientific (Additional file 1: Supplement 1a). Equipment list with suppliers is provided in Additional file 1: Supplement 1b.
d-Mannitol, 0.5 M was prepared one day before use. 9.1 g of d-mannitol was dissolved in 100 mL of dH2O. The pH was adjusted to 7.2 with NaOH and/or HCl. The solution was sterilized with a 0.2-μm filter and stored at the room temperature.
Driselase, 2.5% stock solution was prepared just before use. 0.25 g driselase was dissolved in 10 mL of 0.5 M Mannitol solution (in 15 mL falcon). After that it was vortexed and wrapped with the aluminum foil, incubated on a shaker (40 rpm) for 30 min at 4 °C. This was followed by its centrifugation (2500 × g for 10 min) and filter sterilization using a 0.2-μm filter.
Stock solutions for RM10 and RM1
Final concentration in RM10
Final concentration in RM1
d-Mannitol in GM
6 g/100 mL GM
Dissolve, adjust pH to 6.6 with NaOH/HCl, autoclave.
2.94 g/40 mL dH2O
Dissolve and autoclave.
The algal strain used in this study was Draparnaldia sp. CCAC 6921. The strain originates from a dry bank of the river ‘Rio Picocca’ in Sardinia, Italy. It was isolated from a leaf surface. The strain was identified by sequencing of the nuclear rRNA genes (18S, 5.8S, ITS2 and partial 28S; 2397 bp) according to Ref. . The resulting sequence was most closely related to Draparnaldia glomerata CCAP 418/2, from which it differed by 2 nucleotides in ITS2 and 28S rDNA. The newly determined sequence is available under the accession number LR597279, Project: PRJEB33155 from the European Nucleotide Archive (ENA), . The axenic strain of Draparnaldia sp. CCAC 6921 can be purchased from the Central Collection of Algal Cultures (CCAC; University of Duisburg-Essen).
Draparnaldia sp. was cultivated axenically using aerated liquid culture. The growth medium (GM) was based on Bold’s basal medium . Four vitamins were added, Vitamin B12 (0.6 μg/L), (+)-Biotin (3 μg/L), Thiamine·HCl (300 μg/L), Niacinamide (0.3 μg/L). The pH was adjusted to 6.6. Both filaments and protoplasts were grown under standard conditions as described previously . A general guide for Draparnaldia cultivation, including the long-term storage and the recipe for GM, is provided in Additional file 2.
Protoplasts were isolated according to the protocol originally developed for Physcomitrella patens , with several modifications. The modified version of the protocol is described in the Results.
Regeneration Medium (RM). GM was supplemented with mannitol for osmotic stabilisation of protoplasts and with calcium chloride to promote cell wall regeneration and subsequent division. The optimal concentration of mannitol was determined stepwise. Initially, Draparnaldia filaments were exposed to different concentrations of mannitol (0.2–0.5 M). This revealed that filaments can grow only below 0.35 M mannitol. Therefore, the lowest mannitol concentration capable of maintaining viable protoplasts was determined and chosen as the optimum. For CaCl2, the optimal concentration was determined by resuspending protoplasts in a 6-well culture plate containing 3 mL/well of RM comprising GM, mannitol and one of six concentrations of CaCl2 (0.17, 1, 2.5, 5, 7.5, 10 mM). This range was selected because 0.17 mM is the concentration used in GM of Draparnaldia and 10 mM is one of the highest concentrations used in protoplast regeneration of higher plants . Protoplasts were observed daily. Selection criteria for the optimal calcium chloride concentration included > 85% survival rate of protoplasts, cell wall formation (visualized using the Calcofluor white staining , cell division, filament formation and branching.
Optimal plating density for regeneration. Protoplasts were grown at 4 different densities (102, 103, 104, 105 protoplasts/mL) using the RM developed in step (1). These values reflect optimal densities found across many algal and plant species [33, 34, 35]. Morphological observations were made 5, 10 and 20 days after protoplast inoculation. The plating density was considered ‘optimal’ when all surviving protoplasts were able to regenerate into branched filaments.
Filament regeneration on GM. RM was beneficial for early phases of regeneration. However, the osmoticum prevented restoration of the typical Draparnaldia morphology. To enable full regeneration, filaments were grown in RM until they began to branch (5–7 days after protoplast isolation). Then, the replacement of RM by GM was initiated. To this end, two strategies were tested. First, RM was completely replaced by GM. Second, RM was replaced gradually by removing 50% of the RM and adding the same volume of GM on the day when the first branching occurred. The same procedure was repeated again on the following day.
