Caisová and Jobe Plant Methods (2019) 15:74 https://doi.org/10.1186/s13007-019-0460-6 Plant Methods METHODOLOGY Open Access Regeneration and transient gene expression in protoplasts of Draparnaldia (chlorophytes), an emerging model for comparative analyses with basal streptophytes Lenka Caisová1* and Timothy O. Jobe2 Abstract Background: 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 suc- cessful, 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. Results: We have established the frst transformation protocol for Draparnaldia. This protocol is based on protoplast transformation by electroporation. It includes instructions on protoplast isolation, regeneration and transient transfec- tion. It also provides a list of the efective selective agents for future Draparnaldia transformations. Conclusions: Our protocol opens a way for Draparnaldia functional genomics analyses. Moreover, it also provides an important base for establishment of stable transformation. Keywords: Chlorophytes, Colonization of land, Draparnaldia, Land plants, Model organism, Protoplasts, Streptophytes, Transformation Background resemble mosses. Tis raises the important question of Colonization of land by plants was a major transition why no land plants have evolved from chlorophytes? on Earth. Although it is generally accepted that land To better understand what made streptophytes so plants evolved from freshwater streptophyte algae, their successful, we are currently establishing the freshwater key properties enabling such a transition are still poorly multicellular chlorophyte alga Draparnaldia sp. (Chae- understood [1–5 and citations therein]. To examine these tophorales, Chlorophyceae) as a model for comparative properties several basal land plant and streptophyte algal analyses between these two lineages. For the phyloge- models, such as Anthoceros [6], Chara [7], Closterium netic position of Draparnaldia in the green tree of life [8], Klebsormidium [9], Marchantia [10], Mougeotia [11] see Fig. 1. Draparnaldia possesses a broad range of and Physcomitrella [12] are (or are currently being) estab- adaptations to aquatic and terrestrial habitats. It dis- lished. However, there are also many chlorophytes (a plays complex morphology similar to mosses and some sister lineage to streptophyte algae and land plants) that streptophyte algae: branching flaments, rhizoids with moved to terrestrial habitats and morphologically even apical growth, and tissue specialization [13–15]. It also reproduces in a similar manner as many streptophyte algae, for Draparnaldia life cycle see Fig. 2. Moreo- *Correspondence: [email protected] ver, it is well positioned phylogenetically. It belongs to 1 Centre for Plant Sciences, Faculty of Biological Sciences, University the Chaetophorales (fg. 1 in Ref. [13]), whose species of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK Full list of author information is available at the end of the article range from unbranched flaments with a single-celled © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Caisová and Jobe Plant Methods (2019) 15:74 Page 2 of 14 Chlorophyceae Land plants Chlamydomonas (Chlamydo- Anthoceros, monadales) Marchantia, Draparnaldia Physcomitrella (Chaetophorales) Zygnematophyceae Ulvophyceae Closterium, Mougeotia Chlor Coleochaetales Trebouxiophyceae ophytes Charales Chlorodendrophyceae Chara Streptophytes Klebsormidiales Pedinophyceae Klebsormidium Prasinophytes Mesostigmatales, Chlorokybales flagellated alga Fig. 1 Phylogenetic position of Draparnaldia in the green tree of life. In addition to Draparnaldia, a well-established Chlamydomonas chlorophyte model species as well as several streptohyte models are shown. The tree schematic is based on Ref. [4, 65–67]. Dash-lined lineages are probably not monophyletic attachment to branched flaments with multi-celled studies. (2) Exogenous DNA can be delivered into the rhizoids. Tus, it enables comparison of complex fla- cell using diferent methods, such as electroporation mentous body development between chlorophytes and [22, 23] or polyethylene glycol (PEG)-mediated trans- basal streptophytes. All these features make Drapar- formation [24, 25]. (3) A few reports about protoplast naldia an attractive model to distinguish properties isolation and regeneration in flamentous chlorophytes that are unique to streptophytes from those that are exist [26–28], suggesting that protoplast transforma- common to both chlorophytes and streptophytes. tion might be possible. Here, we present the frst pro- Draparnaldia transcriptome has recently been tocol for protoplast transformation of Draparnaldia. sequenced and will be published in a separate paper. Te protocol consists of four parts: protoplast isolation, In addition, there is a plan for genome sequencing. regeneration, transient transfection via electroporation, However, establishing Draparnaldia as a valuable and identifcation of efective selective agents for future model also requires that it is genetically transform- Draparnaldia transformations. able. From the variety of methods for plant and algal transformation [11, 16–21] we focused on transfor- Methods mation via protoplasts. Te reasons for this choice Chemicals and equipment were threefold: (1) It permits regeneration of the alga All chemicals were of highest purity grade and were from a single cell, which is crucial for developmental purchased from Bayer, Duchefa Biochemie, Merck, Caisová and Jobe Plant Methods (2019) 15:74 Page 3 of 14 US akinetes young germling US germination PS US US US PS asexual asexual zoospore formation akinete formation (aquatic habitat) zoospore US US US young PS US PS zygote microzoospores sexual(?) fusion Fig. 2 Life cycle of Draparnaldia. Two types of asexual reproduction are presented. Zoospores are strictly aquatic reproductive stages and have a distinct Upright Sytem (US) and Prostrate System (PS). Akinetes ( resting stages) are zoospores arrested in a parental flament. They are formed = during drought periods and enable a long-term survival in the terrestrial habitat. However, water availability is required for their germination. The germinating akinetes have only the US, the PS is formed later. Note, that Draparnaldia is also capable of fragmentation (not shown). Fragmentation is initiated with algal transition from the aquatic to the terrestrial habitat. It leads to the flament splitting into two new flaments with a fully developed UP and PS. A general mechanism of the fragmentation process has been described in Ref. [29]. In addition, a sexual reproduction of Draparnaldia has been reported [68], but it has not been confrmed for this specifc strain New England BioLabs, Roth, Serva, Sigma-Aldrich or was adjusted to 7.2 with NaOH and/or HCl. Te solution Termo Fisher Scientifc (Additional fle 1: Supplement was sterilized with a 0.2-μm flter and stored at the room 1a). Equipment list with suppliers is provided in Addi- temperature. tional fle 1: Supplement 1b. Driselase, 2.5% stock solution was prepared just before use. 0.25 g driselase was dissolved in 10 mL of 0.5 M Reagent setup Mannitol solution (in 15 mL falcon). After that it was d-Mannitol, 0.5 M was prepared one day before use. 9.1 g vortexed and wrapped with the aluminum foil, incubated of d-mannitol was dissolved in 100 mL of dH2O. Te pH on a shaker (40 rpm) for 30 min at 4 °C. Tis was followed Caisová and Jobe Plant Methods (2019) 15:74 Page 4 of 14 several modifcations. Te modifed version of the proto- by its centrifugation (2500 × g for 10 min) and flter steri- lization using a 0.2-μm flter. col is described in the Results. Regeneration medium 10 and 1 (RM10 and RM1) was derived from Growth medium (GM) by adding d-man- Protoplast regeneration nitol and calcium chloride (Table 1). Teir names refer Te protocol for protoplast regeneration in liquid to the fnal concentration of calcium chloride. For RM10, medium was developed in three steps. 10 mL of Stock solution 1 was mixed with 196.6 μL of Stock solution 2. For RM1, 9 mL of Stock solution 1 was 1. Regeneration Medium (RM). GM was supplemented mixed with 1 mL of RM10. Te media and stock solu- with mannitol for osmotic stabilisation of proto- tions can be stored at 4 °C for at least 2 weeks. plasts and with calcium chloride to promote cell wall regeneration and subsequent division. Te optimal Draparnaldia origin concentration of mannitol was determined stepwise. Te algal strain used in this study was Draparnaldia sp. Initially, Draparnaldia flaments were exposed to dif- CCAC 6921. Te strain originates from a dry bank of the ferent concentrations of mannitol