Determination of selective agents
Ten selective agents (including zeocin—a selective marker in pChlamy_4 plasmid) were tested in Draparnaldia (see “Results”). The initial screening was performed with 4 concentrations (5, 25, 50, 100 μg/mL), representing low and high values typically used for plants and algae [e.g. 25, 37, 38, 39]. For those agents that were able to kill Draparnaldia, the minimum inhibitory concentration was determined. Experiments used 15-day old regenerated filaments (original plating density 1200 protoplasts/mL) growing in liquid GM in 24-well culture plates. One of the wells was a positive control (GM without selective agents). The percentage of surviving (i.e. green) filaments was examined 7 and 14 days after the application of selective agents.
Data collection and evaluation
All experiments were conducted in three replicates. All observations, except the transformation (see above), were performed directly in the 6- or 24-well culture plates. For establishment of the protoplast regeneration protocol, at least 200 protoplasts or filaments per well were examined. For survival tests of electroporated protoplasts and determination of effective selective agents, all filaments per dish/well were analysed. Standard deviations are shown in each graph and table.
Protocol for protoplast isolation
Before starting, prepare the biomass, material, media and reagents. For instructions of how to grow the liquid aerated culture of Draparnaldia see Additional file 2.
Harvest 3–4 g of fresh biomass of 7–10-day-old aerated culture using the polyester (PET) mesh (pluriSelect) with a pore size of 40 μm.
Note: 3–4 g of biomass corresponds approximately to 60–90 mL of culture, depending on the density. 1 g of fresh biomass yields between 3–4 × 106 protoplasts/mL.
Transfer the harvested biomass into a Petri dish (100 × 20 mm) using forceps.
To introduce plasmolysis, resuspend the biomass in 12 mL 0.5 M mannitol and seal the Petri dish with Parafilm.
Incubate the Petri dish for 35–40 min on rotator (70 rpm shaking, room temperature).
While waiting, prepare 2.5% driselase solution. For instructions see “Methods” section.
Add 4.5 mL of sterile driselase to the Petri dish with biomass and mannitol. Seal the Petri dish with Parafilm and cover it with aluminium foil to protect protoplasts from strong light.
Note: The final concentration of driselase is > 0.68%.
Incubate the Petri dish with the mixture on a rotary shaker (30–40 rpm shaking, room temperature) until the majority of biomass breaks down. This takes about 45–60 min.
Note: Protoplast isolation from older biomass (approximately 15 days old aerated culture) is possible, but not recommended. It takes 2–3 h longer and it is less efficient. It yields about 103–104 protoplast/g of biomass. The same holds true for non-aerated Draparnaldia cultures.
Check the viability of protoplasts under the light microscope. Abundant free-floating protoplasts and remnants of undigested biomass should be present.
Note: An inverted microscope for tissue cultures (e.g. CK × 4, Olympus, Tokyo, Japan) is recommended. It allows to check the status of the protoplasts directly in the Petri dish.
To separate protoplasts from the majority of undigested biomass, filter the mixture from the Petri dish through the polyester (PET) mesh (pluriSelect) with a pore size of 40 μm.
Transfer the filtrate (containing protoplasts and short filaments) back to the Petri dish from Step 10. Seal the Petri dish with Parafilm and cover it with aluminium foil.
Incubate the Petri dish containing the filtrate on the rotary shaker for another 15 min (30–40 rpm shaking, room temperature). This step allows enzymatic digestion of the majority of remaining filament fragments.
To separate protoplast from the residual undigested filaments, filter once more through the polyester (PET) mesh (pluriSelect) with a pore size of 15 μm.
Since many protoplasts remain attached to the Petri dish, rinse the Petri dish with an additional 3 mL 0.5 M mannitol and filter the suspension through the same mesh as used in Step 13.
To separate protoplasts from the remaining cell wall debris, refilter the filtrate from the Step 13 through the polyester (PET) mesh (pluriSelect) with a pore size of 10 μm.
To wash away the rest of driselase, split the filtered protoplast suspension into two 15 mL glass Screw Cap Culture Tubes.
Centrifuge at 50 × g for 10 min with the acceleration and brake set to 3 (Eppendorf Centrifuge 5810R, rotor A-4-62).
Discard the supernatant.
Resuspend the protoplasts in 5 mL 0.5 M mannitol by gently rotating the tubes.
Repeat Steps 17 and 18.
Resuspend the protoplasts in 5 mL 0.5 M mannitol by gently rotating the tubes and combine the contents of both tubes.
Repeat Steps 17 and 18.
Resuspend the protoplasts in 4 mL RM 10 and set aside a small aliquot (about 400 μL) of the protoplast suspension for quantitative and qualitative assessment of protoplasts.
Check the viability of protoplasts using the light microscope (alternatively, use the fluorescein diacetate dye (FDA ). Determine protoplast density using a hemocytometer  or measure the OD750. Confirm the loss of cell wall using Calcofluor white M2R .
Note: In total 15 protoplast isolations have been performed based on this protocol. The viability of protoplast was regularly about 95%. Protoplast density varied from 3–4 × 106 protoplasts/mL. Protoplasts showed no calcofluor white fluorescence.
Next, we established an efficient protocol for regeneration of the isolated protoplasts, i.e. cell wall regeneration and restoration of the original morphology. This was done in three steps.
Influence of calcium chloride on protoplast regeneration in Draparnaldia
Third, we replaced RM by GM. Because the complete replacement of RM was too drastic (> 50% filaments died), gradual replacement (with 100% survival of filaments) was chosen for the protocol (see Steps 4 and 5).
Protocol for protoplast regeneration
Pre-incubate protoplasts for 24 h in 4 mL RM10 in a glass Screw Cap Culture Tube. Gently resuspend the protoplasts at least 3 ×/day.
Note: Do not pre-incubate the protoplasts in Petri dishes or plates. Unlike protoplasts from embryophytes, those of Draparnaldia attach to the surface and cannot be removed without damage. Protoplasts can be kept in RM10 up to 5 days, after that the regeneration rate decreases to about 70%.
Adjust the density of protoplasts in RM1 medium to 102–103/mL.
Note: Use cut filter tips for manipulation with protoplasts. This will eliminate their damage.
Incubate protoplasts in RM1 until the first branches occur. Importantly, protoplasts will not regenerate in a glass tube, they must be incubated in a Petri dish or in a culture plate.
Note: Dish (plate) should not be filled to more than 50% to allow for proper gas exchange. Seal the dish (plate) with Parafilm to avoid contamination and evaporation.
As soon as branching is observed (usually 5–7 days after incubation in RM1) remove 50% of the RM and add the same volume of GM.
Note: The regenerated filaments are firmly attached to the surface using their Prostrate System (PS and dashed line in Fig. 7). Therefore, the majority of them will not be washed out during the media exchange.
Repeat Step 4 on the next day.
Transfection of protoplasts
Protocol for transfection of protoplasts
Pre-incubate protoplasts for 24 h in 4 mL RM10 in a glass Screw Cap Culture Tube. Gently resuspend the protoplasts at least 3 ×/day.
Measure protoplast density by OD750: Pipette 900 μL RM10 into the 1.5 mL semi-micro cuvette (12.5 × 12.5 × 4.5 mm). Add 100 μL of the protoplast suspension and stir gently with a cut filter tip. Perform measurement and throw the sample away.
Note: Using cut filter tips for manipulation with protoplasts will eliminate their damage.
Resuspend protoplasts from Step 1 in RM10 to a final OD750 of 0.5.
Note: Again, use cut filter tips for manipulation with protoplasts.
Incubate protoplasts on ice for 20 min.
Pipette 400 μL of the cooled protoplast suspension with cut filter tip into the 1.5 mL pre-chilled microtube.
Add approximately 1 μg of linearized plasmid and stir gently with a cut filter tip.
Using a cut filter tip, transfer the mixture to a pre-chilled 0.4-cm electroporation cuvette with a 2 mm gap.
Perform electroporation (electrical pulse 300 V, 2 ms).
After electroporation, incubate protoplasts on ice for additional 3 min. While waiting, prepare a Petri dish for protoplast regeneration: Place three sterile square coverslips on the bottom of a 60 × 15 mm Petri dish and add 7 mL of RM1.
Using a cut filter tip, pipette the protoplast suspension out of the cuvette and placed it on the top of coverslips.
Note: Note that if coverslips are not applied protoplasts attach to the surface of the Petri dish and cannot be collected for microscopy without damage.
Seal the Petri dish with Parafilm to prevent evaporation during regeneration.
Regenerate protoplasts under standard conditions as described in Ref. .
Note: Incubation of protoplasts in the dark is not required.
Effects of different selective agents on Draparnaldia (the wild-type)
In this study we developed a protoplast transformation protocol for Draparnaldia.
The yield of protoplasts from a single isolation (106 protoplasts) was comparable with protoplast yields reported for well-established model organisms, such as Physcomitrella patens  or Arabidopsis thaliana [43, 44]. Similarly to those organisms, a high protoplast yield could only be achieved when young plant/algal material was used (see “Results”). Probably older plant/algal cells have more complex rigid cell walls, which are more resistant to enzymatic digestion [45, 46, 47]. To generate young homogenous biomass, we recommend to grow Draparnaldia in aerated liquid culture (Additional file 2). The combination of fresh medium and aeration transfers the majority of cells (more specifically their protoplasts) into zoospores that give rise to new filaments. The intensity of aeration should be adjusted to ensure homogeneous culture suspension, which is important for equal growth of filaments. Note, that Draparnaldia can also theoretically be transformed by using zoospores (flagellates lacking a cell wall), like gametes of seaweed Ulva . However, Draparnaldia zoospores do not have a strong phototaxis and stay motile for only a few minutes before attaching to the substrate. This makes their collection and purification very difficult and thus prevents them from being used as an efficient source for transformation.
Protoplast regeneration was based on the same parameters as regeneration in other plant/algal organisms [16, 19, 46, 48, 49, 50, 51, 52, 53, 54]. These parameters were: optimized osmoticum, calcium chloride and plating density of protoplasts (see “Methods” section). Note, that similar to mosses, no addition of plant hormones was needed. Importantly, Draparnaldia protoplasts can be regenerated in Petri dishes or multiwell culture plates, but not in glass tubes. This problem does not seem to be related to the glass material itself (protoplasts can regenerate on coverslips, see “Results”), but rather to some other conditions such as suboptimal light or gas exchange. Another interesting observation was that the majority of protoplasts differentiated into zoospores prior to their regeneration (see “Results”). In fact, this behaviour mimics natural development of Draparnaldia (compare Figs. 2 and 7) and it can be explained by using young biomass for protoplast isolation. Young biomass is known to be favorable for zoospore formation [13, 55]. Moreover, Draparnaldia zoospores can be easily induced by abiotic stresses, such as medium exchange [13, 56]. Therefore, it is likely that the observed zoospores were either initiated during protoplast isolation (probably when GM was replaced by mannitol) or when protoplasts were transferred into RM.
We also showed that Draparnaldia protoplasts are capable of being transiently transfected by electroporation. Thus, the next goal will be to determine whether they can be stably transformed. Success in achieving this goal will require optimization of the electroporation as well as testing alternative approaches of transgene delivery. This includes alternative procedures for direct uptake of DNA by protoplasts [19, 25], particle bombardment [57, 58] as well as Agrobacterium-mediated conjugative transformation . Also, it will be important to determine whether stable transformation occurs via homologous recombination (like in Physcomitrella, [60, 61]) or random integration (like in most land plants, ).
Finally, we demonstrated that regenerated protoplasts (= the wild type) are sensitive to several commonly used selective agents (see “Results”. This sensitivity to multiple agents is advantageous for at least two reasons. (1) It might allow a number of molecular applications, including gene stacking, generation of T-DNA mutant libraries and subsequent mutant complementation, and assays where multiple reporter genes are required such as BiFC [63, 64]. (2) It also suggests that many available antibiotic/herbicide reporter genes could potentially be used for Draparnaldia transformation.
Here we present the first protoplast transformation protocol for Draparnaldia sp.—the emerging chlorophyte model for comparative analysis with early streptophytes. The protocol yields 106 protoplasts per isolation. It allows full regeneration of > 90% of protoplasts and enables protoplast transient transfection by electroporation. To further expand this toolset, we also identified selective agents that are suitable for selection of future Draparnaldia transformants. This protocol opens a way for functional analyses in Draparnaldia. It also provides the first step towards establishing stable transformation in Draparnaldia. Although our protocol has been established for Draparnaldia, we believe that it can be used as a reference to develop transformation protocols in other chlorophyte as well as streptophyte filamentous algae.
We would like to acknowledge O. Artz (Cologne University, Germany), A. C. Cuming (Leeds University, UK), H. Buschmann (Osnabrück University, Germany) and P. F. Perroud (Marburg University, Germany) for sharing their experience with moss and streptophyte algal transformation. We are grateful to A. C. Cuming and S. Kopriva (Cologne University, Germany) for comments on the manuscript.
LC conceived and designed the study, performed the experiments, wrote the paper. TOJ guided protoplast transfection experiments, performed vector modification, edited the paper. Both authors read and approved the final manuscript.
This work was supported by the German Research Foundation (DFG Grant CA 1321/3-1, to LC).
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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